Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Cellular switch molecule for sperm motility control: a novel target for male contraception and infertility treatments
Reporter and Curator: Sudipta Saha, Ph.D.
Researchers have discovered the cellular switch that boosts the activity of sperm cells so that they can travel to the egg. The finding may lead to new options for male contraception as well as treatments for infertility resulting from problems with sperm mobility.
Inside the male reproductive tract, mature sperm are capable of limited movement. This limited movement, however, is not enough to propel them toward the egg when they enter the female reproductive tract. To begin their journey, they must first be activated by the hormone progesterone, which is released by the egg.
The researchers reported that the molecule to which progesterone must bind is the enzyme alpha/beta hydrolase domain containing protein 2 (ABHD2), found in the sperm cell’s outer membrane. Similarly, strategies to bypass or enhance the enzyme might provide therapies for treating infertility resulting from sperm that lack movement capability.
Before a sperm can transition to the hyper-active phase, calcium must pass through the cell’s outer membrane and enter the flagella, the tail-like appendage the cell uses to propel itself. The sperm protein known as CatSper joins with similar proteins in the flagella to allow the entry of calcium.
When the researchers undertook the current study, it was not known whether progesterone interacted directly with CatSper to trigger the calcium influx, or acted on some other molecule (which, in turn, acted on CatSper). Before treating sperm with progesterone, the researchers exposed them to a chemical that inhibits a particular class of enzymes that they believed could include the candidate molecule that acted on CatSper. The hunch proved correct: the treated cells remained inactive after progesterone exposure, indicating that CatSper was not directly involved.
Working with modified progesterone, the researchers eventually isolated ABHD2 from the sperm tails. When the researchers inactivated ABHD2, exposure to progesterone failed to activate the sperm cells, confirming that ABHD2 is the molecular target for progesterone.
All of the technical terminology aside, this means that the researchers have pinned down the cellular switch that boosts the sperm along to the egg, so by blocking the ABHD2 activity, new male birth control methods could be on the way. Conversely, enhancing the enzyme could lead to new treatments for male infertility.
It will be interesting to see how this discovery impacts future research concerning male birth control and infertility treatments. Perhaps it’s the missing piece of information that will quickly yield an effective new male contraception option.
Vitamin D–Binding Protein and Vitamin D Status of Black Americans and White Americans
CE Powe, MK Evans, J Wenger, AB Zonderman, AH Berg, M Nalls, H Tamez, et al.
N Engl J Med 21 Nov,2013; 369:1991-2000 http://dx.doi.org/10.1056/NEJMoa1306357
Summary
BACKGROUND
Low levels of total 25-hydroxyvitamin D are common among black Americans. Vitamin D–binding protein has not been considered in the assessment of vitamin D deficiency.
METHODS
In the Healthy Aging in Neighborhoods of Diversity across the Life Span cohort of blacks and whites (2085 participants), we measured
levels of total 25-hydroxyvitamin D,
vitamin D–binding protein, and
parathyroid hormone as well as
bone mineral density (BMD).
We genotyped study participants for two common polymorphisms in the vitamin D–binding protein gene (rs7041 and rs4588). We estimated levels of bioavailable 25-hydroxyvitamin D in homozygous participants.
RESULTS
Mean (±SE) levels of both total 25-hydroxyvitamin D and vitamin D–binding protein were lower in blacks than in whites (total 25-hydroxyvitamin D, 15.6±0.2 ng per milliliter vs. 25.8±0.4 ng per milliliter, P<0.001; vitamin D–binding protein, 168±3 μg per milliliter vs. 337±5 μg per milliliter, P<0.001).
Genetic polymorphisms independently appeared to explain 79.4% and 9.9% of the variation in levels of vitamin D–binding protein and total 25-hydroxyvitamin D, respectively.
BMD was higher in blacks than in whites (1.05±0.01 g per square centimeter vs. 0.94±0.01 g per square centimeter, P<0.001).
Levels of parathyroid hormone increased with decreasing levels of total or bioavailable 25-hydroxyvitamin D (P<0.001 for both relationships),
yet within each quintile of parathyroid hormone concentration, blacks had significantly lower levels of total 25-hydroxyvitamin D than whites.
Among homozygous participants, blacks and whites had similar levels of bioavailable 25-hydroxyvitamin D overall (2.9±0.1 ng per milliliter and 3.1±0.1 ng per milliliter, respectively; P=0.71) and
within quintiles of parathyroid hormone concentration.
CONCLUSIONS
Community-dwelling black Americans, as compared with whites, had low levels of total 25-hydroxyvitamin D and vitamin D–binding protein,
resulting in similar concentrations of estimated bioavailable 25-hydroxyvitamin D.
Racial differences in the prevalence of common genetic polymorphisms provide a likely explanation for this observation. (Funded by the National Institute on Aging and others.)
Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome
Larry H Bernstein: Author
and
Reporter: Aviva Lev-Ari, PhD, RN
Mitochondria, the cardiovascular system and metabolic syndrome
Start date
April 24, 2013
End date
April 24, 2013
Venue
London, UK / Kennedy Lecture Theatre, Institute of Child Health
Location
London, UK
Topics
– Mitochondrial ROS metabolism in the heart
– Mitochondrial permeability transition pore
– Mitochondria in vascular smooth muscle
– Therapeutic targets for cardiac disease
Invited speakers
This event has now passed – please visit our Conference calendar for future Abcam events
Confirmed speakers:
Paolo Bernardi, University of Padova, Italy ‘The mitochondrial permeability transition pore: A mystery solved?’
Susan Chalmers, University of Strathclyde Glasgow ‘Mitochondria in vascular smooth muscle: from regulation of calcium signals to control of proliferation’
Andrew Hall, UCL ‘The role of sirtuin 3 in cardiac dysfunction’
Derek Hausenloy, UCL ‘Mitochondrial dynamics as a therapeutic target for cardiac disease’
Guy Rutter, Imperial College London ‘Mitochondria and insulin secretion – links to diabetes’
Michael Murphy, MRC Mitochondiral Biology Unit, Cambridge ‘Exploring mitochondrial ROS metabolism in the heart using targeted probes and bioactive molecules’
Toni Vidal Puig, Institute of Metabolic Science, University of Cambridge ‘Adipose tissue expandability, lipotoxicity and the metabolic syndrome’
Calcium signaling is instrumental for excitation-contraction coupling (ECC). The involvement of mitochondria in establishing rapid cytosolic calcium transients in this process remain debated.
Two models have emerged:
slow integration versus rapid and
ample beat-to-beat changes of
cytosolic calcium transients into the mitochondria matrix.
a brief outline of cardiac calcium signaling »
Mitochondrial Calcium transport mechanisms
Calcium influx can be mediated by:
Mitochondrial Calcium Uniporter (MCU)
Mitochondrial Ryanodine receptor type 1 (mRyR1)
Leucine-zipper-EF-hand-containing transmembrane protein 1 (LETM1)
BALTIMORE — Two new studies contribute further to the debate over the cardiovascular risk associated with supplementary or dietary calcium, each decidedly coming down on the side of no significant risk — to men or women.
“[Based on these findings], clinicians should continue to evaluate calcium intake, encourage adequate dietary intake, and if necessary, use supplements to reach but not exceed recommended intakes,” Douglas C. Bauer, MD, from the University of California, San Francisco, the lead author of the first study, told Medscape Medical News.
Results of both studies were reported at the recent American Society for Bone and Mineral Research (ASBMR) 2013 Annual Meeting.
Dr. Bauer’s observational trial is one of few contemporary studies to evaluate the level of risk among men, who are often poorly represented in calcium studies, he noted. The results showed no association between calcium dietary intake or supplementation and total or cardiovascular mortality. The second study was an updated meta-analysis of calcium supplementation among women and similarly demonstrated no increased risk for ischemic heart disease (with adjudicated outcomes) or total mortality in elderly women. It did draw some criticism for potential bias and contamination, however.
Nevertheless, says Robert Marcus, MD, a retired Stanford University bone specialist, the 2 studies are “powerful. The one involving men had very elegant, accurate reports of death and validated diagnosis of myocardial infarction, and the [study involving women] was also excellent work,” he commented.
“This field has been the subject of an enormous amount of controversy, ambiguity, and confusion for the past several years, and I think the most important thing is to help us come up with what is true,” he said. The quality of data to suggest an adverse effect of calcium is “very poor,” and there is now compelling evidence that there is little to substantiate this, he noted. But despite these reassuring new findings, public anxiety over a potential risk with calcium is unlikely to go away, he believes.
In recommendations issued in 2010, the ASBMR said that most adults 19 years of age and older require about 600 to 800 IUs of vitamin D daily and 1000 to 1200 mg of calcium daily through food and with supplements, if needed.
Contemporary Data on Calcium Intake in Men
The use of calcium supplements, predominantly with vitamin D, is an important therapy for the prevention of osteoporosis and its clinical consequences. But concerns about the cardiovascular safety of calcium have emerged periodically; in 2 alarming meta-analyses published in 2010 and 2011 by Dr. Mark Bolland and colleagues, for example, there was a 27% increase in MI among individuals allocated to calcium supplements in the first study and a 24% increased risk in the second.
More recently, a 40% increase in total mortality and up to a 50% increase in cardiovascular mortality was seen among women from a Swedish mammography cohort with a calcium intake exceeding 1400 mg per day. In that study, the effect on mortality appeared to be especially strong if a high dietary intake of calcium was combined with calcium supplements.
In their new study, Dr. Bauer and his colleagues set out to assess rates of dietary calcium intake, use of supplements, and mortality in a prospective cohort of 5967 men over the age of 65 years in the Osteoporotic Fractures in Men (MrOS) study.
The participants completed extensive surveys at baseline on their dietary calcium intake, and supplementation was verified by a review of pill bottles by trained staff.
Mean dietary calcium intake was 1142 ± 590 mg/day, with more than half — 65% — of participants reporting use of calcium supplements.
Over the 10-year follow-up, among 2022 men who died, 687 deaths were caused by cardiovascular disease. The highest mortality for CVD was seen in the quartile with the lowest intake from calcium supplementation.
And in models that adjusted for age, energy intake, and calcium use, men in the lowest quartile of total calcium intake (less than 621 mg per day) had higher total mortality compared with those in the highest quartile (more than 1565 mg of calcium per day).
Adjustment for additional confounding factors showed no association between calcium dietary intake and total or cardiovascular mortality (P for trend .51 and .79, respectfully). Likewise, there was no association between calcium supplementation and total or cardiovascular mortality.
The authors also conducted an additional analysis of calcium intake and adjudicated cardiovascular disease events in a subcohort of the study, MrOS Sleep, involving 3120 patients who took part in a 7-year follow-up, and again there was no higher risk for cardiovascular events associated with calcium intake.
The study did have is limitations, Dr. Bauer acknowledged, including the observational design, calcium intake being assessed with a food frequency questionnaire, and cause of death not being formally adjudicated. Nevertheless, the findings are important in demonstrating the level of risk among men in a contemporary setting, he pointed out.
“Contrary to the Swedish study of women, we found no evidence that calcium supplementation is harmful to men, even among those with the highest dietary calcium intake,” he concluded, recommending that future studies should include adjudicated outcomes.
Study in Men as Expected, but Female Research Questioned
In the second study reported at the ASBMR meeting, Joshua Lewis, MD, PhD, from the University of Western Australia, Perth, and colleagues reported a meta-analysis of 19 randomized controlled trials involving women over the age of 50 years who had received calcium supplementation for more than a year.
Importantly, the analysis included reports of adjudicated cardiovascular outcomes, which the researchers note is important because gastrointestinal events can be misreported as cardiac ones. They also assessed all-cause mortality.
Among 59,844 participants in the studies, there were 4646 deaths, and the relative risk for death among those randomized to calcium supplements was 0.96 (P = .18).
The relative risk for 3334 ischemic heart disease events among 46,843 participants was 1.02 (P = .053), and the risk for 1097 MI events among 49,048 participants was 1.09 (P =.21).
“The data from this meta-analysis does not support the concept that calcium supplementation with or without vitamin D increases the risk of ischemic heart disease or total mortality in elderly women,” concluded Dr. Lewis.
But bone specialist Ian Reid, MD, from the University of Auckland, New Zealand, who was a coauthor on some of the Bolland studies, said this analysis essentially repeats previous ones, but with the inclusion of 20,000 patients from the Women’s Health Initiative (WHI), many of whom continued to take their own calcium tablets, regardless of whether they were randomized to calcium or placebo.
These “contaminated” WHI data have the potential to mask the effect of calcium, he told Medscape Medical News. In addition, in a study from Denmark also included in the meta-analysis, subjects were not properly blinded to treatment assignment and the calcium and control groups were not comparable at baseline for cardiovascular risk, which introduced “major potential bias,” he added.
Regarding the results from the study in men by Dr. Bauer and colleagues, Dr. Reid said they were not surprising to him. “Generally, people who take calcium supplements have more health-conscious behaviors than those who do not and so have a lower baseline risk of heart disease” that can “obscure small adverse effects of drugs such as calcium,” he observed.
An effect has to be “very substantial” before it can be picked up in an observational study, because of the many confounders that can obscure such an effect, he concluded.
Dr. Bauer, Dr. Lewis, Dr. Reid, and Dr. Marcus have reported no financial relationships. MrOS is supported by funding from the National Institutes of Health.
American Society for Bone and Mineral Research 2013 Annual Meeting. Abstracts 1001 and 1002, presented October 4, 2013.
Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Author, Introduction: Larry H Bernstein, MD, FCAP
Author, Summary: Justin Pearlman, MD, PhD, FACC
and
Article Curator: Aviva Lev-Ari, PhD, RN
Image created by Adina Hazan 06/30/2021
This article is the Part VIII in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells. Calcium has a storage and release cycle that flags activation of important cellular activities that include in particular the cycle activation of muscle contraction both to circulate blood and control its pressure and distribution. Homeostasis – the maintenance of status – requires controlled release of calcium from storage and return of calcium to storage. Such controls are critical both within cells, and for the entire biologic system. Thus the role of kidneys in maintaining the correct total body load of available calcium is just as vital as the subcellular systems of calcium handling in heart muscle and in the muscles that line arteries to control blood flow. The practical side to this knowledge includes not only identifying abnormalities at the cellular as well as system levels, but also identifying better opportunities to characterize disease and to intervene.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Vascular Smooth Muscle Cells: The Cardiovascular Calcium Signaling Mechanism
Section Two:
Cardiomyocytes Cells: The Cardiac Calcium Signaling Mechanism
Section Three:
The Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
Introduction
by Larry H Bernstein, MD, FACC
Introduction
This discussion in two Sections brings to a conclusion the two main aspects of calcium signaling and transient current induction in the cardiovascular system – involving vascular smooth muscle and cardiomyocyte. In this first Section, it extends the view of smooth muscle beyond the vascular smooth muscle to contraction events in gastrointestinal tract, urinary bladder, and uterus, but by inference, to ductal structures (gallbladder, parotid gland, etc.). This discussion also reinforces the ECONOMY of the evolutionary development of these functional MOTIFS, as a common thread is used again, and again, in specific contexts. The main elements of this mechanistic framework are:
the endoplasmic (sarcoplasmic) reticulum as a strorage depot for calcium needed in E-C coupling
the release of Ca(2+) into the cytoplasm
the generation of a voltage and current with contraction of the muscle cell unit
the coordination of smooth muscle cell contractions (in waves)
this appears to be related to the Rho/Rho kinase pathway
there is also a membrane depolarization inherent in the activation mechanism
whether there is an ordered relationship between the calcium release and the membrane polarization, and why this would be so, in not clear
three different models of calcium release are shown from the MJ Berridge classification article below in Figure 1.
Model C is of special interest because of the focus on cytosolic (Ca+) ion transfers involving the interstitial cells of Cajal (Ramin e’ Cajal) through gap junctions
Classification of Smooth Muscle Ca2+ Activation Mechanisms
Excitation–contraction coupling in SMCs occurs through two main mechanisms. Many SMCs are activated by Ca2+ signalling cascades (Haddock & Hill, 2005; Wray et al. 2005). In addition, there is a Rho/Rho kinase signaling pathway that acts by altering the Ca2+ sensitivity of the contractile system (Somlyo & Somlyo, 2003). Since the latter appears to have more of a modulatory function,most attention will focus on how Ca2+ signalling is activated. Since membrane depolarization is a key element for the activation of many SMCs,much attention will focus on the mechanisms responsible for depolarizing the membrane. However, there are other SMCs where activation depends on the periodic release of Ca2+ from internal stores. These different Ca2+ activation mechanisms fall into the following three main groups (Fig. 1).
Figure 1. The three main mechanisms responsible for generating the Ca2+ transients that trigger smooth
muscle cell (SMC) contraction
A, receptor-operated channels (ROCs) or a membrane oscillator induces the membrane depolarization (_V) that
triggers Ca2+ entry and contraction.
B, a cytosolic Ca2+ oscillator induces the Ca2+ signal that drives contraction.
C, a cytosolic Ca2+ oscillator in interstitial cells of Cajal (ICCs) or atypical SMCs induces the membrane depolarization
that spreads through the gap junctions to activate neighbouring SMCs. Reproduced from Berridge (2008), with permission
SOURCE for Figure 1: J Physiol 586.21 M. J. Berridge Smooth muscle cell calcium activation mechanisms 5048
Many SMCs are activated by membrane depolarization (_V) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). Themembrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction. The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is described in mechanism C.
Mechanism B.
The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.
Mechanism C.
A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (_V) that triggers contraction through mechanism A. In the following sections, some selected SMC types will illustrate how these signalling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca2+ signals are activated.
Vascular, Lymphatic and Airway Smooth Muscle Cells
Vascular, lymphatic and airway smooth muscle, which generate rhythmical contractions over an extended period of time, have an endogenous pacemaker mechanism driven by a cytosolic Ca2+ oscillator. In addition, these SMCs also respond to neurotransmitters released from the neural innervation. In the case of mesenteric arteries, the perivascular nerves release both ATP and noradrenaline (NA). The ATP acts first to produce a small initial contraction that is then followed by a much larger contraction when NA initiates a series of Ca2+ transients (Lamont et al. 2003). Such agonist-induced Ca2+ oscillations are a characteristic feature of the activation mechanisms of vascular (Iino et al. 1994; Lee et al. 2001; Peng et al. 2001; Perez & Sanderson, 2005b; Shaw et al. 2004) and airway SMCs (Kuo et al. 2003; Perez & Sanderson, 2005a; Sanderson et al. 2008). In some blood vessels, a specific tone is maintained by the spatial averaging of asynchronous oscillations. However, there are some vessels where the oscillations in groups of cells are synchronized resulting in the pulsatile contractions known as vasomotion (Mauban et al. 2001; Peng et al. 2001; Lamboley et al. 2003; Haddock & Hill, 2005). Such vasomotion is also a feature of lymphatic vessels (Imtiaz et al. 2007). Another feature of this oscillatory activity is that variations in transmitter concentration are translated into a change in contractile tone through a mechanism of frequency modulation (Iino et al. 1994; Kuo et al. 2003; Perez & Sanderson, 2005a,b). Frequency modulation is one of the mechanisms used for encoding and decoding signalling information through Ca2+ oscillations (Berridge, 2007).
The periodic pulses of Ca2+ that drive these rhythmical SMCs are derived from the internal stores through the operation of a cytosolic Ca2+ oscillator (Haddock & Hill, 2005; Imtiaz et al. 2007; Sanderson et al. 2008). The following general model, which applies to vascular, lymphatic, airway and perhaps also to corpus cavernosum SMCs, attempts to describe the nature of this oscillator and how it can be induced or modulated by neurotransmitters. A luminal loading Ca2+ oscillation mechanism (Berridge & Dupont, 1994; Berridge, 2007) forms the basis of this cytosolic oscillator model that depends upon the following sequential series of events (Fig. 5).
Figure 5. Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release
of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave
The oscillator is induced/modulated by neurotransmitters such as acetylcholine (ACh), 5-hydroxytryptamine (5-HT),
noradrenaline (NA) and endothelin-1 (ET-1), which act through inositol 1,4,5-trisphosphate (InsP3) that initiates
the oscillatory mechanism. The sequence of steps 1–9 is described in the text. Reproduced from Berridge (2008),
with permission.
SOURCE for Figure 5: J Physiol 586.21 M. J. Berridge Smooth muscle cell calcium activation mechanisms 5053
For Disruption of Calcium Homeostasis in Vascular Smooth Muscle Cells, see
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
The heart relies on a complex network of cells to maintain appropriate function. Cardiomyocytes, the contracting cells in the heart, exist in a three-dimensional network of endothelial cells, vascular smooth muscle, and an abundance of fibroblasts as well as transient populations of immune cells. Gap junctions electrochemically coordinate the contraction of individual cardiomyocytes, and their connection to the extracellular matrix (ECM) transduces force and coordinates the overall contraction of the heart. Intracellularly, repeating units of actin and myosin form the backbone of sarcomere structure, the basic functional unit of the cardiomyocyte (Fig. 1). The sarcomere itself consists of ∼20 proteins; however, more than 20 other proteins form connections between the myocytes and the ECM and regulate muscle contraction (Fig. 1 B). Given the complexity of the coordinated efforts of the many proteins that exist in multimeric complexes, dysfunction occurs when these interactions are disrupted.
Figure 1. Anatomy of the cardiac sarcomere(A) Diagram of the basic organization of the sarcomere. The sarcomere forms the basic contractile unit in the cardiomyocytes of the heart. Thin filaments composed of actin are anchored at the Z line and form transient sliding interactions with thick filaments composed of myosin molecules. The M Line, I Band, and A Band are anatomical features defined by their components (actin, myosin, and cytoskeletal proteins) and appearance in polarized light. Titin connects the Z line with the M line and contributes to the elastic properties and force production of the sarcomere through its extensible region in the I Band. Coordinated shortening of the sarcomere creates contraction of the cardiomyocyte. (B) Representation of the major proteins of the cardiac sarcomere. Attachment to the ECM is mediated by costameres composed of the dystroglycan–glycoprotein complex and the integrin complex. Force transduction and intracellular signaling are coordinated through the costamere. The unique roles of each of these proteins are critical to appropriate function of the heart. T-cap, titin cap; MyBP-C, myosin-binding protein C; NOS, nitric oxide synthase.
Although the heart may functionally tolerate a variety of pathological insults, adaptive responses that aim to maintain function eventually fail, resulting in a wide range of functional deficits or cardiomyopathy. Although a multitude of intrinsic and extrinsic stimuli promote cardiomyopathies, the best described causes are the >900 mutations in genes expressed in the cardiomyocyte (Fig. 1 B; Wang et al., 2010). Mutations in most of these genes cause a diverse range of cardiomyopathies, many with overlapping clinical phenotypes. Mutations in sarcomeric genes are usually inherited in an autosomal-dominant manner and are missense mutations that are incorporated into sarcomeres (Seidman and Seidman, 2001). Thus far >400 mutations in 13 sarcomeric proteins including β-myosin heavy chain (β-MyHC), α-cardiac actin, tropomyosin, and troponin have been associated with cardiomyopathy (www.cardiogenomics.med.harvard.edu). Table I summarizes these mutated proteins.
Ca2+ regulation and calcineurin signaling
Ca2+ concentrations inside the cardiomyocyte are critically important to actin–myosin interactions. Ca2+ is sequestered within the sarcoplasmic reticulum and the sarcomere itself, which serves as an intracellular reserve that is released in response to electrical stimulation of the cardiomyocyte. After contraction, sarco/endoplasmic reticulum Ca2+-ATPase sequesters the Ca2+ back into the sarcoplasmic reticulum to restore Ca2+balance. There is a clear correlation between force production and perturbation of Ca2+regulation, alterations of which might directly induce pathological, anatomical, and functional alterations that lead to heart failure via activation of GPCRs (Minamisawa et al., 1999).
Ca2+ in the cytosol can be increased to modulate sarcomere contractility by signaling through Gαq recruitment and activation of PLCβ. Ca2+ released from the sarcoplasmic reticulum activates calmodulin, which phosphorylates calcineurin, a serine/threonine phosphatase. Upon activation, calcineurin interacts with and dephosphorylates nuclear factor of activated T cells (NFAT), which then translocates into the nucleus. Calcineurin activation exacerbates hypertrophic signals and expedites the transition to a decompensatory state. Indeed, cardiac-specific overexpression of calcineurin or NFAT leads to significant cardiac hypertrophy that progresses rapidly to heart failure (Molkentin et al., 1998). Administration of antagonists of calcineurin attenuates the hypertrophic response of neonatal rat ventricular myocytes to stimuli such as phenylephrine (PE) and angiotensin II (Taigen et al., 2000).
Mechanotransduction and signaling in the cardiomyocyte
The responses of cardiomyocytes to systemic stress or genetic abnormalities are modulated by mechanosensitive mechanisms within the cardiomyocyte (Molkentin and Dorn, 2001; Seidman and Seidman, 2001; Frey and Olson, 2003). A complex network of proteins that connects the sarcomere to the ECM forms the basis of the mechanotransduction apparatus. For example, components of the costamere complex, which form the connection between the sarcomere and the ECM via integrins, initiate intracellular signaling and subsequently alter contractile properties and transcriptional regulation in response to membrane distortion. Mechanosensitive ion channels are also implicated in signal initiation in response to systemic stress (Le Guennec et al., 1990;Zhang et al., 2000; de Jonge et al., 2002). These channels are likely responsible for acute changes that might initiate other longer-term responses in the heart but are nonetheless important to consider when examining possible transducers of systemic and tissue alterations to the cardiomyocyte.
Changes in wall stress induce signaling pathways that are associated with the development of cardiac pathology. The many intracellular signaling pathways that mediate responses to increased demand on the heart have been extensively reviewed elsewhere (Force et al., 1999; Molkentin and Dorn, 2001; Heineke and Molkentin, 2006). Here, we focus on pathways that are intimately involved in pathogenesis (Fig. 4). Although their effects in compensatory responses early in pathology initially increase function by promoting growth and contractility, persistent responses eventually compromise function.
Figure 4.Signaling pathways associated with cardiac hypertrophy.
Although many pathways are associated with cardiomyopathy, up-regulation of transcription and induction of apoptosis are major mediators of pathogenic responses in the heart. The GPCR-associated pathway (dark red) can be activated by ET-1 and AngII, which are released in response to reduced contractility, and mediates contractile adaptation through increased calcium release from the sarcoplasmic reticulum. Increased intracellular calcium activates calmodulin and induces activation of the transcription factor MEF2. Incorporation into the sarcomere of mutant proteins that exhibit reduced ATP efficiency inhibits the sequestration of calcium from the cytosol and further enhances increases in intracellular calcium concentration. GPCR signaling is also associated with activation of the Akt signaling pathway (light green) that induces fetal gene expression and the cardiac hypertrophic response through inhibition of GSK3β. Apoptotic pathways (light blue) are induced by cytochrome c (CytC) release from mitochondria and activation of death receptors (like FasR) by cytokines such as TNF. Calcium overload and myocyte loss significantly contribute to reduced contractility in many forms of cardiomyopathy. ET-1, endothelin-1; HDAC, histone deacetylase; NFAT, nuclear factor of activated T cells; MEF-2, myocyte enhancer factor 2; SERCA, sarco/endoplasmic reticulum calcium-ATPase; cFLIP, cellular FLICE-inhibitory protein; AngII, angiotensin II; FasR, Fas receptor.
For Disruption of Calcium Homeostasis in Cardiomyocyte Cells, see
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
. ↵ Adams, J.W., D.S. Migita, M.K. Yu, R. Young, M.S. Hellickson, F.E. Castro-Vargas, J.D. Domingo, P.H. Lee, J.S. Bui, S.A. Henderson. 1996. Prostaglandin F2 alpha stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes. J. Biol. Chem. 271:1179–1186. doi:10.1074/jbc.271.2.1179 Abstract/FREE Full Text
. ↵ Akyürek, O., N. Akyürek, T. Sayin, I. Dinçer, B. Berkalp, G. Akyol, M. Ozenci, D. Oral. 2001. Association between the severity of heart failure and the susceptibility of myocytes to apoptosis in patients with idiopathic dilated cardiomyopathy. Int. J. Cardiol. 80:29–36. doi:10.1016/S0167-5273(01)00451-X CrossRefMedline
. ↵ Ashrafian, H., M.P. Frenneaux. 2007. Metabolic modulation in heart failure: the coming of age. Cardiovasc. Drugs Ther. 21:5–7. doi:10.1007/s10557-007-6000-z CrossRefMedline
. ↵ Basso, C., D. Corrado, F.I. Marcus, A. Nava, G. Thiene. 2009. Arrhythmogenic right ventricular cardiomyopathy. Lancet. 373:1289–1300. doi:10.1016/S0140-6736(09)60256-7 CrossRefMedline
. ↵ Berko, B.A., M. Swift. 1987. X-linked dilated cardiomyopathy. N. Engl. J. Med. 316:1186–1191. doi:10.1056/NEJM198705073161904 Medline
. ↵ Buvoli, M., M. Hamady, L.A. Leinwand, R. Knight. 2008. Bioinformatics assessment of beta-myosin mutations reveals myosin’s high sensitivity to mutations. Trends Cardiovasc. Med. 18:141–149. doi:10.1016/j.tcm.2008.04.001 CrossRefMedline
. ↵ Bybee, K.A., T. Kara, A. Prasad, A. Lerman, G.W. Barsness, R.S. Wright, C.S. Rihal. 2004. Systematic review: transient left ventricular apical ballooning: a syndrome that mimics ST-segment elevation myocardial infarction. Ann. Intern. Med. 141:858–865. Abstract/FREE Full Text
. ↵ Chin, T.K., J.K. Perloff, R.G. Williams, K. Jue, R. Mohrmann. 1990. Isolated noncompaction of left ventricular myocardium. A study of eight cases. Circulation. 82:507–513. doi:10.1161/01.CIR.82.2.507 Abstract/FREE Full Text
. ↵ Communal, C., K. Singh, D.R. Pimentel, W.S. Colucci. 1998. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation. 98:1329–1334. Abstract/FREE Full Text
. ↵ Corrado, D., C. Basso, G. Thiene, W.J. McKenna, M.J. Davies, F. Fontaliran, A. Nava, F. Silvestri, C. Blomstrom-Lundqvist, E.K. Wlodarska, et al. 1997. Spectrum of clinicopathologic manifestations of arrhythmogenic right ventricular cardiomyopathy/dysplasia: a multicenter study. J. Am. Coll. Cardiol. 30:1512–1520. doi:10.1016/S0735-1097(97)00332-X Abstract
. ↵ Cregler, L.L. 1989. Progression from hypertrophic cardiomyopathy to dilated cardiomyopathy. J. Natl. Med. Assoc. 81:820: 824–826. Search Google Scholar
. ↵ D’Angelo, D.D., Y. Sakata, J.N. Lorenz, G.P. Boivin, R.A. Walsh, S.B. Liggett, G.W. Dorn II. 1997. Transgenic Galphaq overexpression induces cardiac contractile failure in mice. Proc. Natl. Acad. Sci. USA. 94:8121–8126. doi:10.1073/pnas.94.15.8121 Abstract/FREE Full Text
. ↵ Dávila-Román, V.G., G. Vedala, P. Herrero, L. de las Fuentes, J.G. Rogers, D.P. Kelly, R.J. Gropler. 2002. Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 40:271–277. doi:10.1016/S0735-1097(02)01967-8 Abstract/FREE Full Text
. ↵ Davis, J., H. Wen, T. Edwards, J.M. Metzger. 2007. Thin filament disinhibition by restrictive cardiomyopathy mutant R193H troponin I induces Ca2+-independent mechanical tone and acute myocyte remodeling. Circ. Res. 100:1494–1502. doi:10.1161/01.RES.0000268412.34364.50 Abstract/FREE Full Text
. ↵ de Jonge, H.W., D.H. Dekkers, B.C. Tilly, J.M. Lamers. 2002. Cyclic stretch and endothelin-1 mediated activation of chloride channels in cultured neonatal rat ventricular myocytes. Clin. Sci. 103(Suppl 48):148S–151S. Medline
. ↵ Deinum, J., J.M. van Gool, M.J. Kofflard, F.J. ten Cate, A.H. Danser. 2001. Angiotensin II type 2 receptors and cardiac hypertrophy in women with hypertrophic cardiomyopathy. Hypertension. 38:1278–1281. doi:10.1161/hy1101.096114 Abstract/FREE Full Text
. ↵ Dolci, A., R. Dominici, D. Cardinale, M.T. Sandri, M. Panteghini. 2008. Biochemical markers for prediction of chemotherapy-induced cardiotoxicity: systematic review of the literature and recommendations for use. Am. J. Clin. Pathol. 130:688–695. doi:10.1309/AJCPB66LRIIVMQDR Abstract/FREE Full Text
. ↵ Edwards, B.S., R.S. Zimmerman, T.R. Schwab, D.M. Heublein, J.C. Burnett Jr. 1988. Atrial stretch, not pressure, is the principal determinant controlling the acute release of atrial natriuretic factor. Circ. Res. 62:191–195. Abstract/FREE Full Text
. ↵ Esposito, G., S.V. Prasad, A. Rapacciuolo, L. Mao, W.J. Koch, H.A. Rockman. 2001. Cardiac overexpression of a G(q) inhibitor blocks induction of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase activity in in vivo pressure overload. Circulation. 103:1453–1458. Abstract/FREE Full Text
. ↵ Flavigny, J., M. Souchet, P. Sébillon, I. Berrebi-Bertrand, B. Hainque, A. Mallet, A. Bril, K. Schwartz, L. Carrier. 1999. COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes. J. Mol. Biol. 294:443–456. doi:10.1006/jmbi.1999.3276 CrossRefMedline
. ↵ Force, T., R. Hajjar, F. Del Monte, A. Rosenzweig, G. Choukroun. 1999. Signaling pathways mediating the response to hypertrophic stress in the heart. Gene Expr. 7:337–348. Medline
. ↵ Freedom, R.M., S.J. Yoo, D. Perrin, G. Taylor, S. Petersen, R.H. Anderson. 2005. The morphological spectrum of ventricular noncompaction. Cardiol. Young. 15:345–364. doi:10.1017/S1047951105000752 CrossRefMedline
. ↵ Frey, N., E.N. Olson. 2003. Cardiac hypertrophy: the good, the bad, and the ugly. Annu. Rev. Physiol. 65:45–79. doi:10.1146/annurev.physiol.65.092101.142243 CrossRefMedline
. ↵ Geng, Y.J., Y. Ishikawa, D.E. Vatner, T.E. Wagner, S.P. Bishop, S.F. Vatner, C.J. Homcy. 1999. Apoptosis of cardiac myocytes in Gsalpha transgenic mice. Circ. Res. 84:34–42. Abstract/FREE Full Text
. ↵ Gill, C., R. Mestril, A. Samali. 2002. Losing heart: the role of apoptosis in heart disease—a novel therapeutic target? FASEB J. 16:135–146. doi:10.1096/fj.01-0629com Abstract/FREE Full Text
. ↵ Grogan, M., M.M. Redfield, K.R. Bailey, G.S. Reeder, B.J. Gersh, W.D. Edwards, R.J. Rodeheffer. 1995. Long-term outcome of patients with biopsy-proved myocarditis: comparison with idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 26:80–84. doi:10.1016/0735-1097(95)00148-S Abstract
. ↵ Gupta, A., N.S. Aberle II, J. Ren, A.C. Sharma. 2005. Endothelin-converting enzyme-1-mediated signaling in adult rat ventricular myocyte contractility and apoptosis during sepsis. J. Mol. Cell. Cardiol. 38:527–537. doi:10.1016/j.yjmcc.2005.01.002 CrossRefMedline
. ↵ Haq, S., G. Choukroun, Z.B. Kang, H. Ranu, T. Matsui, A. Rosenzweig, J.D. Molkentin, A. Alessandrini, J. Woodgett, R. Hajjar, et al. 2000. Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy. J. Cell Biol. 151:117–130. doi:10.1083/jcb.151.1.117 Abstract/FREE Full Text
. ↵ Herron, T.J., K.S. McDonald. 2002. Small amounts of alpha-myosin heavy chain isoform expression significantly increase power output of rat cardiac myocyte fragments. Circ. Res. 90:1150–1152. doi:10.1161/01.RES.0000022879.57270.11 Abstract/FREE Full Text
. ↵ Herron, T.J., R. Vandenboom, E. Fomicheva, L. Mundada, T. Edwards, J.M. Metzger. 2007. Calcium-independent negative inotropy by beta-myosin heavy chain gene transfer in cardiac myocytes. Circ. Res. 100:1182–1190. doi:10.1161/01.RES.0000264102.00706.4e Abstract/FREE Full Text
. ↵ Hoogerwaard, E.M., P.A. van der Wouw, A.A. Wilde, E. Bakker, P.F. Ippel, J.C. Oosterwijk, D.F. Majoor-Krakauer, A.J. van Essen, N.J. Leschot, M. de Visser. 1999. Cardiac involvement in carriers of Duchenne and Becker muscular dystrophy. Neuromuscul. Disord. 9:347–351. doi:10.1016/S0960-8966(99)00018-8 CrossRefMedline
. ↵ Huang, X.P., J.F. Du. 2004. Troponin I, cardiac diastolic dysfunction and restrictive cardiomyopathy. Acta Pharmacol. Sin. 25:1569–1575. Medline
. ↵ Huang, Y., R.P. Hickey, J.L. Yeh, D. Liu, A. Dadak, L.H. Young, R.S. Johnson, F.J. Giordano. 2004. Cardiac myocyte-specific HIF-1alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J. 18:1138–1140. doi:10.1096/fj.03-1377com Abstract/FREE Full Text
. ↵ Iacovoni, A., R. De Maria, A. Gavazzi. 2010. Alcoholic cardiomyopathy. J. Cardiovasc. Med. (Hagerstown). 11:884–892. doi:10.2459/JCM.0b013e32833833a3 CrossRefMedline
. ↵ Ingwall, J.S., R.G. Weiss. 2004. Is the failing heart energy starved? On using chemical energy to support cardiac function. Circ. Res. 95:135–145. doi:10.1161/01.RES.0000137170.41939.d9 Abstract/FREE Full Text
. ↵ Iwai-Kanai, E., K. Hasegawa, M. Araki, T. Kakita, T. Morimoto, S. Sasayama. 1999. alpha- and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes. Circulation. 100:305–311. Abstract/FREE Full Text
. ↵ Kalsi, K.K., R.T. Smolenski, R.D. Pritchard, A. Khaghani, A.M. Seymour, M.H. Yacoub. 1999. Energetics and function of the failing human heart with dilated or hypertrophic cardiomyopathy. Eur. J. Clin. Invest. 29:469–477. doi:10.1046/j.1365-2362.1999.00468.x CrossRefMedline
. ↵ Kamisago, M., S.D. Sharma, S.R. DePalma, S. Solomon, P. Sharma, B. McDonough, L. Smoot, M.P. Mullen, P.K. Woolf, E.D. Wigle, et al. 2000. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N. Engl. J. Med. 343:1688–1696. doi:10.1056/NEJM200012073432304 CrossRefMedline
. ↵ Kaneda, T., C. Naruse, A. Kawashima, N. Fujino, T. Oshima, M. Namura, S. Nunoda, S. Mori, T. Konno, H. Ino, et al. 2008. A novel beta-myosin heavy chain gene mutation, p.Met531Arg, identified in isolated left ventricular non-compaction in humans, results in left ventricular hypertrophy that progresses to dilation in a mouse model. Clin. Sci. 114:431–440. doi:10.1042/CS20070179 CrossRefMedline
. ↵ Kantor, P.F., M.A. Robertson, J.Y. Coe, G.D. Lopaschuk. 1999. Volume overload hypertrophy of the newborn heart slows the maturation of enzymes involved in the regulation of fatty acid metabolism. J. Am. Coll. Cardiol. 33:1724–1734. doi:10.1016/S0735-1097(99)00063-7 Abstract/FREE Full Text
. ↵ Karam, S., M.J. Raboisson, C. Ducreux, L. Chalabreysse, G. Millat, A. Bozio, P. Bouvagnet. 2008. A de novo mutation of the beta cardiac myosin heavy chain gene in an infantile restrictive cardiomyopathy. Congenit. Heart Dis. 3:138–143. doi:10.1111/j.1747-0803.2008.00165.x CrossRefMedline
. ↵ Katritsis, D., P.T. Wilmshurst, J.A. Wendon, M.J. Davies, M.M. Webb-Peploe. 1991. Primary restrictive cardiomyopathy: clinical and pathologic characteristics. J. Am. Coll. Cardiol. 18:1230–1235. doi:10.1016/0735-1097(91)90540-P Abstract
. ↵ Keeling, P.J., Y. Gang, G. Smith, H. Seo, S.E. Bent, V. Murday, A.L. Caforio, W.J. McKenna. 1995. Familial dilated cardiomyopathy in the United Kingdom. Br. Heart J. 73:417–421. doi:10.1136/hrt.73.5.417 Abstract/FREE Full Text
. ↵ Kinnunen, P., O. Vuolteenaho, H. Ruskoaho. 1993. Mechanisms of atrial and brain natriuretic peptide release from rat ventricular myocardium: effect of stretching. Endocrinology. 132:1961–1970. doi:10.1210/en.132.5.1961 Abstract/FREE Full Text
. ↵ Knowlton, K.U., M.C. Michel, M. Itani, H.E. Shubeita, K. Ishihara, J.H. Brown, K.R. Chien. 1993. The alpha 1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy. J. Biol. Chem. 268:15374–15380. Abstract/FREE Full Text
. ↵ Kojima, M., I. Shiojima, T. Yamazaki, I. Komuro, Z. Zou, Y. Wang, T. Mizuno, K. Ueki, K. Tobe, T. Kadowaki, et al. 1994. Angiotensin II receptor antagonist TCV-116 induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intracellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro. Circulation. 89:2204–2211. Abstract/FREE Full Text
. ↵ Krown, K.A., M.T. Page, C. Nguyen, D. Zechner, V. Gutierrez, K.L. Comstock, C.C. Glembotski, P.J. Quintana, R.A. Sabbadini. 1996. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J. Clin. Invest. 98:2854–2865. doi:10.1172/JCI119114 Medline
. ↵ Kuwahara, K., Y. Saito, M. Takano, Y. Arai, S. Yasuno, Y. Nakagawa, N. Takahashi, Y. Adachi, G. Takemura, M. Horie, et al. 2003. NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function. EMBO J. 22:6310–6321. doi:10.1093/emboj/cdg601 CrossRefMedline
. ↵ Le Guennec, J.Y., N. Peineau, J.A. Argibay, K.G. Mongo, D. Garnier. 1990. A new method of attachment of isolated mammalian ventricular myocytes for tension recording: length dependence of passive and active tension. J. Mol. Cell. Cardiol. 22:1083–1093. doi:10.1016/0022-2828(90)90072-A CrossRefMedline
. ↵ Lopaschuk, G.D., M.A. Spafford, D.R. Marsh. 1991. Glycolysis is predominant source of myocardial ATP production immediately after birth. Am. J. Physiol. 261:H1698–H1705. Medline
. ↵ Lowes, B.D., W. Minobe, W.T. Abraham, M.N. Rizeq, T.J. Bohlmeyer, R.A. Quaife, R.L. Roden, D.L. Dutcher, A.D. Robertson, N.F. Voelkel, et al. 1997. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. J. Clin. Invest. 100:2315–2324. doi:10.1172/JCI119770 Medline
. ↵ Luckey, S.W., L.A. Walker, T. Smyth, J. Mansoori, A. Messmer-Kratzsch, A. Rosenzweig, E.N. Olson, L.A. Leinwand. 2009. The role of Akt/GSK-3beta signaling in familial hypertrophic cardiomyopathy. J. Mol. Cell. Cardiol. 46:739–747. doi:10.1016/j.yjmcc.2009.02.010 CrossRefMedline
. ↵ Maass, A.H., M. Buvoli. 2007. Cardiomyocyte preparation, culture, and gene transfer. Methods Mol. Biol. 366:321–330. doi:10.1007/978-1-59745-030-0_18 CrossRefMedline
. ↵ Marian, A.J. 2000. Pathogenesis of diverse clinical and pathological phenotypes in hypertrophic cardiomyopathy. Lancet. 355:58–60. doi:10.1016/S0140-6736(99)06187-5 CrossRefMedline
. ↵ Maron, B.J., A. Pelliccia. 2006. The heart of trained athletes: cardiac remodeling and the risks of sports, including sudden death. Circulation. 114:1633–1644. doi:10.1161/CIRCULATIONAHA.106.613562 FREE Full Text
. ↵ Maron, B.J., P.F. Nichols III, L.W. Pickle, Y.E. Wesley, J.J. Mulvihill. 1984. Patterns of inheritance in hypertrophic cardiomyopathy: assessment by M-mode and two-dimensional echocardiography. Am. J. Cardiol. 53:1087–1094. doi:10.1016/0002-9149(84)90643-X CrossRefMedline
. ↵ Maron, B.J., J.M. Gardin, J.M. Flack, S.S. Gidding, T.T. Kurosaki, D.E. Bild. 1995. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation. 92:785–789. Abstract/FREE Full Text
. ↵ Matsui, T., L. Li, J.C. Wu, S.A. Cook, T. Nagoshi, M.H. Picard, R. Liao, A. Rosenzweig. 2002. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J. Biol. Chem. 277:22896–22901. doi:10.1074/jbc.M200347200 Abstract/FREE Full Text
. ↵ Menon, S.C., B.W. Eidem, J.A. Dearani, S.R. Ommen, M.J. Ackerman, D. Miller. 2009. Diastolic dysfunction and its histopathological correlation in obstructive hypertrophic cardiomyopathy in children and adolescents. J. Am. Soc. Echocardiogr. 22:1327–1334. doi:10.1016/j.echo.2009.08.014 CrossRefMedline
. ↵ Mestroni, L., C. Rocco, D. Gregori, G. Sinagra, A. Di Lenarda, S. Miocic, M. Vatta, B. Pinamonti, F. Muntoni, A.L. Caforio, et al.; Heart Muscle Disease Study Group. 1999. Familial dilated cardiomyopathy: evidence for genetic and phenotypic heterogeneity. J. Am. Coll. Cardiol. 34:181–190. doi:10.1016/S0735-1097(99)00172-2 Abstract/FREE Full Text
. ↵ Michael, A., S. Haq, X. Chen, E. Hsich, L. Cui, B. Walters, Z. Shao, K. Bhattacharya, H. Kilter, G. Huggins, et al. 2004. Glycogen synthase kinase-3beta regulates growth, calcium homeostasis, and diastolic function in the heart. J. Biol. Chem. 279:21383–21393. doi:10.1074/jbc.M401413200 Abstract/FREE Full Text
. ↵ Michels, V.V., P.P. Moll, F.A. Miller, A.J. Tajik, J.S. Chu, D.J. Driscoll, J.C. Burnett, R.J. Rodeheffer, J.H. Chesebro, H.D. Tazelaar. 1992. The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N. Engl. J. Med. 326:77–82. doi:10.1056/NEJM199201093260201 Medline
. ↵ Minamisawa, S., M. Hoshijima, G. Chu, C.A. Ward, K. Frank, Y. Gu, M.E. Martone, Y. Wang, J. Ross Jr, E.G. Kranias, et al. 1999. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell. 99:313–322. doi:10.1016/S0092-8674(00)81662-1 CrossRefMedline
. ↵ Miyata, S., W. Minobe, M.R. Bristow, L.A. Leinwand. 2000. Myosin heavy chain isoform expression in the failing and nonfailing human heart. Circ. Res. 86:386–390. Abstract/FREE Full Text
. ↵ Mogensen, J., T. Kubo, M. Duque, W. Uribe, A. Shaw, R. Murphy, J.R. Gimeno, P. Elliott, W.J. McKenna. 2003. Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations. J. Clin. Invest. 111:209–216. CrossRefMedline
. ↵ Molkentin, J.D., J.R. Lu, C.L. Antos, B. Markham, J. Richardson, J. Robbins, S.R. Grant, E.N. Olson. 1998. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 93:215–228. doi:10.1016/S0092-8674(00)81573-1 CrossRefMedline
. ↵ Nagata, K., R. Liao, F.R. Eberli, N. Satoh, B. Chevalier, C.S. Apstein, T.M. Suter. 1998. Early changes in excitation-contraction coupling: transition from compensated hypertrophy to failure in Dahl salt-sensitive rat myocytes. Cardiovasc. Res. 37:467–477. doi:10.1016/S0008-6363(97)00278-2 Abstract/FREE Full Text
. ↵ Narula, J., N. Haider, R. Virmani, T.G. DiSalvo, F.D. Kolodgie, R.J. Hajjar, U. Schmidt, M.J. Semigran, G.W. Dec, B.A. Khaw. 1996. Apoptosis in myocytes in end-stage heart failure. N. Engl. J. Med. 335:1182–1189. doi:10.1056/NEJM199610173351603 CrossRefMedline
. ↵ Narula, J., P. Pandey, E. Arbustini, N. Haider, N. Narula, F.D. Kolodgie, B. Dal Bello, M.J. Semigran, A. Bielsa-Masdeu, G.W. Dec, et al. 1999. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc. Natl. Acad. Sci. USA. 96:8144–8149. doi:10.1073/pnas.96.14.8144 Abstract/FREE Full Text
. ↵ Neubauer, S. 2007. The failing heart—an engine out of fuel. N. Engl. J. Med. 356:1140–1151. doi:10.1056/NEJMra063052 CrossRefMedline
. ↵ O’Neill, B.T., E.D. Abel. 2005. Akt1 in the cardiovascular system: friend or foe? J. Clin. Invest. 115:2059–2064. doi:10.1172/JCI25900 CrossRefMedline
. ↵ Ohler, A., J. Weisser-Thomas, V. Piacentino, S.R. Houser, G.F. Tomaselli, B. O’Rourke. 2009. Two-photon laser scanning microscopy of the transverse-axial tubule system in ventricular cardiomyocytes from failing and non-failing human hearts. Cardiol. Res. Pract. 2009:802373. Medline
. ↵ Olivetti, G., R. Abbi, F. Quaini, J. Kajstura, W. Cheng, J.A. Nitahara, E. Quaini, C. Di Loreto, C.A. Beltrami, S. Krajewski, et al. 1997. Apoptosis in the failing human heart. N. Engl. J. Med. 336:1131–1141. doi:10.1056/NEJM199704173361603 CrossRefMedline
. ↵ Palmiter, K.A., M.J. Tyska, D.E. Dupuis, N.R. Alpert, D.M. Warshaw. 1999. Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms. J. Physiol. 519:669–678. doi:10.1111/j.1469-7793.1999.0669n.x Abstract/FREE Full Text
. ↵ Parvatiyar, M.S., J.R. Pinto, D. Dweck, J.D. Potter. 2010. Cardiac troponin mutations and restrictive cardiomyopathy. J. Biomed. Biotechnol. 2010:350706. doi:10.1155/2010/350706 Medline
. ↵ Peddy, S.B., L.A. Vricella, J.E. Crosson, G.L. Oswald, R.D. Cohn, D.E. Cameron, D. Valle, B.L. Loeys. 2006. Infantile restrictive cardiomyopathy resulting from a mutation in the cardiac troponin T gene. Pediatrics. 117:1830–1833. doi:10.1542/peds.2005-2301 Abstract/FREE Full Text
. ↵ Ritter, M., E. Oechslin, G. Sütsch, C. Attenhofer, J. Schneider, R. Jenni. 1997. Isolated noncompaction of the myocardium in adults. Mayo Clin. Proc. 72:26–31. doi:10.4065/72.1.26 Abstract/FREE Full Text
. ↵ Rodeheffer, R.J., I. Tanaka, T. Imada, A.S. Hollister, D. Robertson, T. Inagami. 1986. Atrial pressure and secretion of atrial natriuretic factor into the human central circulation. J. Am. Coll. Cardiol. 8:18–26. doi:10.1016/S0735-1097(86)80086-9 Abstract
. ↵ Rose, E.A., A.C. Gelijns, A.J. Moskowitz, D.F. Heitjan, L.W. Stevenson, W. Dembitsky, J.W. Long, D.D. Ascheim, A.R. Tierney, R.G. Levitan, et al.; Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure (REMATCH) Study Group. 2001. Long-term use of a left ventricular assist device for end-stage heart failure. N. Engl. J. Med. 345:1435–1443. doi:10.1056/NEJMoa012175 CrossRefMedline
. ↵ Sack, M.N., T.A. Rader, S. Park, J. Bastin, S.A. McCune, D.P. Kelly. 1996. Fatty acid oxidation enzyme gene expression is downregulated in the failing heart. Circulation. 94:2837–2842. Abstract/FREE Full Text
. ↵ Sadoshima, J., S. Izumo. 1993. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 12:1681–1692. Medline
. ↵ Sakata, Y., B.D. Hoit, S.B. Liggett, R.A. Walsh, G.W. Dorn II. 1998. Decompensation of pressure-overload hypertrophy in G alpha q-overexpressing mice. Circulation. 97:1488–1495. Abstract/FREE Full Text
. ↵ Sasse-Klaassen, S., B. Gerull, E. Oechslin, R. Jenni, L. Thierfelder. 2003. Isolated noncompaction of the left ventricular myocardium in the adult is an autosomal dominant disorder in the majority of patients. Am. J. Med. Genet. A. 119A:162–167. doi:10.1002/ajmg.a.20075 Medline
. ↵ Schram, K., S. De Girolamo, S. Madani, D. Munoz, F. Thong, G. Sweeney. 2010. Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes. Cell. Mol. Biol. Lett. 15:551–563. doi:10.2478/s11658-010-0027-z CrossRefMedline
. ↵ Schwartz, K., Y. Lecarpentier, J.L. Martin, A.M. Lompré, J.J. Mercadier, B. Swynghedauw. 1981. Myosin isoenzymic distribution correlates with speed of myocardial contraction. J. Mol. Cell. Cardiol. 13:1071–1075. doi:10.1016/0022-2828(81)90297-2 CrossRefMedline
. ↵ Seidman, J.G., C. Seidman. 2001. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell. 104:557–567. doi:10.1016/S0092-8674(01)00242-2 CrossRefMedline
. ↵ Semsarian, C., M.J. Healey, D. Fatkin, M. Giewat, C. Duffy, C.E. Seidman, J.G. Seidman. 2001. A polymorphic modifier gene alters the hypertrophic response in a murine model of familial hypertrophic cardiomyopathy. J. Mol. Cell. Cardiol. 33:2055–2060. doi:10.1006/jmcc.2001.1466 CrossRefMedline
. ↵ Shen, W.K., W.D. Edwards, S.C. Hammill, K.R. Bailey, D.J. Ballard, B.J. Gersh. 1995. Sudden unexpected nontraumatic death in 54 young adults: a 30-year population-based study. Am. J. Cardiol. 76:148–152. doi:10.1016/S0002-9149(99)80047-2 CrossRefMedline
. ↵ Shiojima, I., K. Sato, Y. Izumiya, S. Schiekofer, M. Ito, R. Liao, W.S. Colucci, K. Walsh. 2005. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J. Clin. Invest. 115:2108–2118. doi:10.1172/JCI24682 CrossRefMedline
. ↵ Silberbach, M., T. Gorenc, R.E. Hershberger, P.J. Stork, P.S. Steyger, C.T. Roberts Jr. 1999. Extracellular signal-regulated protein kinase activation is required for the anti-hypertrophic effect of atrial natriuretic factor in neonatal rat ventricular myocytes. J. Biol. Chem. 274:24858–24864. doi:10.1074/jbc.274.35.24858 Abstract/FREE Full Text
. ↵ Simpson, P., A. McGrath, S. Savion. 1982. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ. Res. 51:787–801. Abstract/FREE Full Text
. ↵ Smith, C.S., P.A. Bottomley, S.P. Schulman, G. Gerstenblith, R.G. Weiss. 2006. Altered creatine kinase adenosine triphosphate kinetics in failing hypertrophied human myocardium. Circulation. 114:1151–1158. doi:10.1161/CIRCULATIONAHA.106.613646 Abstract/FREE Full Text
. ↵ Stanley, W.C., G.D. Lopaschuk, J.G. McCormack. 1997. Regulation of energy substrate metabolism in the diabetic heart. Cardiovasc. Res. 34:25–33. doi:10.1016/S0008-6363(97)00047-3 FREE Full Text
. ↵ Taigen, T., L.J. De Windt, H.W. Lim, J.D. Molkentin. 2000. Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy. Proc. Natl. Acad. Sci. USA. 97:1196–1201. doi:10.1073/pnas.97.3.1196 Abstract/FREE Full Text
. ↵ Teekakirikul, P., S. Eminaga, O. Toka, R. Alcalai, L. Wang, H. Wakimoto, M. Nayor, T. Konno, J.M. Gorham, C.M. Wolf, et al. 2010. Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β. J. Clin. Invest. 120:3520–3529. doi:10.1172/JCI42028 CrossRefMedline
. ↵ Torre-Amione, G., S. Kapadia, J. Lee, J.B. Durand, R.D. Bies, J.B. Young, D.L. Mann. 1996. Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation. 93:704–711. Abstract/FREE Full Text
. ↵ Vikstrom, K.L., S.M. Factor, L.A. Leinwand. 1996. Mice expressing mutant myosin heavy chains are a model for familial hypertrophic cardiomyopathy. Mol. Med. 2:556–567. Medline
. ↵ Wang, L., J.G. Seidman, C.E. Seidman. 2010. Narrative review: harnessing molecular genetics for the diagnosis and management of hypertrophic cardiomyopathy. Ann. Intern. Med. 152:513–520: W181. Abstract/FREE Full Text
. ↵ Weiford, B.C., V.D. Subbarao, K.M. Mulhern. 2004. Noncompaction of the ventricular myocardium. Circulation. 109:2965–2971. doi:10.1161/01.CIR.0000132478.60674.D0 FREE Full Text
. ↵ Yamaji, K., S. Fujimoto, Y. Ikeda, K. Masuda, S. Nakamura, Y. Saito, C. Yutani. 2005. Apoptotic myocardial cell death in the setting of arrhythmogenic right ventricular cardiomyopathy. Acta Cardiol. 60:465–470. doi:10.2143/AC.60.5.2004965 CrossRefMedline
. ↵ Yang, Z., N.E. Bowles, S.E. Scherer, M.D. Taylor, D.L. Kearney, S. Ge, V.V. Nadvoretskiy, G. DeFreitas, B. Carabello, L.I. Brandon, et al. 2006. Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ. Res. 99:646–655. doi:10.1161/01.RES.0000241482.19382.c6 Abstract/FREE Full Text
. ↵ Yousef, Z.R., P.W. Foley, K. Khadjooi, S. Chalil, H. Sandman, N.U. Mohammed, F. Leyva. 2009. Left ventricular non-compaction: clinical features and cardiovascular magnetic resonance imaging. BMC Cardiovasc. Disord. 9:37. doi:10.1186/1471-2261-9-37 CrossRefMedline
. ↵ Zhang, Y.H., J.B. Youm, H.K. Sung, S.H. Lee, S.Y. Ryu, W.K. Ho, Y.E. Earm. 2000. Stretch-activated and background non-selective cation channels in rat atrial myocytes. J. Physiol. 523:607–619. doi:10.1111/j.1469-7793.2000.00607.x Abstract/FREE Full Text
The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Author and Curator: Larry H Bernstein, MD, FCAP
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
This is the Part IV of a series on the cytoskeleton and structural shared thematics in cellular movement and cellular dynamics. The last two are specific to the heart, and the third was renal tubular caicium exchange and the effects of Na+ and hormones.
In Part I, Identification of Biomarkers that are Related to the Actin Cytoskeleton
The prior articles discussed common management motifs across cell-types that are essential for cell division, embryogenesis, cancer metastasis, osteogenesis, musculoskeletal function, vascular compliance, and cardiac contractility. This second article concentrates on specific functionalities for cardiac contractility based on Ca++ signaling in excitation-contraction coupling, addressing modifications specific to cardiac muscle and not to skeletal muscle. In Part I there was discussion of the importance of Ca2+ signaling on innate immune system, and the roles of calcium in immunology will be further expanded in a third article of the series.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Observations of Tissues Dependent on Electrical Impulses and Differences in Calcium-Efflux Mechanisms
Voice of Justin Pearlman
Skeletal muscles are named for muscle bundles attached to skeleton elements, including head and neck, thorax, and the long bones of limbs, but the same structural and neuronally controlled muscle type is also in the abdomenal wall and the scalp, face, and eyes (for eye motion), each serving the function of movement on demand. The skeletal element these muscles attach to are tendons (fibrous tissue), often anchored to bone before and after an articulation (joint). There are several features that distinguish skeletal muscle from smooth muscle and from myocardium (heart muscle). Skeletal muscles are striated. They have fast-twitch and slow-twitch fibers in various proportions. They are under voluntary neural control, not autonomic (involuntary).
In distinction, smooth muscles line arterial blood vessels, lymphatics, the urinary bladder, the gastrointestinal tract, the respiratory tract, and also the uterus, the pili of the skin (goose bumps), and are in the eyes to control pupil diameter and lens focus. They are controlled by autonomic innervation.
The myocardium, or heart muscle, is distinct in many ways. The heart muscle has a unique architecture with Z-bands. The heart muscle a syncytium of cardiac muscle made of cardiomyocytes, which means instead of a bundle of separate cells each distinctly bounded by a cell membrane, the entire heart muscle can be viewed as a single multinucleated cell (or merger of cells). Skeletal muscle has multinucleated cells also from the merger of multiple blast cells, but unlike the heart there are distinct cell boundaries between skeletal myocytes, known as myofibers. The heart has fiber layers with different orientations (spiral clockwise and counterclockwise arrangement of muscle fibers) that result in multiple types of motion, but technically all of the heart muscle fibers are part of a single conglomerate cell. The motions of the heart include: translation, tilting, shortening, thickening, narrowing, twisting, rotating, lengthening and widening. The heart cell contracts and has innervation to the AV node and the SA node, with both sympathetic and parasymptathetic innervation.
All three types of muscle apply a basic Motif of proteins that change length in response to a calcium signal. The calcium is stored is sacks called the sarcoplasmic reticulum. The calcium is released into the main fluid of the cell (the cytoplasm), where it controls different functions. Even in skeletal muscle there is a difference between thigh and thorax, and we know from comparative ornithology that the enzymology and energy metabolism of the wings of birds that soar, hawks and eagles, differs from the chicken, or the turkey.
Key features are illustrated below.
Figure 1….. skeletal muscle vs heart calcium channels.
receptors voltage gated Ca(2) channel
We see in Figure 1 that both the skeletal muscle and the cardiomyocyte have a Ryanodyne receptor that is the flow device for carrying the Ca(2+) ions from the sarcoplasm into the cytoplasm. In the skeletal muscle there is a dihydropyridine receptor. The heart muscle is voltage gated. The interaction with calmodulin (not shown) via Calcium/calmodulin-dependent Protein Kinase Type II delta = CaMKI, II – IV. CaMKII has isoforms a, b, c, d – and CaMKIId has two splice variants (cytoplasmic and nuclear). These will be discussed fully in the fifth of the series. Take note of the fact the CaMKII isoform is found only in the heart. So we have here molecules with similar structure, but not completely homologous. Structure and function have made small, requiring significant adaptations.
Figure 2. A cardiomycyte structure with the sarcomere and calcium efflux into the cytoplasn, and with the mitochondrion available for Ca(2+) exchange with the cytoplasm, and with Ca(2+), Na(+) and K(+) channels contiguous with the extracellular space.
RyR
The arterial endothelium is functionally protected by eNOS converting arginine to citrulline. This does not occur with adult form of urea cycle (Krebs Henseleit) disorder, as there is no substrate. iNOS, a nitric oxide isoform present in macrophages that invade through intercellular spaces into the underlying matrix. A large study presented at the European Society of Cardiology (ESC) 2013 Congress has indicated that there is not a relationship of tight control of type 2 diabetes and cardiovascular events, even though we know that there is a relationship between diabetes and
insulin resistance
endothelial activation
inflammatory markers
homocysteine
Adipokines interact in type 2 diabetes with inflammatory cytokines for development of insulin resistance, and these are markers of arterial vascular disease. But the association of diabetes with heart disease, long considered valid, has come into some dispute. Recently, saxagliptin was associated with a significant 27% increased risk of hospitalizations for heart failure in the Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus (SAVOR-TIMI 53) study, a component of the prespecified secondary end point. In the Examination of Cardiovascular Outcomes with Alogliptin versus Standard of Care in Patients with Type 2 Diabetes Mellitus and Acute Coronary Syndrome (EXAMINE) study, there was no increased risk of heart failure with alogliptin. While saxagliptin and alogliptin significantly reduced glycated hemoglobin levels, there was some debate about the role of the drugs, which are dipeptidyl peptidase-4 (DPP-4) inhibitors, in clinical practice. There is some disappointment with respect to the diabetes issue, but that might be remedied by improvement based on the appropriate combination of biomarkers for prediction asnd monitoring at the earliest onset. Dr William White said alogliptin lowers the glycemic index significantly, and such reductions can reduce the risk of microvascular complications. We know from the prior literature that it might take five years-plus before we determine a microvascular benefit. A serious problem in the validity of the results was that statistically, saxagliptin met the primary end point of noninferiority, with the drug no worse than placebo. Glycated hemoglobin levels were reduced with saxagliptin, down from 8.0% at baseline to 7.7% at the end of the trial (p<0.001 vs placebo). In addition, more patients in the saxagliptin arm had glycated hemoglobin levels reduced to less than 7.0%. The relevant question is what the effect was for patients who achieved a glycated Hb of < 7.7%, which makes the p-value meaningless for an 0.3% change overall.
Implications of ca(2+) handling dysfunction
A. if the dysfuction is in smooth muscle – effect on arterial elasticity
B. if the dysfunction is in cardiomyocytes – Ventricular contractility & arrhythmias
We now review the calcium cycling of smooth muscle based on extracted work at MIT and Harvard Medical School, and at the University of Iowa. The work focuses on the disordered Ca(2+) signaling that plays a large role in the development of “arterial stiffness”, not disregarding the competing roles of endothelial nitric oxide and the inflammatory cell mediated oxidative stress related iNOS in the arterial circulation, and the preference for stress points at the junction of arteries. Disordered Ca(2+) in vascular smooth muscle leads to ischemic arterial disease, vascular rigidity from loss of flexibility, which can lead to ischemic myocardial damage.
Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells
Calcium ions (Ca2+) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion to be a ubiquitous 2nd messenger that carries information essential for cellular functions as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca2+ signal greatly differ from one cell type to another. In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca2+ signal. In each VSMC phenotype (synthetic/proliferating1 and contractile2 [1], tonic or phasic), the Ca2+ signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca2+ handling molecules (Figure 1).
1Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
2Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
in contractile VSMCs, the initiation of contractile events is driven by membrane depolarization; and the principal entry-point for extracellular Ca2+ is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca2+ is the store-operated calcium (SOC) channel. Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), has a critical role in determining the frequency of SR Ca2+ release by controlling the velocity of Ca2+ upload into the sarcoplasmic reticulum (SR) and the Ca2+ sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs
Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile response is initiated by extracellular Ca2* influx due to activation of Receptor Operated Ca2* channels (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca2* influx leads to large SR Ca2* release due to the activation of IP3R or RyR clusters (“Ca2*-induced Ca2*release” phenomenon). Cytosolic Ca2* is rapidly reduced by SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca2* and setting the sensitivity of RyR or IP3R for the next spike. Contraction of VSMCs occurs during oscillatory Ca2* transient. Middle panel: schematic representation of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima. Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca2*. Calcium is weakly reduced by SR calcium pumps (only SERCA2b, having low turnover and low affinity to Ca2* is expressed). Store depletion leads to translocation of SR Ca2* sensor STIM1 towards PM, resulting in extracellular Ca2* influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca2* transient is critical for activation of proliferation-related transcription factors ‘NFAT). Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca2*/calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5-trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca2* ATPase; SR – sarcoplasmic reticulum.
General aspects of calcium cycling and signaling in vascular smooth muscle cells
Besides maintaining vascular tone in mature vessels, VSMCs also preserve blood vessel integrity. VSMCs are instrumental for vascular remodeling and repair via proliferation and migration. Interestingly, Ca2* plays a central role in both physiological processes. In VSMCs, calcium signaling involves a cross-regulation of Ca2* influx, sarcolemmal membrane signaling molecules and Ca2* release and uptake from the sarco/endo/plasmic reticulum and mitochondria, which plays a central role in both vascular tone and integrity.
Calcium handling by the plasma membrane’s calcium channels and pumps
Membrane depolarization is believed to be a key process for the activation of calcium events in mature VSMCs. Thus, much attention has been given to uncovering the various mechanisms responsible for triggering this depolarization. Increased intra-vascular pressure of resistance arteries stimulates gradual membrane depolarization in VSMCs, increasing the probability of opening L-type high voltage-gated Ca2* channels (Cav1.2) (LTCC). Alternatively, the calcium-dependent contractile response can be induced through the activation of specific membrane receptors coupled to phospholipase C (PLC) isoforms3. The various isoforms of transient receptor potential (TRP) ion channel family, particularly TRPC3, TRPC6 and TRPC7 possibly activated directly by diacyl glycerol (DAG), can also contribute to initial plasma membrane Ca2* influx and subsequent membrane depolarization.
Among voltage-insensitive calcium influx pathways, the store-operated Ca2* channels (SOC), maintain a long-term cellular Ca2* signal. They are activated upon a decrease of internal store Ca2* concentration resulting from a Ca2* release via the opening of SR Ca2* release channels. SOC has two essential regulatory components, the SR/ER located Ca2* sensor STIM1 (stromal interaction molecule) and the Ca2* channels Orai. Upon decrease of [Ca2*] in the reticulum (<500µM), Ca2* dissociates from STIM1; then STIM1 molecules oligomerize and translocate to specialized cortical reticulum compartments adjacent to the plasma membrane. There, the STIM1 cytosolic activating domains bind to and cluster the Orai proteins into an opened archaic Ca2* channel known as Ca2*-release activated Ca2* channel (CRAC).
All isoforms of PLC, catalyze the hydrolysis of phosphatidylinositol4,5-biphosphate (PIP2) to produce the intracellular messengers IP3 increase and diacylglycerol (DAG); both of which promote cytosolic Ca2* rise through activation of plasma membrane or sarcoplasmic reticulum calcium channels.
The CRAC is responsible for the “2h cytosolic Ca2* increase” required to induce VSMCs proliferation.
The calcium signal is terminated by membrane hyper-polarization and cytosolic Ca2+ removal. First, calcium sparks resulting from the opening of sub-plasmalemmal clusters of RyR activate large-conductance Ca2+ sensitive K+ (BK) channels. Then, the resulting spontaneous transient outward currents (STOC) hyperpolarize the membrane and decrease the open probability of L-type Ca2+ channels. Cytosolic calcium is extruded at the level of plasma membrane by plasma membrane Ca2+ ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). The principal amount of cytosolic Ca2+ (> 70%) is re-uploaded to the internal store.
Calcium handling by the sarco/endoplasmic reticulum’s calcium channels and pumps
The initial entry of Ca2+ through plasma membrane channels triggers large Ca2+ release from the internal store via the process of Ca2+-induced Ca2+-release (CICR). The mechanism responsible for initiating Ca2+ release depends on Ca2+ sensitive SR calcium channels, the ryanodin receptor (RyR)5 or the IP3 receptor (IP3R). Indeed, IP3R and RyR are highly sensitive to cytosolic Ca2+ concentrations and when cytosolic Ca2+ concentration ranges from nM to µM, they open up. On the contrary, a higher cytosolic Ca2+ concentration (from µM to mM) closes them. In other words, cytosolic Ca2+ increase first exerts a positive feedback and facilitates SR channels opening whereas a further increase has an opposite effect and actually inhibits the SR channels opening. Importantly enough to be mentioned, RyR phosphorylation by the second messenger cyclic ADP ribose (cADPR) and protein kinase A (PKA) enhances Ca2+ sensitivity, the phosphorylation induced by the protein kinase C (PKC) decreases RyR sensitivity to Ca2+.
Sarco/Endoplasmic Ca2+ATPases (SERCA), the only calcium transporters expressed within sarco/endoplasmic reticulum (SR), serve to actively return calcium into this organelle. In mammals, three SERCA genes ATP2A1, ATP2A2 and ATP2A3 coding for SERCA1, SERCA2 and SERCA3 isoforms respectively have been identified [35]. Each gene gives rise to a different SERCA isoform through alternative splicing (Figure 2); they all have discrete tissue distributions and unique regulatory properties, providing a potential focal point within the cell for the integration of diverse stimuli to adjust and fine-tune calcium homeostasis in the SR/ER. In VSMCs, SERCA2a and the ubiquitous SERCA2b isoforms are expressed; besides vascular smooth muscle, SERCA2a is preferentially expressed in cardiac and skeletal muscles. SERCA2b differs from SERCA2a by an extension of 46 amino acids. Diversity of SERCA isoforms in the same cell suggests that each of them could be responsible for controlling unique cell functions.
RyR are structurally and functionally analogous to IP3R, although they are approximately twice as large and have twice the conductance of IP3R [27]; RyR channels are sensitive to store loading and IP3R channels are sensitized by the agonist-dependent formation of IP3.
SERCA2’s activity depends on its interaction with phospholamban and is inhibitory in its de-phosphorylated form. PKA phosphorylation of phospholamban results in its dissociation from SERCA2, thus activating the Ca2+ pumps. Cyclic ADP-ribose was also reported to stimulate SERCA pump activity.
As previously mentioned, SR Ca2+ content controls the sensitivity of SR Ca2+ channels, RyR and IP3R, as well as functioning of SOC-mediated Ca2+ entry, thereby determining the type of intracellular calcium transient. Since SOCs opening depends on Ca2+ content of the store, one may suggest that SERCA participates to its regulation. Consistent with this, SOCs open up when the leak of Ca2+ from intracellular stores is not compensated with SERCA activity; SERCA inhibitors such as thapsigargin which prevent Ca2+ uptake are commonly used to chemically induce SOC currents; several works have established that SERCA can cluster with STIM1 and Orai1 in various cellular types.
Mechanisms of cytosolic Ca2+ oscillations in VSMC
Ca2+ oscillations are one of the ways that VSMCs respond to agonists. These Ca2+ oscillations are maintained during receptor occupancy and are driven by an endogenous pacemaker mechanism, called the cellular Ca2+ oscillator. Ca2+ oscillators were classified into two main types, the membrane oscillators and the cytosolic oscillators.
Membrane oscillators are those which generate oscillations at the cell membrane by successive membrane depolarization. In most small resistance arteries, inhibitors of plasma membrane voltage-dependent channels reduce or even abolish the membrane potential oscillations which precede rhythmical contractions. This suggests that rhythmic extracellular Ca2+ influx can be required for calcium oscillatory transient. Besides, membrane oscillators greatly depend on Ca2+ entry in order to provide enough Ca2+ to charge up the intracellular stores for each oscillatory cycle.
Cytosolic oscillators do not depend on the cell membrane to generate oscillations. Instead, they arise from intracellular store membrane instability. The pacemaker mechanism of cytosolic Ca2+ oscillator is based on the velocity of luminal Ca2+ loading and luminal Ca2+ content. The mechanism responsible for initiating Ca2+ release depends either on RyRs or IP3R activation. As soon as stores are sufficiently charged with Ca2+, the SR Ca2+ channels become sensitive to cytosolic Ca2+ and can participate to the process of Ca2+-induced Ca2+-release, which is responsible for orchestrating the regenerative release of Ca2+ from the SR/ER. Importantly, extracellular Ca2+ influx is not required for cytosolic oscillator function. Indeed, the Ca2+ oscillations can be observed in the absence of extracellular Ca2+.
In mature vessels, VSMCs mainly exhibit a tonic or phasic contractile phenotype. In contractile VSMCs extracellular calcium influx predominantly takes place through the voltage-dependent L-type calcium channel, LTCC9 (Figure 3). Extracellular Ca2* influx causes a small increase of cytosolic Ca2* generated by the opening of IP3R clusters, called puff and/or RyR2 clusters, called spark. These local rises of cytosolic Ca2* generate a larger SR Ca2* release through the Ca2*-induced Ca2* release phenomenon. Elevation of free cytosolic calcium triggers VSMC contraction.
In contractile VSMCs, NFAT can be activated by sustained Ca2* influx (persistent Ca2* sparklets) mediated by clusters of L-type Ca2* channels operating in a high open probability mode
Steady state increase in cytosolic Ca2* triggers tonic contraction; oscillatory type of Ca2* transient triggers phasic contraction. It is worth mentioning that accumulating evidence indicate that SR Ca2*ATPase functioning/location within the cell (which greatly influences the velocity of calcium upload) determines the mode of Ca2* transient in VSMCs. Consistent with this, i) “phasic” VSMCs display a greater number of peripherally located SR than “tonic” VSMCs; indeed “tonic” VSMCs exhibit centrally located SR; (rev in [61, 77]); ii) drugs which interfere with the IP3 pathway or intracellular stores abolish spontaneous vaso-motion; iii) blocking SERCA strongly inhibits the Ca2* oscillations, demonstrating that they are induced by SR Ca2* release; this latter argument is further supported by the fact that oscillations are present even in the absence of extracellular Ca2*
SERCA2a has a higher catalytic turnover when compared to SERCA2b due to a higher rate of de-phosphorylation and a lower affinity for Ca2+; ii) SER-CA2a is absent in synthetic VSMCs, which only exhibit tonic contraction, iii) transferring the SERCA2a gene to synthetic cultured VSMCs modifies the agonist-induced calcium transient from steady-state to oscillatory mode. Therefore, one might suggest that the physiological role of SERCA2a in VSMCs consists of controlling the “cytosolic oscillator”, thereby determining phasic vs tonic type of smooth muscle contraction.
SERCA2a as a potential target for treating vascular proliferative diseases
Abundant proliferation of VSMCs is an important component of the chronic inflammatory response associated to atherosclerosis and related vascular occlusive diseases (intra-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Great efforts have been made to prevent/reduce trans-differentiation and proliferation of synthetic VSMCs. Anti-proliferative therapies including the use of pharmacological agents and gene therapy approaches are, until now, considered as a suitable approach in the treatment of these disorders. Indeed, coronary stenting is the only procedure that has been proven to reduce the incidence of late restenosis after percutaneous transluminal coronary angioplasty. Nevertheless, post-interventional intra-stent restenosis, characterized by the re-narrowing of the arteries caused by VSMC proliferation, occurs in 10 to 20 % of patients. These disorders remain the major limitation of revascularization by percutaneous transluminal angioplasty and artery bypass surgery. The use of drug-eluting stents (stent eluting anti-proliferative drug) significantly reduces restenosis but impairs the re-endothelialization process and subsequently often induces late thrombosis. In human, trans-differentiation of contractile VSMCs towards a synthetic/proliferating inflammatory/migratory phenotype after percutaneous transluminal angioplasty appears to be a fundamental process of vascularproliferative disease.
Concluding remarks
Over the last decade, great progress has been made in the understanding of the various intracellular molecular mechanisms in VSMCs which control calcium cycling and excitation/contraction or excitation/transcription coupling. VSMCs employ a great variety of Ca2+ signaling systems that are adapted to control their different contractile functions. Alterations in the expressions of Ca2+ handling molecules are closely associated with VSMC phenotype modulation. Furthermore, these changes in expression are inter-connected and each acquired or lost Ca2+ signaling molecule represents a component of signaling module functioning as a single unit.
In non-excitable synthetic VSMCs, calcium cycling results from the protein module ROC/IP3R/STIM1/ORAI1 which controls SOC influx. Agonist stimulation of synthetic VSMCs translates into a sustained increase in cytosolic Ca2+. This increase is required for the activation of NFAT downstream cellular signaling pathways inducing proliferation, migration and possibly an inflammatory response. Calcium cycling in excitable contractile VSMCs is governed by the protein module composed of ROC/LTCC/RyR2/SERCA2a and controls the contractile response.
Author details
Larissa Lipskaia
Mount Sinai School of Medicine, Department of Cardiology, New York, NY, USA
Isabelle Limon
Univ Paris 6, UR4 stress inflammation and aging, Paris, France
B. cardiomyocyte or smooth muscle. Let’s look a little further.
CaM kinase and disordering of intracellular calcium homeostasis , molecular link to arrhythmias
Mark E. Anderson, MD, PhD, Professor of Medicine and Pharmacology, University of Iowa, Iowa City, IADr. Anderson has presented a large body of work done at Vanderbilt University and University of Iowa Medical Schools for over a decade. The major hypothesis is that in the aftermath of a heart attack, the structural and electrical remodeling renders the heart prone to arrhythmias . The signaling molecule called calmodulin (CaM) kinase is a key and the work suggests that drugs that block CaM kinase activity might make good anti-arrhythmic medications. CaM kinase is a molecule that is intricately involved in calcium signaling and regulation. CaM kinase regulates calcium entry into the cell and calcium storage and release inside the cell.
Calcium enters heart cells through proteins called L-type calcium channels, donut-like pores in the cell membrane that open and close. If these channels stay open and let too much calcium into the cell, the risk of arrhythmia increases. Studies have shown that CaM kinase activity is increased in animal models and human heart disease. Dr. Anderson poses the question – does CaM kinase — which we know is elevated in heart disease — drive arrhythmias? The question is driven by their findings that the addition of activated CaM kinase allowed more calcium than normal to flow into isolated heart cells. The investigators measured the opening and closing of single calcium channels using a technique called patch-clamp electrophysiology. Then they added an already-activated form of CaM kinase to the preparation. When we added the activated CaM kinase, the calcium channels opened like crazy,” Anderson said. “In fact, they were more likely to open and stay open for long periods of time.
They also showed that cardiac cells with added CaM kinase had electrical changes called early afterdepolarizations (EADs). EADs are believed to be the triggering cause of arrhythmias in cardiomyopathy, hypertrophy, and long QT syndrome. The investigators implanted tiny telemeters into the mice and recorded electrocardiograms (ECGs) , which revealed not only the electrical changes expected in diseased hearts, Anderson said, but also an increased tendency for arrhythmias. Next, they treated the mice with a drug that blocks CaM kinase activity significantly suppressed the arrhythmias. They also found that cardiac cells isolated from the mice and found spontaneous EADs, which disappeared when the cells were treated with the CaM kinase-blocking drug. The evidence all points to CaM kinase driving arrhythmias.
They have demonstrated that CaM kinase is also important for calcium-activated gene expression and that it may be involved in the changes that occur in association with cardiac hypertrophy and heart failure. Anderson suggests that CaM kinase could be the link to explain why calcium channels open more frequently in heart failure, why people in heart failure have arrhythmias. He postulates that it would good to have a target that addresses both phenotypic disorders — the arrhythmia phenotype and the heart failure phenotype — and CaM kinase may be that target. Further, he observes that with the exception of so-called beta blockers, none of the current anti-arrhythmic drugs have been shown to reduce the mortality rate. More recent work in Iowa has identified a new link – a link between the inflammation in heart muscle following a heart attack and the enzyme calcium/calmodulin-dependent protein kinase II or CaM kinase II.
CaM kinase II, a pivotal enzyme that registers changes in calcium levels and oxidative stress and translates these signals into cellular effects, including changes in heart rate, cell proliferation and cell death. CaM kinase II also regulates gene expression — which genes are turned on or off at any given time. We have seen how Inhibition of CaM kinase II in mice protects the animals’ hearts against some of the damaging effects of a heart attack. A study compared a large number of genes that were expressed in the protected mice compared to the non-protected control mice. A particularly interesting finding was that a cluster of inflammatory genes was differently expressed depending on whether CaM kinase II was active or inhibited. Specifically, the research showed that heart attack triggered increased expression of a set of pro-inflammatory genes, and inhibition of CaM kinase II substantially reduced this effect.
The main research themes pursued by the Anderson laboratory are
Oxidative activation of CaMKII;
CaMKII signaling to ion channels;
The role of CaMKII in inflammation;
The role of CaMKII in cardiac pacemaker cells;
The role of CaMKII in cell survival.
Keywords: Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium, Calcium-Calmodulin-Dependent Protein Kinases, Calcium Channels, L-Type, Calmodulin, Arrhythmia, Ion channel, Hypertrophy, Cell Signaling, Signal Transduction
Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II.
Ana Sierra; Asipu Sivaprasadarao; Peter M Snyder; Ekaterina Subbotina; Michel Vivaudou; Zhiyong Zhu; Leonid V Zingman; et al.
Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.
Grueter, CE, Abiria, SA, Wu, Y, Anderson, ME, Colbran, RJ.
Biochemistry, 47(6), 1760-7, 2008.
Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies.
Werdich, AA, Lima, EA, Dzhura, I, Singh, MV, Li, J, Anderson, ME, Baudenbacher, FJ.
Am J Physiol Heart Circ Physiol, 294(5), H2352-62, 2008.
Conserved Regulation of Cardiac Calcium Uptake by Peptides Encoded in Small Open Reading Frames
Emile G. Magny1, Jose Ignacio Pueyo1, Frances M.G. Pearl1,2, MA Cespedes1, et al.
1 School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK.
2 Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK Science http:/dx.doi.org/10.1126/science.1238802
Small Open Reading Frames (smORFs) are short DNA sequences able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others showed previously that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport in the Drosophila heart influencing regular muscle contraction. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, where they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.
Excitation-contraction coupling in the heart: the state of the question.
Recent developments have led to great progress toward determining the mechanism by which calcium is released from the sarcoplasmic reticulum in the heart. The data support the notion of calcium-induced calcium release via a calcium-sensitive release channel. Calcium release channels have been isolated and cloned. This situation creates a paradox, as it has also been found that calcium release is smoothly graded and closely responsive to sarcolemmal membrane potential, properties that would not be expected of calcium-induced calcium release, which has intrinsic positive feedback. There is, therefore, no quantitative understanding of how the properties of the calcium release channel can lead to the macroscopic physiology of the whole cell. This problem could, in principle, be solved by various schemes involving heterogeneity at the ultrastructural level. The simplest of these require only that the sarcolemmal calcium channel be located in close proximity to one or more sarcoplasmic reticulum release channels. Theoretical modeling shows that such arrangements can, in fact, resolve the positive feedback paradox. An agenda is proposed for future studies required in order to reach a specific, quantitative understanding of the functioning of calcium-induced calcium release.
The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function
Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca(2+) transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.
Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites
The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca(2+) was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca(2+) (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca(2+) on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low [trans Ca(2+)] (20 microM), increasing [cis Ca(2+)] from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating [cis Ca(2+)] above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca(2+)] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca(2+) enhanced the sensitivity of the channel to activating cytosolic Ca(2+), and it essentially reversed the inhibition of the channel by high cytosolic Ca(2+). Potentiation of Po by increased luminal Ca(2+) occurred irrespective of whether the electrochemical gradient for Ca(2+) supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca(2+) through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca(2+) acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca(2+)-sensitive site(s) at the luminal face of the channel or associated protein.
Contemporary Definitions and Classification of the Cardiomyopathies
AHA Scientific Statement: Council on Clin. Cardiol.; HF and Transplant. Committee; Quality of Care and Outcomes Res. and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention
BJ Maron, Chair; JA Towbin; G Thiene; C Antzelevitch; D Corrado; D Arnett; AJ Moss; et al.
Circulation. 2006; 113: 1807-1816 http://dx.doi.org/10.1161/CIRCULATIONAHA.106.174287
Classifications of heart muscle diseases have proved to be exceedingly complex and in many respects contradictory. Indeed, the precise language used to describe these diseases is profoundly important. A new contemporary and rigorous classification of cardiomyopathies (with definitions) is proposed here. This reference document affords an important framework and measure of clarity to this heterogeneous group of diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular genetics in cardiology, as well as the introduction of several recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy.
Ryanopathy: causes and manifestations of RyR2 dysfunction in heart failure
Belevych AE, Radwański PB, Carnes CA, Györke S.
College of Medicine, The Ohio State University, Columbus, OH.
Cardiovasc Res. 2013; 98(2):240-7. http://dx.doi.org/10.1093/cvr/cvt024.
Epub 2013 Feb 12. PMID: 23408344 PMCID: PMC3633158 [Available on 2014/5/1]
The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodeling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
Up-regulation of Sarcoplasmic Reticulum Ca(2+) Uptake Leads to Cardiac Hypertrophy, Contractile Dysfunction and Early Mortality in mice deficient in CASQ2
Kalyanasundaram A, Lacombe VA, Belevych AE, Brunello L, Carnes CA, Janssen PM, … Gyørke S.
Department of Physiology and Cell Biology, College of Medicine, Ohio State University, Columbus, OH.
Cardiovasc Res. May 2013; 98(2):297-306. http://dx.doi.org/10.1093/cvr/cvs334. Epub 2012 Nov 6.
Aberrant Ca(2+) release (i.e. Ca(2+) ‘leak’) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptors (RyR2) is linked to heart failure (HF). Does SR-derived Ca(2+) can actually cause HF? We ask whether and by what mechanism combining dysregulated RyR2 function with facilitated Ca(2+) uptake into SR exacerbates abnormal SR Ca(2+) release and induces HF.
We crossbred mice deficient in expression of cardiac calsequestrin (CASQ2) with mice overexpressing the skeletal muscle isoform of SR Ca(2+)ATPase (SERCA1a). The new double-mutant strains displayed early mortality, congestive HF with left ventricular dilated hypertrophy, and decreased ejection fraction. Intact right ventricular muscle preparations from double-mutant mice preserved normal systolic contractile force but were susceptible to spontaneous contractions. Double-mutant cardiomyocytes while preserving normal amplitude of systolic Ca(2+) transients displayed marked disturbances in diastolic Ca(2+) handling in the form of multiple, periodic Ca(2+) waves and wavelets. Dysregulated myocyte Ca(2+) handling and structural and functional cardiac pathology in double-mutant mice were associated with increased rate of apoptotic cell death. Qualitatively similar results were obtained in a hybrid strain created by crossing CASQ2 knockout mice with mice deficient in phospholamban.
We demonstrate that enhanced SR Ca(2+) uptake combined with dysregulated RyR2s results in sustained diastolic Ca(2+) release causing apoptosis, dilated cardiomyopathy, and early mortality. Further, up-regulation of SERCA activity must be advocated with caution as a therapy for HF in the context of abnormal RyR2 function.
Comment in
Mind the store: modulating Ca(2+) reuptake with a leaky sarcoplasmic reticulum. [Cardiovasc Res. 2013] PMID: 23135969 [PubMed – in process] PMCID: PMC3633154 [Available on 2014/5/1]
Myocardial Delivery of Stromal Cell-Derived Factor 1 in Patients With Ischemic Heart Disease: Safe and Promising Circ. Res.. 2013;112:746-747
Circulation Research Thematic Synopsis: Cardiovascular Genetics Circ. Res.2013;112:e34-e50,
Ryanodine Receptor Phosphorylation and Heart Failure: Phasing Out S2808 and ³Criminalizing² S2814 ,
By the time the heart reaches the pathological state clinically recognized as heart failure (HF), it has undergone profound and often irreversible alterations in structure and function at the molecular, cellular and organ level. Although the etiologies of HF are diverse:
hypertension,
myocardial infarction,
atherosclerosis,
valvular insufficiency,
mutations in genes encoding sarcomeric proteins
Some alterations are commonly found in most forms of HF, and they may account for the maladaptive structural remodeling and systolic dysfunction that characterize this syndrome.
At the cellular level, there are well documented changes in
ionic channel density and function (electrical remodeling),
increased ROS production,
mitochondrial dysfunction,
imbalanced energy intake and consumption,
genetic reprogramming,
altered excitation-contraction coupling,
and in general, dysregulation of a multitude of other processes and pathways that are essential for proper cardiac function. Combined, this myriad of alterations leads to
loss in contractility and
loss ejection fraction,
ventricular wall remodeling,
increased vascular resistance, and
dysregulated fluid homeostasis.
In this issue of Circulation Research, Respress et al.2 report that preventing phosphorylation of cardiac ryanodine receptors (RyR2) at a single residue, S2814, is sufficient to avert many of these alterations and improve cardiac function in HF. The results presented here follow a string of papers that touch on the delicate and controversial subject of ryanodine receptor phosphorylation and HF. They offer a new twist to a contentious story and attempt to reconcile many apparently contradicting results, but key issues remain.
Calcium “Leak” in HF
It appears that suppressing the dysfunction of a select group of biological and molecular signaling pathways may substantially improve or even reverse the cardiac deterioration observed in HF. For example, correcting the characteristically depressed sarcoplasmic reticulum (SR) calcium content of failing cardiomyocytes is a target of HF gene therapy. SR calcium “leak”, an operational term that indicates increased and untimely calcium release by RyR2s, also appears common to several models of HF. Therefore, stemming off calcium “leak” may prevent the progression of cardiac malfunction in HF patients. However, a rationalized therapy towards this aim must be founded on the precise knowledge of the mechanisms leading to calcium leak. Marks group, in a landmark publication in 2000 (ref. 6) and later in multiple other high-impact factor papers (many of them co-authored by Wehrens 7-10) postulated that RyR2 “hyperphosphorylation” at S2808 by PKA was the primary mechanism leading to increased calcium “leak” in HF. This idea was initially appealing and fueled intensive research in the subject, but many groups failed to reproduce central tenets of this hypothesis. (11 and 12) The controversies surrounding the Marks-Wehrens hypothesis of increased calcium leak by hyperphosphorylation of RyR2-S2808 have been recently and comprehensibly reviewed by Bers.13 Here I will focus on the modifications to this hypothesis as derived from the new findings of Respress et al.2 Emerging points from these new findings will be the demotion of S2808, to intervene not as universal player in HF but only in selective forms of this syndrome, and the role of S2814 as pre-eminent generator of calcium leak that leads to arrhythmias and exacerbates other forms of HF. The “criminalization” of S2814 has begun in earnest.
CaMKII Effect on Calcium Leak and the Role of S2808 and S2814
Many studies have provided evidence that persistent CaMKII activity can lead to cardiac arrhythmias and promote HF.14-16 Animals and patients with congestive HF display increased levels of CaMKII,17,18 and overexpression of AC3-I, a peptide inhibitor of CaMKII, delays the onset of HF in mice.19 There is also good agreement4,20 (although not universal21) that CaMKII, and not PKA, increases calcium leak, and therefore, it is likely that the arrhythmogenic and deleterious activity of CaMKII in HF may be associated with this effect. Obviously, if PKA does not cause calcium leak directly, this by itself imposes insurmountable constraints on the Marks-Wehrens hypothesis that posits that PKA phosphorylation of RyR2-S2808 is responsible for the high calcium leak of HF. With the focus now on CaMKII, the obligated question is then, by what mechanisms CaMKII increases calcium leak from the SR? To increase calcium leak, the cell must either increase SR calcium content, and/or increase the activity of the RyR2 (albeit the latter alone would have only transient effects due to autoregulatory mechanisms22). Since both PKA and CaMKII increase SR calcium load by phosphorylating phospholamban (but at different residues) and relieving the inhibition it exerts on SERCA2a, the differential effect of these kinases must result from the regulation they exert on RyR2s. Wehrens group offers here2 at least a partial explanation of this complex mechanism and, along with previous papers co- authored with Marks, these groups set specific roles for S2808 and S2814 on regulation of RyR2 activity and their protective effect (or lack thereof) in HF. In their view, PKA exclusively phosphorylates S2808 and dissociates FKBP12.6, which destabilizes the closed state of the channel and increases RyR2 activity, whereas CaMKII (almost) exclusively phosphorylates S2814, has no effect on FKBP12.6 binding, and equally activates RyR2s. In this issue, Respress et al.2 report that preventing phosphorylation of S2814 (by genetic substitution of Ser by Ala, S2814A) protects against non-ischemic (pressure overload) HF but has no effect on ischemic HF; conversely, and against other data by the same groups, S2808 phosphorylation was not significantly different in non-ischemic HF, implying that it is relevant only in ischemic HF. This clean targeting of RyR2 phospho-epitopes by PKA and CaMKII and their nice “division of labor” for pathogenicity in distinct forms of HF would really simplify phosphorylation schemes and reconcile apparent contradictions. However, as is generally the case, the proposal appears oversimplified and almost too good to be true. Let’s discuss each of the premises on which the Respress et al.2 results have been interpreted and the problems associated with these premises.
One kinase = one site = one effect. Is it really that simple?
The RyR2 is a huge protein. It is assembled as a tetrameric complex of ~2 million Da, with each subunit composed of ~5,000 amino acids.
Using canonical phosphorylation consensus and high confidence values, the RyR2 may be phosphorylated in silico at more than 100 sites by the combined action of PKA,
CaMKII,
PKG, and
PKC, to name a few.11
Granted, a “potential” phosphorylation site is very different than a demonstrated, physiologically-relevant phosphorylation site and it is possible that many of the predicted residues are not phosphorylated in vivo. Even then, several groups have demonstrated that CaMKII phosphorylates RyR2 with stoichiometry of at least 3 or 4 to 1 with respect to PKA.23-26 This fact is by itself compelling evidence that there are multiple phosphorylation sites in RyR2. Now, let’s make the optimistic assumption that all the PKA sites have already been mapped, and that S2808 and S2030 (ref. 27) are the only PKA sites. Taking into account the CaMKII:PKA phosphorylation ratio (3:1 or 4:1), this would then yield a minimum of ~6 – 8 CaMKII phosphorylation sites (per channel subunit!). In this perspective, it is almost disingenuous to label S2808 as “the” PKA site, and we may purposely deceive ourselves when we label S2814 “the” CaMKII site. Against this sense of pessimism and intractability, let’s not forget that S2808 was actually discovered as a CaMKII site.24 It is possible then that the number of CaMKII sites is smaller if only S2030 remains as a bona fide PKA site. Still, neither scheme supports one CaMKII site per channel subunit.
But let’s go along for a moment with the possibility, however unlikely, that PKA phosphorylates S2808 only, and CaMKII phosphorylates S2814 only. When calling these sites by their distinctive numbers, it is easy to forget that these phospho-sites are only 6 residues apart, that is, a minuscule proportion (~0.000003%) in the context of the whole channel protein. How can the same reaction (phosphorylation) that occurs at sites so close to one another be differentially transmitted to the very distant gating domains of the channel? If these residues were lining the pore of the channel, where critical differences emerge by substituting one residue but not the neighboring one, then it would be easier to understand how S2808 and S2814 could transmit distinct signals. But both are part of a “phosphorylation hot spot”, a cytoplasmic loop that contains additional potential phospho-sites11 and that has been mapped to the external surface of the channel.28 Marks and Wehrens groups have shown that phosphorylation of S2808A by CaMKII or of S2814A by PKA fully activate the channel.7,9 At face value, this means that knocking out one phospho-residue does not cripple this “hot spot” and that phosphorylation of at least one residue in this external loop enables it to transmit conformational changes to the gating domains of the channel. Seen in this structural context in which the “hot spot” works in unison upon phosphorylation of at least one residue, it is very difficult (but not impossible) to accommodate the notion that phosphorylation of S2808 or S2814 alone dictates the differential response of the RyR2 to PKA and CaMKII.
An Alternative Model to explain Differential PKA and CaMKII Effects
An alternative model to explain the differential effect of PKA and CaMKII to elicit calcium leak from RyR2 that takes into account other phospho-sites is needed. Before formulating it, let’s consider some important points. First, it is not difficult to assume that the role of the “phosphorylation hot spot” is to readily pick up signals from different kinases. The multi-valence of this “hot spot” is demonstrated so far by the fact that S2808 may be phosphorylated by CaMKII24,25,26 and by PKA,6,25,26 and its eagerness to undergo phosphorylation by the fact that S2808 is at least ~50% phosphorylated even at basal state25-27,29,30 and phospho-signals from these sites may be readily detected upon β-adrenergic stimulation of the heart.30,31Second, if we accept the Shannon and Bers results that CaMKII, and not PKA, elicits calcium leak from the SR,4,20 this obligatorily means that PKA phosphorylation of S2808 is not responsible for eliciting calcium leak (in direct conflict with the Marks-Wehrens hypothesis). In support of this notion, studies by the Houser and Valdivia groups have provided evidence that preventing S2808 phosphorylation has negligible impact on the β-adrenergic response of the heart and on the progression of non-ischemic and ischemic HF.30-32 Third, another PKA site, S2030, largely ignored in the Marks-Wehrens scheme, has been mapped and shown to activate channel openings27 and although its place in the larger context of RyR2 phosphorylation has not been determined yet, I think it is illogical to assume that its existence is futile and that it contributes nothing to regulation of the channel. Thus, according to the preceding discussion, it is almost unsustainable to postulate that the differential effects of CaMKII and PKA to elicit calcium leak stems from their effects on the RyR2 “phosphorylation hot spot” alone. Instead, I would like to posit an alternative model that integrates findings by many of the above-referenced groups (Fig. 1). In this model, the surface domain of the RyR2 comprising residues 2804-2814 (mouse nomenclature) is an eager target for phosphorylation by PKA, CaMKII and probably other kinases (4 Ser/Thr).11,24-26,29 Phosphorylation of this “hot spot” by either PKA or CaMKII (or both) “primes” the RyR2 for subsequent signals and is probably responsible for the coordinated openings in response to fast calcium stimuli detected in single channel recordings33 and in cellular settings34 (but this has yet to be demonstrated). The differential effect of PKA and CaMKII on RyR2 activity would then depend on the integrated response of the phosphorylated “hot spot” and of additional phosphorylation sites. For example, phosphorylation of S2808 and S2030 by PKA could coordinate channel openings in response to fast calcium stimuli, and phosphorylation of S2814 and other CaMKII site(s) could open RyR2s at diastolic [Ca2+], which would translate in calcium leak. Examples of proteins acting as molecular switchboards in response to various degrees of phosphorylation are not unprecedented.35 In fact, RyR2s are activated by phosphorylation and dephosphorylation as well36,37 and their relative degree of phosphorylation determines a final functional output.38 It is therefore conceivable that the complex response of RyR2s to any type of phosphorylation and the variable results obtained by investigators apparently using the same experimental conditions may be due to the variable degree of phosphorylation in which the RyR2s were found. Of course, until the 3D structure of the RyR2 is solved and we understand the mechanism by which the “phosphorylation hot spot” and other phospho-sites “talk” to the channel’s gating domains this structurally-based model will remain speculative, but it at least takes into consideration compelling evidence on the existence of various phosphorylation sites and departs substantially from the simplified notion of one kinase = one site = one effect.
Fig. 1 Models of RyR2 modulation by phosphorylation
Models of RyR2 modulation by phosphorylation. In the Marks-Wehrens model (A), S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic but is not relevant in ischemic HF, only in nonischemic HF. In the multiphosphorylation site model (B), S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.
In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated Ca2+ release from intracellular Ca2+ stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and “leaky,” leading to arrhythmias and other pathology. This “leaky RyR2 hypothesis” is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory.
In the signalling scheme outlined in Figure1 of this commentary, which prevailed until the end of the last century, the two major determinants of intracellular Ca2+ transients and thereby the contractile force of the heart were (a) the size of the Ca2+ current entering via the LTCC (well exemplified by the negative inotropic effects of LTCC blockers) and (b) the activity of SERCA and thus the Ca2+ load of the SR. The critical role of the latter was convincingly demonstrated by the fact that Plb–/– mice, which have maximal SERCA activity, exhibit higher basal force and reduced inotropic response to isoprenaline (1).
In the Marks-Wehrens model, S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic, but is not relevant in ischemic HF, only in non-ischemic HF. In the multi-phosphorylation site model, S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.
Appealing as Marks’ theory is, the concept has been challenged and remains controversial (Tables1 and 2). On the one hand, some theoretical considerations argue against it. For example, it seems counterintuitive that phosphorylation at a single residue in a protein of more than 5,000 amino acids could profoundly affect channel open probability. Second, S2808, the proposed site of phosphorylation by PKA, is located in an area distant from the FKBP12.6/RyR2 interaction site (3), making it somewhat unlikely that phosphorylation affects FKPB12.6 binding. Third, it seems unlikely and to contradict experimental results (4) that an isolated increase in RyR2 open probability has more than a transient consequence on Ca2+ handling, because an isolated increase in Ca2+release from the RyR2 will automatically lead to reduced Ca2+ load in the SR and therefore fast normalization of Ca2+ transients (autoregulation).
More concerning than theoretical considerations are numerous reports that failed to reproduce important aspects of the data that support the leaky RyR2 hypothesis and the critical importance of S2808 (Tables (Tables11and and2).2). (a) Phosphorylation of RyR2 at S2808 has been found by others to be either not altered in heart failure at all or to be only moderately increased (5–8). Others have reported that 75% of the available RyR2 S2808 sites are phosphorylated under normal conditions, making a 9-fold change in chronic heart failure somewhat unlikely (9). (b) Whereas general consensus exists that β-adrenergic stimulation increases spontaneous Ca2+ release (the “Ca2+ leak”) from the SR, the role of RyR2 phosphorylation and FKBP12.6 dissociation remains controversial. Importantly, PKA had no effect on Ca2+release in permeabilized Plb–/– mouse myocytes, i.e., cells in which the SR is maximally loaded with Ca2+ and one would have expected a particularly strong effect of increasing RyR2 open probability.
Now, let’s go back to the results of Respress et al.2 and consider them in this light. They found that preventing phosphorylation of S2814 alone mitigates non-ischemic HF induced by transverse aortic constriction (TAC) in mice. This implies that other CaMKII sites are not necessary to mitigate the CaMKII-induced calcium leak that they propose is responsible for the deleterious effect in WT mice subjected to TAC. If phosphorylation of the “hot spot” is compulsory to prime the RyR2 to process and discriminate other phosphorylation signals, then other residues in that “hot spot” must have been phosphorylated to fulfill this need. Surprisingly, S2808 was not significantly phosphorylated in this setting. This leaves a very difficult conundrum: if S2808 was not phosphorylated significantly and the other CaMKII sites are not necessary to stop calcium leak, how then can we explain the results of Respress et al.2? Of course there are always alternatives, and we would be inconsistent if we rigidly adhere to one model and fell into the dogmatism we are criticizing. The conclusions of Respress et al.2 are in line with their findings, but at this point the numbers do not add up and it’s obvious that the great complexity of this process (RyR2 phosphorylation) precludes simplified and neatly organized schemes. As a clear example of this, in the landmark paper by Marks group,6 S2808 was found substantially hyperphosphorylated in tachypacing-induced failing dogs, also a non-ischemic model of HF. This does not fit well in the current scheme of Wehrens where S2808A protects against ischemic HF, but has no prominent role in non-ischemic HF.
In summary, CaMKII and PKA may have specific roles in calcium leak and, since they both increase SR calcium load, their differential effect likely resides on their effect on RyR2s. However, the effect of PKA- or CaMKII-phosphorylation of RyR2s does not appear solved yet. Starting in 2000 and up to the present day, Marks and Wehrens have provided high-quality data in prominent journals aggressively pursuing the notion that PKA phosphorylates S2808 only, that CaMKII phosphorylates S2814 only, and that these sites alone integrate multiple signals to open RyR2s. Many key aspects of their general hypothesis including dissociation of FKBP12.6 by PKA phosphorylation of S2808, subconductance states as hallmarks of phosphorylation, and the prominent role of S2808 as promoter of arrhythmias and HF have not been confirmed by several groups. The present paper by the Wehrens group modifies slightly the original claim that S2808 was involved in ischemic and non-ischemic forms of HF and continues to shift the lion’s share of pathogenicity to S2814. However, as discussed above, the Marks-Wehrens model largely ignores compelling data on the presence of multiple phosphorylation sites and the complexity they add to the finely graded response of RyR2s to phosphorylation.
2. Respress JL, van Oort RJ, Li N, Rolim N, Dixit S, Dealmeida A, Voigt N, Lawrence WS, Skapura DG, Skårdal K, Wisloff U, Wieland T, Ai X, Pogwizd SM, Dobrev D, Wehrens XH. Role of RyR2 Phosphorylation at S2814 During Heart Failure Progression. Circ Res. 2012;xx:xx–xx. [in the issue; printer, please update] [PMC free article] [PubMed]
6. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000;101(4):365–376. [PubMed]
7. Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A. 2006;103:511–518. [PMC free article] [PubMed]
36. Lokuta AJ, Rogers TB, Lederer WJ, Valdivia HH. Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. J Physiol. 1995;487:609–622. [PMC free article] [PubMed]
37. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Gyorke S. Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes. J Physiol. 2003;552(Pt 1):109–118. [PMC free article] [PubMed]
38. Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at Ser-2809 by protein kinase A produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res. 2006; 98:1506–1513. [PubMed]
The Cardiac Ryanodine Receptor (calcium release channel) – Emerging role in Heart Failure and Arrhythmia Pathogenesis
The cardiac sarcoplasmic reticulum calcium release channel, commonly referred to as the ryanodine receptor, is a key component in cardiac excitation–contraction coupling, where it is responsible for the release of calcium from the sarcoplasmic reticulum. As our knowledge of the ryanodine receptor has advanced an appreciation that this key E–C coupling component may have a role in the pathogenesis of human cardiac disease has emerged. Heart failure and arrhythmia generation are both pathophysiological states that can result from deranged excitation–contraction coupling. Evidence is now emerging that hyperphosphorylation of the cardiac ryanodine receptor is an important event in chronic heart failure, contributing to impaired contraction and the generation of triggered ventricular arrhythmias.
Furthermore the therapeutic benefits of β blockers in heart failure appear to be partly explained through a reversal of this phenomenon. Two rare inherited arrhythmogenic conditions, which can cause sudden death in children, have also been shown to result from mutations in the cardiac ryanodine receptor. These conditions,
catecholaminergic polymorphic ventricular tachycardia and
arrhythmogenic right ventricular cardiomyopathy (subtype 2),
further implicate the ryanodine receptor as a potentially arrhythmogenic substrate and suggest this channel may offer a new therapeutic target in the treatment of both cardiac arrhythmias and heart failure.
Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes
D Terentyev, S Viatchenko-Karpinski, I Gyorke, R Terentyeva and S Gyorke
Texas Tech University Health Sciences Center, Lubbock, TX
J Physiol 2003; 552(1), pp. 109–118. http:/dx.doi.org/10.1113/jphysiol.2003.046367
Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation–contraction coupling; furthermore, abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca(2+) sparks and SR Ca(2+) load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca(2+) sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca(2+) stores, as determined by application of caffeine. These changes in Ca(2+) release and SR Ca(2+) load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR mediated SR Ca(2+) release leading to depleted SR Ca(2+) stores in cardiac myocytes.
In heart muscle cells, the process of excitation–contraction (EC) coupling is mediated by Ca(2+) influx through sarcolemmal L-type Ca(2+) channels activating Ca(2+) release channels (ryanodine receptors, RyRs) in the sarcoplasmicreticulum (SR). Once activated, the RyR channels allow Ca(2+) to be released from the SR into the cytosol to induce contraction. This mechanism is known as Ca(2+)-induced calcium release (CICR) (Fabiato, 1985; Bers, 2002).
During relaxation, most of the Ca(2+) is resequestered into the SR by the Ca(2+)-ATPase. The amount of Ca(2+) released and the force of contraction depend on the magnitude of the Ca(2+) trigger signal, the functional state of the RyRs and the amount of Ca(2+) stored in the SR. Reversible phosphorylation of proteins composing the EC coupling machinery plays an important role in regulation of cardiac contractility (Bers, 2002). Thus, during stimulation of the b-adrenergic pathway, phosphorylation of several target proteins, including the L-type Ca(2+) channels, RyRs and phospholamban, by protein kinase A (PKA) leads to an overall increase in SR Ca2+ release and contractile force in heart cells (Callewaert et al. 1988, Spurgeon et al. 1990; Hussain & Orchard, 1997; Zhou et al. 1999; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). PKA-dependent phosphorylation of the L-type Ca(2+) channels increases the Ca2+ current (ICa), increasing both the Ca2+ trigger for SR Ca2+ release and the SR Ca(2+) content (Callewaert et al. 1988; Hussain & Orchard, 1997; Del Principe et al. 2001). Phosphorylation of phospholamban (PLB) relieves the tonic inhibition dephosphorylated PLB exerts on the SR Ca(2+)-ATPase (SERCA) resulting in enhanced SR Ca(2+) accumulation and enlarged Ca(2+) release (Kranias et al. 1985; Simmermann & Jones, 1998). With regard to the RyR, despite clear demonstration of phosphorylation of the channel in biochemical studies (Takasago et al. 1989; Yoshida et al. 1992), the consequences of this reaction to channel function have not been clearly defined. RyR phosphorylation by PKA and Ca(2+)–calmodulin dependent protein kinase (CaMKII) has been reported to increase RyR activity in lipid bilayers (Hain et al. 1995; Marx et al. 2000; Uehara et al. 2002). Moreover, it has been reported that in heart failure (HF), hyperphosphorylation of RyR causes the release of FK-506 binding protein (FKBP12.6) from the RyR, rendering the channel excessively leaky for Ca(2+) (Marx et al. 2000). However, other studies have reported no functional effects (Li et al. 2002) or even found phosphorylation to reduce RyR channel steady-state open probability (Valdivia et al. 1995; Lokuta et al. 1995).
The Action of Protein Kinases is Opposed by Dephosphorylating Phosphatases.
Three types of protein: phosphatases (PPs), referred to as
PP1,
PP2A and
PP2B (calcineurin),
have been shown to influence cardiac performance (Neumann et al. 1993; Rusnak & Mertz, 2000). Overall, according to most studies phosphatases appear to downregulate SR Ca(2+) release and contractile performance (Neumann et al. 1993; duBell et al. 1996, 2002; Carr et al. 2002; Santana et al. 2002). Furthermore, PP1 and PP2A activities appear to be increased in heart failure (Neumann, 2002; Carr et al. 2002). However, again the precise mode of action of these enzymes on intracellular Ca(2+) handling in normal and diseased hearts remains poorly understood. In the present study, we have investigated the effects of protein phosphatases PP1 and PP2A on local Ca(2+) release events, Ca(2+) sparks, in cardiac cells. Our results show that phosphatases activate RyR mediated SR Ca(2+) release leading to depletion of SR Ca(2+) stores. These results provide novel insights into the mechanisms and potential role of protein phosphorylation/dephosphorylation in regulation of Ca(2+) signaling in normal and diseased hearts.
RESULTS
Effects of PP1 and PP2A on Ca2+ Sparks and SR Ca(2+) Content.
PP1 caused an early transient potentiation of Ca2+ spark frequency followed by a delayed inhibition of event occurrence.
PP1 produced similar biphasic effects on the magnitude and spatio-temporal characteristics of Ca(2+) sparks
Specifically, during the potentiatory phase (1 min after addition of the enzyme), PP1 significantly increased the amplitude, rise-time, duration and width of Ca(2+) sparks; during the inhibitory phase (5 min after addition of the enzyme), all these parameters were significantly suppressed by PP1.
The SR Ca(2+) content decreased by 35 % or 69 % following the exposure of myocytes to either 0.5 or 2Uml_1 PP1, respectively (Fig. 1C).
Qualitatively similar results were obtained with phosphatase PP2A. Similar to the effects of PP1, PP2A (5Uml_1) produced a transient increase in Ca(2+) spark frequency (~4-fold) followed by a depression of event occurrence and decreased SR Ca(2+) content (by 82 % and 65 %, respectively). Also similar to the action of PP1, PP2A increased the amplitude and spatio-temporal spread (i.e. rise-time, duration and width) of Ca(2+) sparks at 1 min and suppressed the same parameters at 5 min of exposure to the enzyme (Table 1). Together, these results suggest that phosphatases enhance spark-mediated SR Ca2+ release, leading to decreased SR Ca(2+) content.
Preventive effects of calyculin A and okadaic acid
Preventive effects of ryanodine
PP1-mediated RyR dephosphorylation
The cardiac RyR is phosphorylated at Ser-2809 (in the rabbit sequence) by both PKA and CAMKII (Witcher et al. 1991; Marx et al. 2000). Although additional phosphorylation sites may exist on the RyR (Rodriguez et al. 2003), Ser-2809 is believed to be the only site that is phosphorylated by PKA, and RyR hyperphosphorylation at this site has been reported in heart failure (Marx et al. 2000). To test whether indeed phosphatases dephosphorylated the RyR in our permeabilized myocyte experiments we performed quantitative immunoblotting using an antibody that specifically recognizes the phosphorylated form of the RyR at Ser-2809 (Rodriguez et al. 2003). Myocytes exhibited a significant level of phosphorylation under baseline conditions. Maximal phosphorylation was 201 % of control. When exposed to 2Uml_1 PP1, RyR phosphorylation was 58 % of the control basal condition. Exposing to a higher PP1 concentration (10Uml_1) further reduced RyR phosphorylation to 22% of control. Thus, consistent with the results of our functional measurements, PP1 decreased RyR phosphorylation in cardiac myocytes.
A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 2Uml_1 PP1. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP1 in the same cell. The Ca(2+) transients were elicited by a whole bath application of 10 mM caffeine. B, averaged spark frequency at early (1 min) and late (5 min) times following the addition of either 0.5 or 2Uml_1 of PP1 to the bathing solution. C, averaged SR Ca(2+) content for 0.5 or 2Uml_1 of PP1 measured before and 5 min after exposure to the enzyme. Data are presented as means ± S.E.M. of 6 experiments in different cells.
A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 5Uml_1 PP2A. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP2A in the same cell. B and C, averaged spark frequency (B) and SR Ca(2+) content (C) for the same conditions as in A. Data are presented as means ± S.E.M. of 6 experiments in different cells.
DISCUSSION
In the present study, we have investigated the impact of physiologically relevant exogenous protein phosphatases PP1 and PP2A on RyR-mediated SR Ca(2+) release (measured as Ca(2+) sparks) in permeabilized heart cells. Our principal finding is that phosphatases stimulated RyR channels leading to depleted SR Ca(2+) stores. These results have important ramifications for understanding the mechanisms and role of protein phosphorylation/dephosphorylation in modulation of Ca(2+) handling in normal and diseased heart.
Modulation of SR Ca2+ release by Protein Phosphorylation/Dephophorylation
Since protein dephosphorylation clearly resulted in increased functional activity of the Ca(+)release channel, our results imply that a reverse, phosphorylation reaction should reduce RyR activity. If indeed such effects take place, why do they not manifest in inhibition of Ca(+)sparks? One possibility is that enhanced Ca(+) uptake by SERCA masks or overcomes the effects phosphorylation may have on RyRs. In
addition, the potential inhibitory influence of protein phosphorylation on RyR activity in myocytes could be countered by feedback mechanisms involving changes in luminal Ca(+)(Trafford et al. 2002; Gyorke et al. 2002). In particular, reduced open probability of RyRs would be expected to lead to increased Ca2+ accumulation in the SR; increased intra-SR [Ca(2+)] in turn would increase activity of RyRs at their luminal Ca(2+) regulatory sites as demonstrated for the RyR channel inhibitor tetracaine (Gyorke et al. 1997; Overend et al. 1997). Thus potentiation of SERCA combined with the intrinsic capacity of the release mechanism to self-regulate could explain at least in part why PKA-mediated protein phoshorylation results in maintained potentiation of Ca(2+) sparks despite a potential initial decrease in RyR activity.
Role of altered RyR Phosphorylation in Heart Failure
Marx et al. (2000) have proposed that enhanced levels of circulating catecholamines lead to increased phosphorylation of RyR in heart failure. Based on biochemical observations as well as on studying properties of single RyRs incorporated into artificial lipid bilayers, these investigators have hypothesized that hyperphosphorylation of RyRs contributes to pathogenesis of heart failure by making the channel excessively leaky due to dissociation of FKBP12.6 from the channel. We show that the mode of modulation of RyRs by phosphatases does not support this hypothesis as dephosphorylation caused activation instead of inhibition of activity of RyR channels in a relatively intact setting. Interestingly, our results provide the basis for a different possibility in which dephophosphorylation of RyR rather than its phosphorylation causes depletion of SR Ca(2+) stores by stimulating RyRs in failing hearts. It has been reported that PP1 and PP2 activities are increased in heart failure (Huang et al. 1999; Neumann et al. 1997; Neuman, 2002). Furthermore, overexpression of PP1 or ablation of the endogenous PP1 inhibitor, l-1, results in depressed contractile performance and heart failure (Carr et al. 2002). Our finding that PP1 causes depletion of SR Ca(2+) stores by activating RyRs could account for, or contribute to, these results.
DelPrincipe F, Egger M, Pignier C & Niggli E (2001). Enhanced E-C coupling efficiency after beta-stimulation of cardiac myocytes. Biophys J 80, 64a.
Gyorke I & Gyorke S (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J 75, 2801–2810.
Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S & Wiesner TF (2002). Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7, d1454–d1463.
Gyorke I, Lukyanenko V & Gyorke S (1997). Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol 500, 297–309.
MacDougall LK, Jones LR & Cohen P (1991). Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196, 725–734.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365–376.
Rodriguez P, Bhogal MS & Colyer J (2003). Stoichiometric phosphorylation of cardiac ryanodine receptor on serine-2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (in press).
Proc Natl Acad Sci U S A. 2010 August 3; 107(31): E124.
The ryanodine receptor/calcium-release channel (RyR2) on the sarcoplasmic reticulum (SR) is the source of Ca2+ required for myocardial excitation–contraction (EC) coupling. During stress (i.e., exercise), contractility of the cardiac muscle is increased largely because of phosphorylation and activation of key proteins that regulate SR Ca2+ release. These include the voltage-gated calcium channel (Cav1.2) on the plasma membrane through which Ca2+ enters the cardiomyocyte, the sarco/endoplasmic reticulum calcium ATPase (SERCA2a)/phospholamban complex that pumps Ca2+ into the SR, and the RyR2 channel that releases Ca2+ from the SR, all of which are activated by phosphorylation.
For the past 10 y, Eisner et al. (1) have advanced the idea that activation of the RyR2 channel (e.g., by phosphorylation) cannot play a role in regulating systolic Ca2+ release and cardiac contractility. They base their position on an experiment in which they used caffeine to activate the RyR2 channel and showed that Ca2+ release was increased but after a few beats, returned to baseline (1). However, their experiment is not a good model for the physiological response to stress in which the three key regulators of EC coupling are all activated by the same signal (i.e., phosphorylation) such that there is increased Ca2+ influx, increased SR Ca2+ uptake, and increased SR Ca2+ release.
In the Eisner caffeine experiment, RyR2 was activated, but the Cav1.2 and SERCA2a were not. Selective activation of RyR2 is not physiological, and the outcome of their experiment was predictable. Caffeine-induced activation of RyR2 resulted in a transient increase in SR Ca2+ release, but because there was no concomitant increase in Ca2+ influx or SR Ca2+ uptake, the increase in SR Ca2+ release could not be sustained. However, on the basis of this experiment, Eisner et al. (1) concluded that activation of RyR2 plays no role in stress-induced increased cardiac contractility.
We have shown that, during stress, the increased heart rate results in a rate-dependent activation of CaMKII that phosphorylates and activates RyR2. We showed the essential role of this rate-dependent activation of RyR2 by CaMKII by showing that genetically engineered mice, lacking the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), exhibit blunted increases in systolic Ca2+-transient amplitudes and contractile responses as heart rate increases (2). We also showed that a reduction in the amount of CaMKII in the RyR2 complex in failing hearts results in defective regulation of the channel, which could explain the loss of the rate-dependent increase in contractility in heart failure.
Eisner et al. (3) challenge all of our findings based on their caffeine experiment. However, our experiments have been conducted under physiological conditions in which all three components involved in Ca2+signaling during muscle contraction are activated, not just one. The only perturbation that we have introduced is to ablate the CaMKII phosphorylation site on RyR2 using a single amino acid substitution. This results in a blunted contractile response, leading us to conclude that CaMKII phosphorylation of RyR2 does indeed play a key role in enhancing contractility as the heart rate increases.
Cardiac Ryanodine Receptor Function and Regulation in Heart Disease
Cardiac Engineering: From Genes and Cells to Structure and Function 2004; 1015(1), pp 144–159
The cardiac ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR) controls intracellular Ca2+ release and muscle contraction in the heart. Ca2+ release via RyR2 is regulated by several physiological mediators. Protein kinase (PKA) phosphorylation dissociates the stabilizing FKBP12.6 subunit (calstabin2) from the RyR2 complex, resulting in increased contractility and cardiac output. Congestive heart failure is associated with
elevated plasma catecholamine levels, and
chronic stimulation of β-adrenergic receptors
leads to PKA hyperphosphorylation of RyR2 in failing hearts.
PKA hyperphosphorylation results in calstabin2-depleted RyR2 that displays altered channel gating and
may cause aberrant SR Ca2+ release,
depletion of SR Ca2+ stores, and
reduced myocardial contractility in heart failure.
Calstabin2-depleted RyR2 may also trigger cardiac arrhythmias that cause sudden cardiac death. In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), RyR2 missense mutations cause reduced calstabin2 binding to RyR2. Increased RyR2 phosphorylation and pathologically increased calstabin2 dissociation during exercise results in aberrant diastolic calcium release, which may trigger ventricular arrhythmias and sudden cardiac death. In conclusion, heart failure and exercise-induced sudden cardiac death have been linked to defects in RyR2-calstabin2 regulation, and this may represent a novel target for the prevention and treatment of these forms of heart disease
The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure
T Zhang, LS Maier, ND Dalton, S Miyamoto, J Ross, DM Bers, JH Brown
University of California, San Diego, La Jolla, Calif; and Loyola University, Chicago, Ill.
Circ Res. 2003;92:912-919. http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5
Recent studies have demonstrated that transgenic (TG) expression of either Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIδB but also a cytoplasmic isoform, CaMKII δC. In the present study, we demonstrate that expression of the δC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic δC isoform of CaMKII can lead to phosphorylation of Ca(2+) regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2(2+) handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIδC can mediate phosphorylation of Ca(2+) regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδC activation in the pathogenesis of dilated cardiomyopathy and heart failure.
Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca2+ signals that phosphorylate a wide range of substrates and thereby affect Ca(2+)-mediated cellular responses.1 The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.1 The CaMKII subunits α, β, γ, and δ show different tissue distributions,1 with the δisoform predominating in the heart.4–7 Splice variants of the δisoform, characterized by the presence of a second variable domain,4,7 include δB, which contains a nuclear localization signal (NLS), and δC, which does not. CaMKII composed of δB subunits localizes to the nucleus, whereas CaMKIIδC localizes to the cytoplasm.4,8,9
CaMKII has been implicated in several key aspects of acute cellular Ca(2+) regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and phospholamban (PLB).10–14 Phosphorylation of RyR has been suggested to alter the channel open probability,14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca(2+) uptake.14 It is also likely that CaMKII phosphorylates the L-type Ca2 channel complex or an associated regulatory protein and thus mediates Ca2 current (ICa) facilitation.16-18 and the development of early after-depolarizations and arrhythmias.19 Thus, CaMKII has significant effects on E-C coupling and cellular Ca2 regulation. Nothing is known about the CaMKII isoforms regulating these responses.
Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte Ca2homeostasis.20 CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy21,22 and heart failure,23 and in cardiac tissue from patients with dilated cardiomyopathy.24,25
Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,27 but this isoform is not detectable in the heart,4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).29
Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,30 later shown to be associated with an increase in activated CaMKII31; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKIIδB, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.32 To determine whether in vivo expression of the cytoplasmic CaMKIIδC can phosphorylate cytoplasmic Ca2regulatory proteins and induce hypertrophy or heart failure, we generated transgenic (TG) mice that expressed the δC isoform of CaMKII under the control of the cardiac specific α-myosin heavy chain (MHC) promoter. Our findings implicate CaMKIIδC in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that this occurs at least in part via alterations in Ca2handling proteins.33
Results
Expression and Activation of CaMKIIδC Isoform After TAC
To determine whether CaMKII was regulated in pressureoverload–induced hypertrophy, CaMKIIδ expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC. A selective increase (1.6-fold) in the lower band of CaMKIIδwas observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A). To confirm that CaMKIIδC was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKIIδC isoform. These experiments revealed that mRNA levels for CaMKIIδC were increased 1 to 7 days after TAC (Figure 1B). In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca2-independent activity.
A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIδB and δC (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIδB plus δ9, and the bottom band (56 kDa) corresponds to CaMKIIδC. *P0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIδC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKIIγ subunit (uppermost), CaMKIIδB plus δ9 (58 kDa), and CaMKIIδC (56 kDa). Quantitation is based on the sum of all of the bands. *P0.05 vs control.
A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKIIδTG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat antirabbit IgG antibody (1:100 dilution). CaMKIIδB localization to the nucleus in CaMKIIδB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKIIδprotein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG () and their littermates () were immunoblotted with an anti-CaMKIIδ antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n5 for each group). **P0.01 vs WT.
Generation and Identification of CaMKIIδC Transgenic Mice
TG mice expressing HA-tagged rat wild-type CaMKIIδC under the control of the cardiac-specific α-MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number),
The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement. The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart.
Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls
(Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca2/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly, the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.
Cardiac Overexpression of CaMKIIδC Induces Cardiac Hypertrophy and Dilated Cardiomyopathy
There was significant enlargement of hearts from CaMKIIδC TGM mice by 8 to 10 weeks (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKIIδC TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7 + 0.1 pL for TGM (n = 96) versus 28.6 + 0.1 pL for WT littermates (n=94; P0.001).
Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL. None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed. Contractile function was significantly decreased.
C, Decreased contractile function in ventricular myocytes isolated from 12-week old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P0.05 vs WT.
Figure 7. Phosphorylation of PLB in CaMKIIδC TG mice.
Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n = 6 to 8 mice per group; *P0.05 vs WT.
Cardiac Overexpression of CaMKIIδC Results in Changes in the Phosphorylation of Ca2 Handling Proteins
To assess the possible involvement of phosphorylation of Ca2cycling proteins in the phenotypic changes observed in the CaMKIIC TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B).
To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3 + 1.3 versus 27.7 + 4.6 pL; P0.14). Also, twitch Ca2 transient amplitude was not yet significantly depressed, and mean δ [Ca2+]i (1 Hz) was only 20% lower (192 + 36 versus 156 + 13 nmol/L; P0.47) versus 50% lower in TGM at 13 weeks.33
The in vivo phosphorylation of RyR2, determined by back phosphorylation, was significantly (2.10.3-fold; P0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2 + 0.3-fold; P0.05) in 4- to 5-week-old TGM mice (Figure 8C).
The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKIIδC TG hearts, can be attributed to the increased activity of CaMKII.
Discussion
CaMKII is involved in the dynamic modulation of cellular Ca2 regulation and has been implicated in the development of cardiac hypertrophy and heart failure.14 Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.27,32 However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca(2+) handling, because they localize to the nucleus.
First, we demonstrate that the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC. Second, we demonstrate that two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions. Moreover, CaMKIIδis found to associate physically with the RyR in the heart. Finally, our data indicate that heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca(2+) handling.
Differential Regulation of CaMKIIδ Isoforms in Cardiac Hypertrophy
The isoform of CaMKII that predominates in the heart is the δ isoform.4–7 Neither the α nor the β isoforms are expressed and there is only a low level of expression of the γ isoforms.39 Both δB and δC splice variants of CaMKIIδ are present in the adult mammalian myocardium36,40 and expressed in distinct cellular compartments.4,8,9
We suggest that the CaMKIIδisoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKIIδC is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that CaMKIIδC is regulated at the transcriptional level in response to
TAC. In addition, activation of both CaMKIIδB and CaMKIIδC, as indexed by autophosphorylation, increases as early as 2 days after TAC. Activation of CaMKIIδB by TAC is relevant to our previous work indicating its role in hypertrophy.9,32 The increased expression, as well as activation of the CaMKIIδC isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.
In conclusion, we demonstrate here that CaMKIIδC can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article33 suggest that altered phosphorylation of Ca(2+) cycling proteins is a major component of the observed decrease in contractile function in CaMKIIδC TG mice. The early occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKIIδC TG mice suggest that CaMKIIδC plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure. These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.
Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.
DR Witcher, RJ Kovacs, H Schulman, DC Cefali, LR Jones
Krannert Institute of Cardiology and the Indiana University School of Medicine, Indianapolis,
Stanford University School of Medicine, Stanford.
Journal of Biological Chemistry 07/1991; 266(17):11144-52. · 4.77 Impact http://www.jbc.org/content/266/17/11144.full.pdf
Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca(2+) release channel by CaM kinase may provide a unique mechanism for regulating intracellular (Ca2+) release.
The Ca(2+) release from the SR causes an increase in Ca(2+) concentration which leads to muscle contraction (1). Recently, the sites of Ca(2+) release have been identified and purified from both cardiac (2-4) and skeletal muscle SR (5- 7) and shown to be the same as the ryanodine receptors or high molecular weight proteins. The structures attach the transverse tubules to the junctional SR both in intact tissues and isolated membrane fractions (1, 8-10). Although the Ca(2+) release channels from cardiac and skeletal muscle show many similarities such as nearly identical
myoplasmic 3- EGTA,
Ca2+ conductances (2-7),
protease sensitivities (11, E ) ,
calmodulin-binding capabilities (ll), and
modulation by allosteric regulators such as Ca2+, Mg2+, ATP, and calmodulin (13-15),
they also exhibit several differences in protein structure and function. Quantitative differences have been noted on the effects of modulators on ryanodine binding to the two proteins (16-18), as well as on Ca(2+) channel kinetics. In addition, the cardiac ryanodine smaller apparent molecular weight than the skeletal muscle receptor on SDS-PAGE (ll), and monoclonal antibodies can be made which react with the cardiac receptor but not the skeletal receptor (16).
Recent work on characterization receptors has culminated in elucidation of structures of the proteins by sequencing of their cDNAs (19-21). Consistent with the differences between the two protein iso- forms noted above, the cardiac and skeletal muscle receptors have been found to be the products of different genes, with overall amino acid identities of 66% (21). Both protein isoforms are very large, containing approximately 5,000 amino acids and exhibiting predicted molecular weights of 564,711 for the cardiac protein (21) and 565,223 (19) or 563,584 (20) for the skeletal muscle protein. In the native state, ryanodine receptors are arranged as tetramers (1-7). In an earlier study (22), we demonstrated that the canine cardiac high molecular weight protein (or ryanodine receptor; Ref. 3) was an excellent substrate CaM kinase (23,24) endogenous to junctional SR membranes. In the work described here, we show that phosphorylation of the cardiac receptor by CaM kinase occurs at a single site, which is not substantially phosphorylated in the skeletal muscle receptor, and that phosphorylation ryanodine receptor at this site activates the Ca2+ channel.
Our data are the first to support the hypothesis (21), that the modulator-binding sites of the cardiac ryanodine receptor are contained within residues 2619-3016. (13, 14). The ryanodine receptor is compared with the primary structure for the multifunctional of the cardiac model of Otsu et al. (21).
Preferential Phosphorylation Receptor-(Fig. 1, arrowheads) is phosphorylated in junctional vesicles by an endogenous calmodulin-requiring proteinase and this phosphorylation is stimulated several fold when exogenous CaM kinase is added. In contrast, the ryanodine receptor in canine fast and vesicles, which migrates with weight on SDS-PAGE (2, 11, 16), is not significantly phosphorylated by either endogenous or exogenous protein kinase (Fig. 1, small arrows).
Similar results were obtained with rabbit skeletal muscle SR vesicles. The identity of the skeletal muscle ryanodine receptor in these studies (Fig. 1, small arrow) was confirmed by immunoblotting with a skeletal muscle isoform-specific antibody (supplied by K. Campbell, University of Iowa). We did detect a low level of phosphorylation of a protein in slow skeletal muscle samples migrating slightly faster than the cardiac receptor, but this protein did not cross-react with skeletal muscle (or cardiac, see below) antibodies, suggesting that it is unrelated to the ryanodine receptor. CaM kinase-catalyzed phosphorylation of the cardiac ryanodine receptor was always at least 10-fold greater than skeletal receptor phosphorylation. These results demonstrate that the skeletal muscle ryanodine receptor phosphorylation is insignificant compared to cardiac protein phosphorylation. Consistent with our results, Otsu et al. (21) have recently shown that, the cardiac isoform receptor is absent from fast and slow skeletal muscle. Phosphorylation of the cardiac ryanodine receptor by cAMP kinase also occurs, but phosphorylation by added cAMP kinase is no greater than that achieved with endogenous CaM kinase. (Fig. 2). In contrast, the amount of exogenous CaM kinase increases receptor phosphorylation 4-fold, to a maximal level of 26 pmol of P/mg of SR protein (Fig. 2). We observed no significant phosphorylation of canine fast and slow or rabbit skeletal muscle ryanodine. Maximal ryanodine binding (3) in these preparations ranged between 5 and 6 pmol/mg of protein, a value nearly identical to the level of receptor phosphorylation achieved with exogenous cAMP kinase (see CaM kinase), but one-fourth the value achieved with added CaM kinase. Since the functional unit release channel contains only one high affinity ryanodine- binding site/tetramer (4), our results suggest that the endogenous CaM kinase is capable of phosphorylating only one-fourth of the available sites, whereas the exogenous kinase can fully phosphorylate the receptor (below) of the Cardiac Ryanodine. The canine Slow skeletal muscle SR receptor of the ryanodine it was recently reported is phosphorylated 1/20th by the of the CaM kinase.
TABLE 1
Immunoprecipitation of Ryanodine receptors from CHAPS-solubilized canine SR membranes. Values are expressed for aliquots of the following fractions: S, solubilized receptors after treatment of membranes with 2% CHAPS; B, bound fraction, containing ryanodine receptors immunoprecipitated from CHAPS superna- tant; F, free fraction, containing ryanodine receptors not immunoprecipitated. Total binding was measured using 20 nM [3H]ryanodine. For nonspecific binding, 10 PM cold ryanodine was added. FIG. 7.
Effect of ATP and calmodulin on the cardiac Ca(2+) release channel. Holding potential was 0 mV, with upward current deflections representing movement of Ba(2+) from the trans to the cis chamber. Gaussian distributions were fit to the peaks of activity in the histograms. Signals were filtered at 300 Hz (low pass Bessel) and digitized at 1 KHz (Axotape, Axon Instruments) for * off-line analysis. In the control (A), p(open) was 0.26. Addition of 1 mM ATP (B) produced prolonged openings of the channel, increasing p(0pen) to 0.81. Subsequent addition of calmodulin (C) decreased p(open) to 0.12, producing long closures and brief aborted openings.
Sequencing of the Cardiac Phosphorylation Site. In order to sequence the phosphorylation site of the cardiac ryanodine receptor, we phosphorylated junctional SR membranes on large scale with added CaM kinase and purified the phosphorylated denatured ryanodine receptor to homogeneity in one step using SDS-gel filtration chromatography (Fig. 3). The purified cardiac ryanodine receptor was digested with trypsin, and the radioactive peptides recovered using Fe(3+) affinity chromatography (30,37). 90% of the loaded radioactivity was recovered in the pH 8.6 and 10 eluates from the Fe column (Fig. 4). These fractions were then combined and subjected to reverse-phase chromatography, yielding a single major radioactive peptide peak eluting at approximately 24% acetonitrile (Fig. 4, inset).
Gas-phase sequencing of the radioactive tryptic peptide gave a single sequence of 18 consecutive residues, which corresponded exactly to residues 2807-2824 reported for the rabbit cardiac ryanodine receptor from cDNA cloning (Fig. 5) (21). When CNBr and endoproteinase Lys-C were used to cleave the receptor, another “P-labeled peptide was isolated and sequenced, which matched with residues 2800-2811 of the rabbit cardiac ryanodine receptor (Fig. 5).
Serine 2809 within the phosphorylated tryptic peptide is situated on the carboxyl-terminal side of 2 arginine residues. The fact that R-R-X-S and R-X-X-S/T are minimal consensus phosphorylation sequences (38,39) for CAMP kinase and CaM kinase, respectively, makes this residue the likely phosphorylation site. Consistent with this, the ratio threitol-serine to phenylthiohydantoin-serine recovered dur- ing cycle 3 of sequencing of this peptide was 10 times greater than that recovered during cycles 6 and 9. It is known that dithiothreitol-serine is the predominant breakdown product of phosphoserine (40, 41). Phosphoamino acid analysis revealed that this peptide contained only phosphoserine; more- over, >90% of the 3’Pi was released from the peptide by cycle 10 (40, 42), demonstrating that no serine residue downstream of this region was significantly labeled.
Based on these results, we conclude that serine 2809 is the amino acid phosphorylated by CaM kinase. When only endogenous CaM kinase was used to phosphorylate the cardiac ryanodine receptor, the same labeled tryptic peptide was recovered and sequenced in four separate runs. Thus, although exogenously added kinase gives a 4-fold stimulation of receptor phosphorylation (Fig. 2), no new sites are phosphorylated. The reason for the low level of phosphorylation obtained with endogenous CaM kinase remains undefined.
Cardiac Electrophysiological Dynamics From the Cellular Level to the Organ Level
Daisuke Sato and Colleen E. Clancy
Department of Pharmacology, University of California – Davis, Davis, CA.
Biomedical Engineering and Computational Biology 2013:5: 69–75
Figure 1. (Top): APD and DI. (Bottom): The physiological mechanism of APD alternans involves recovery from inactivation of ICaL. [see http://dx.doi.org/10.4137/BECB.S10960]
Figure 2. APD restitution and dynamical mechanism of APD alternans. [see http://dx.doi.org/10.4137/BECB.S10960] Review Series. Genetic Causes of Human Heart Failure
Hiroyuki Morita, Jonathan Seidman and Christine E. Seidman
Harvard Medical School, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Boston, MA
J Clin Invest. 2005;115(3):518–526. http://dx.doi.org/10.1172/JCI24351.
Correspondence to: Christine E. Seidman, Department of Genetics, Harvard Medical School, Boston, MA. Ph: (617) 432-7871; E-mail: cseidman@genetics.med.harvard.edu
Factors that render patients with cardiovascular disease at high risk for heart failure remain incompletely defined. Recent insights into molecular genetic causes of myocardial diseases have highlighted the importance of single-gene defects in the pathogenesis of heart failure. Through analyses of the mechanisms by which a mutation selectively perturbs one component of cardiac physiology and triggers cell and molecular responses, studies of human gene mutations provide a window into the complex processes of cardiac remodeling and heart failure. Knowledge gleaned from these studies shows promise for defining novel therapeutic targets for genetic and acquired causes of heart failure.
Introduction
Heart failure currently affects 4.8 million Americans, and each year over 500,000 new cases are diagnosed. In 2003 heart failure contributed to over 280,000 deaths and accounted for 17.8 billion health care dollars (1).
Heart failure almost universally arises in the context of antecedent cardiovascular disease:
atherosclerosis,
cardiomyopathy,
myocarditis,
congenital malformations, or
valvular disease.
The study of single-gene mutations that trigger heart failure provides an opportunity for defining important molecules involved in these processes. Although these monogenic disorders account for only a small subset of overall heart failure cases, insights into the responses triggered by gene mutations are likely to also be relevant to more common etiologies of heart failure.
Early Manifestation – Heart Failure – Ventricular Remodeling.
One of 2 distinct morphologies occurs: left ventricular hypertrophy (increased wall thickness without chamber expansion) or dilation (normal or thinned walls with enlarged chamber volumes).
Each is associated with specific hemodynamic changes. Systolic function is normal, but diastolic relaxation is impaired in hypertrophic remodeling; diminished systolic function characterizes dilated remodeling. Clinical recognition of these cardiac findings usually prompts diagnosis of hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). There is now considerable evidence that many different gene mutations can cause these pathologies (Figure 1), and with these discoveries has come recognition of distinct histopathologic features that further delineate several subtypes of remodeling. The current compendia of genes that remodel the heart already suggest a multiplicity of pathways by which the human heart can fail.
To facilitate a discussion, we have grouped known cardiomyopathy genes according to the probable functional consequences of mutations on
force generation and transmission,
metabolism,
calcium homeostasis, or
transcriptional control.
Gene mutations in one functional category inevitably have an impact on multiple myocyte processes, and, the eventual delineation of signals between functional groups may be critical to understanding cardiac decompensation and heart failure development.
Human gene mutations can cause cardiac hypertrophy (blue), dilation (yellow), or both (green). In addition to these two patterns of remodeling, particular gene defects produce hypertrophic remodeling with glycogen accumulation (pink) or dilated remodeling with fibrofatty degeneration of the myocardium (orange). Sarcomere proteins denote β-myosin heavy chain, cardiac troponin T, cardiac troponin I, α-tropomyosin, cardiac actin, and titin. Metabolic/storage proteins denote AMP-activated protein kinase γ subunit, LAMP2, lysosomal acid α 1,4–glucosidase, and lysosomal hydrolase α-galactosidase A. Z-disc proteins denote MLP and telethonin. Dystrophin-complex proteins denote δ-sarcoglycan, β-sarcoglycan, and dystrophin. Ca2+ cycling proteins denote PLN and RyR2. Desmosome proteins denote plakoglobin, desmoplakin, and plakophilin-2.
Force generation and propagation. Generation of contractile force by the sarcomere and its transmission to the extracellular matrix are the fundamental functions of heart cells. Inadequate performance in either component prompts cardiac remodeling (hypertrophy or dilation), produces symptoms, and leads to heart failure. Given the importance of these processes for normal heart function and overt clinical manifestations of deficits in either force generation or transmission, it is not surprising that more single-gene mutations have been identified in molecules involved in these critical processes than in those of other functional classes.
Human mutations affecting contractile and Z-disc proteins. The schematic depicts one sarcomere,
the fundamental unit of contraction encompassing the protein segment between flanking Z discs. Sarcomere thin filament proteins are composed of actin and troponins C, T, and I. Sarcomere thick filament proteins include myosin heavy chain, myosin essential and regulatory light chains, myosin-binding protein-C and titin. The sarcomere is anchored through titin and actin interactions with Z disc proteins α-actinin, calsarcin-1, MLP, telethonin (T-cap), and ZASP. Human mutations (orange text) in contractile proteins and Z-disc proteins can cause HCM or DCM.
Sarcomere protein mutations. Human mutations in the genes encoding protein components of the sarcomere cause either HCM or DCM. While progression to heart failure occurs with both patterns of remodeling, the histopathology, hemodynamic profiles, and biophysical consequences of HCM or DCM mutations suggest that distinct molecular processes are involved.
Over 300 dominant mutations in genes encoding β-cardiac myosin heavy chain (MYH7), cardiac myosin-binding protein-C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), essential myosin light chain (MYL3), regulatory myosin light chain (MYL2), α-tropomyosin (TPM1), cardiac actin (ACTC), and titin (TTN) have been reported to cause HCM (Figure 2) (2, 3). Recent reports of comprehensive sequencing of sarcomere protein genes in diverse patient populations indicate that MYBPC3 and MYH7 mutations are most frequent (4, 5). Sarcomere gene mutations that cause HCM produce a shared histopathology with enlarged myocytes that are disorganized and die prematurely, which results in increased cardiac fibrosis.
The severity and pattern of ventricular hypertrophy,
age at onset of clinical manifestations, and
progression to heart failure
are, in part, dependent on the precise sarcomere protein gene mutation. For example, TNNT2 mutations are generally associated with a high incidence of sudden death despite only mild left ventricular hypertrophy (6, 7). While only a small subset (10–15%) of HCM patients develop heart failure, this end-stage phenotype has a markedly poor prognosis and often necessitates cardiac transplantation. Accelerated clinical deterioration has been observed with MYH7 Arg719Trp, TNNT2 Lys273Glu, TNNI3 Lys183del, and TPM1 Glu180Val mutations (8–11).
Most HCM mutations encode defective polypeptides containing missense residues or small deletions; these are likely to be stably incorporated into cardiac myofilaments and to produce hypertrophy because normal sarcomere function is disturbed. Many HCM mutations in MYBPC3 fall within carboxyl domains that interact with titin and myosin; however, the exact biophysical properties altered by these defects remain unknown (Figure 2). HCM mutations in myosin are found in virtually every functional domain, which suggests that the biophysical consequences of these defects may vary. Genetic engineering of some human myosin mutations into mice has indicated more consistent sequelae. Isolated single-mutant myosin molecules containing different HCM mutations
had increased actin-activated ATPase activity and
showed greater force production and
faster actin-filament sliding,
biophysical properties that may account for hyperdynamic contractile performance observed in HCM hearts and that suggest a mechanism for premature myocyte death in HCM (12–14). Uncoordinated contraction due to
heterogeneity of mutant and normal sarcomere proteins,
increased energy consumption, and
changes in Ca2+ homeostasis
could diminish myocyte survival and trigger replacement fibrosis. With insidious myocyte loss and increased fibrosis, the HCM heart transitions from hypertrophy to failure.
Mice that are engineered to carry a sarcomere mutation replicate the genetics of human disease; heterozygous mutations cause HCM. One exception is a deletion of proximal myosin-binding protein-C sequences; heterozygous mutant mice exhibited normal heart structure while homozygous mutant mice developed hypertrophy (15). Remarkably, while most heterozygous mouse models with a mutation in myosin heavy chain, myosin-binding protein-C, or troponin T developed HCM (16–18), homozygous mutant mice (19, 20) developed DCM with fulminant heart failure and, in some cases, premature death. These mouse studies might indicate that HCM, DCM, and heart failure reflect gradations of a single molecular pathway. Alternatively, significant myocyte death caused by homozygous sarcomere mutations may result in heart failure. Human data suggest a more complicated scenario. The clinical phenotype of rare individuals who carry homozygous sarcomere mutations in either MYH7 (21) or in TNNT2 (22) is severe hypertrophy, not DCM. Furthermore, individuals with compound heterozygous sarcomere mutations exhibit HCM, not DCM. The absence of ventricular dilation in human hearts with 2 copies of mutant sarcomere proteins is consistent with distinct cellular signaling programs that remodel the heart into hypertrophic or dilated morphologies.
DCM sarcomere protein gene mutations affect distinct amino acids from HCM-causing mutations, although the proximity of altered residues is remarkable. The histopathology of sarcomere DCM mutations is quite different from those causing HCM, and is remarkably nonspecific. Degenerating myocytes with increased interstitial fibrosis are present, but myocyte disarray is notably absent. There are 2 mechanisms by which sarcomere mutations may cause DCM and heart failure: deficits of force production and deficits of force transmission. Diminished force may occur in myosin mutations (e.g., MYH7 Ser532Pro) that alter actin-binding residues involved in initiating the power stroke of contraction. Impaired contractile force may also occur in DCM troponin mutations (TNNT2 ΔLys210, ref. 23; and TNNI3 Ala2Val, ref. 24) that alter residues implicated in tight binary troponin interactions. Because troponin molecules modulate calcium-stimulated actomyosin ATPase activity, these defects may cause inefficient ATP hydrolysis and therein decrease contractile power.
Other DCM sarcomere mutations are more likely to impair force transmission (Figure 2). For example, a myosin mutation (at residue 764) located within the flexible fulcrum that transmits movement from the head of myosin to the thick filament is likely to render ineffectual the force generated by actomyosin interactions (23). DCM TPM1 mutations (25) are predicted to destabilize actin interactions and compromise force transmission to neighboring sarcomere. Likewise, ACTC mutations (26) that impair binding of actin to Z-disc may compromise force propagation. TTN mutations provide quintessential evidence that deficits in force transmission cause DCM and heart failure. By spanning the sarcomere from Z-disc to M-line, this giant muscle protein assembles contractile filaments and provides elasticity through serial spring elements. Titin interacts with α-actinin and telethonin (T-cap) at the Z-disc, with calpain3 and obscurin at the I-band (the extensible thin filament regions flanking Z-discs), and with myosin-binding protein-C, calmodulin, and calpain3 at the M-line region. Human mutations identified in
the Z-disc–I-band transition zone (27),
in the telethonin and α-actinin–binding domain, and
in the cardiac-specific N2B domain (an I-band subregion; ref. 28) each cause DCM and heart failure.
Intermediate filaments and dystrophin-associated glycoprotein mutations. Intermediate filaments function as cytoskeletal proteins linking the Z-disc to the sarcolemma. Desmin is a type III intermediate filament protein, which, when mutated, causes skeletal and cardiac muscle disease (Figure 3). The hearts of mice deficient in desmin (29) are more susceptible to mechanical stress, which is consistent with the function of intermediate proteins in force transmission.
Figure 3
Human mutations (orange text) in components of myocyte cytoarchitecture cause DCM and heart failure. Force produced by sarcomeric actin-myosin interactions is propagated through the actin cytoskeleton and dystrophin to the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin). Desmosome proteins plakoglobin, desmoplakin, and plakophilin-2, provide functional and structural contacts between adjacent cells and are linked through intermediate filament proteins, including desmin, to the nuclear membrane, where lamin A/C is localized. (Adapted from ref. 96.)
Through dystrophin and actin interactions, the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin) provides stability to the sarcomere and transmits force to the extracellular matrix. Human mutations in these proteins cause muscular dystrophy with associated DCM and heart failure (Figure 3). Skeletal muscle manifestations can be minimal in female carriers of X-linked dystrophin defects, and some individuals present primarily with heart failure (30). In the mouse experiment, coxsackievirus B3–encoded protease2A, which can cleave dystrophin, was shown to produce sarcolemmal disruption and cause DCM, which suggests that dystrophin is also involved in the pathologic mechanism of DCM and heart failure that follow viral myocarditis (31).
While deficiencies of proteins that link the sarcomere to the extracellular matrix are likely to impair force transmission, recent studies of mice engineered to carry mutations in these molecules indicate other mechanisms for heart failure. A model of desmin-related cardiomyopathies (32) uncovered striking intracellular aggresomes, electron dense accumulations of heat shock and chaperone protein, α-B-crystalline, desmin, and amyloid in association with sarcomeres. While particularly abundant in the amyloid heart, aggresomes were also found in some DCM and HCM specimens, which suggests that excessive degenerative processing induced by myocyte stress or gene mutation may be toxic to sarcomere function.
Analyses of δ-sarcoglycan null mice (33) also yielded unexpected disease mechanisms, primary coronary vasospasm and myocardial ischemia. Selective restoration of δ-sarcoglycan to the cardiac myocytes extinguished this pathology, thereby implicating chronic ischemia as a contributing factor to heart failure development in patients with sarcoglycan mutations.
Mutations in intercalated and Z-disc proteins. To generate contraction, one end of each actin thin filament must be immobilized. The Z-disc defines the lateral boundary of the sarcomere, where actin filaments, titin, and nebulette filaments are anchored. Metavinculin provides attachment of thin filaments to the plasma membrane and plays a key role in productive force transmission. Two metavinculin gene mutations cause DCM by disruption of disc structure and actin-filament organization (34).
Other Z-disc protein constituents may also function as mechano-stretch receptors (35). Critical components include α-actinin, which aligns actin and titin from neighboring sarcomeres and interacts with muscle LIM protein (MLP encoded by CSRP3), telethonin (encoded by TCAP), which interacts with titin and MLP to subserve overall sarcomere function, and Cypher/Z-band alternatively spliced PDZ-motif protein (Cypher/ZASP), a striated muscle-restricted protein that interacts with α-actinin–2 through a PDZ domain and couples to PKC-mediated signaling via its LIM domains (Figure 2). Mutations in these molecules cause either DCM (35, 36) or HCM (37, 38) and predispose the affected individuals to heart failure. Genetically engineered mice with MLP deficiency (39) help to model the mechanism by which mutations in distinct proteins cause disease. Without MLP, telethonin is destabilized and gradually lost from the Z-disc; as a consequence, MLP-deficient cardiac papillary muscle shows an impairment in tension generation following the delivery of a 10% increase in passive stretch of the muscle and a loss of stretch-dependent induction of molecular markers (e.g., brain natriuretic peptide), which suggests that an MLP-telethonin–titin complex is an essential component of the cardiac muscle mechanical stretch sensor machinery. An important question is how signaling proteins (e.g., Cyper/ZASP) within the Z-disc translate mechanosensing into activation of survival or cell death pathways.
Lamin A/C mutations. The inner nuclear-membrane protein complex contains emerin and lamin A/C. Defects in emerin cause X-linked Emery-Dreifuss muscular dystrophy, joint contractures, conduction system disease, and DCM. Dominant lamin A/C mutations exhibit a more cardiac-restricted phenotype with fibrofatty degeneration of the myocardium and conducting cells, although subclinical involvement of skeletal muscles and contractures are sometimes apparent. The remarkable electrophysiologic deficits (progressive atrioventricular block and atrial arrhythmias) observed in mutations of lamin A/C and emerin indicate the particular importance of these proteins in electrophysiologic cells. A recent study of lamin A/C mutant mice showed evidence of marked nuclear deformation, fragmentation of heterochromatin, and defects in mechanotransduction (40, 41), all of which likely contribute to reduced myocyte viability. The similarities of cardiac histopathology (fibrofatty degeneration) observed in mutations of the nuclear envelope and desmosomes raise the possibility that these structures may both function as important mechanosensors in myocytes (Figure 3).
Desmosome protein mutations. Arrhythmogenic right ventricular cardiomyopathy (ARVD) identifies an unusual group of cardiomyopathies characterized by progressive fibrofatty degeneration of the myocardium, electrical instability, and sudden death (42). While right ventricular dysplasia predominates, involvement of the left ventricle also occurs. Progressive myocardial dysfunction is seen late in the course of disease, often with right-sided heart failure. ARVD occurs in isolation or in the context of Naxos syndrome, an inherited syndrome characterized by prominent skin (palmar-plantar keratosis), hair, and cardiac manifestations. Mutations in protein components of the desmosomes (Figure 3) (plakoglobin, ref. 43; desmoplakin, refs. 44, 45; and plakophilin-2, ref. 46) and in the cardiac ryanodine receptor (RyR2) (ref. 47; discussed below) cause syndromic and nonsydromic ARVD. Desmosomes are organized cell membrane structures that provide functional and structural contacts between adjacent cells and that may be involved in signaling processes. Whether mutations in the desmosomal proteins render cells of the heart (and skin) inappropriately sensitive to normal mechanical stress or cause dysplasia via another mechanism is unknown.
Energy production and regulation
Mitochondrial mutations. Five critical multiprotein complexes, located within the mitochondria, synthesize ATP by oxidative phosphorylation. While many of the protein components of these complexes are encoded by the nuclear genome, 13 are encoded by the mitochondrial genome. Unlike nuclear gene mutations, mitochondrial gene mutations exhibit matrilineal inheritance. In addition, the mitochondrial genome is present in multiple copies, and mutations are often heteroplasmic, affecting some but not all copies. These complexities, coupled with the dependence of virtually all tissues on mitochondrial-derived energy supplies, account for the considerable clinical diversity of mitochondrial gene mutations (Figure 4). While most defects cause either dilated or hypertrophic cardiac remodeling in the context of mitochondrial syndromes such as Kearns-Sayre syndrome, ocular myopathy, mitochondrial encephalomyopathy with lactic-acidosis and stroke-like episodes (MELAS), and myoclonus epilepsy with ragged-red fibers (MERFF) (48), there is some evidence that particular mitochondrial mutations can produce predominant or exclusive cardiac disease (49, 50). An association between heteroplasmic mitochondrial mutations and DCM has been recognized (51).
Figure 4
Human gene mutations affecting cardiac energetics and metabolism. Energy substrate utilization is directed by critical metabolic sensors in myocytes, including AMP-activated protein kinase (AMPK), which, in response to increased AMP/ATP levels, phosphorylates target proteins and thereby regulates glycogen and fatty acid metabolism, critical energy sources for the heart. Glycogen metabolism involves a large number of proteins including α-galactosidase A (mutated in Fabry disease) and LAMP2 (mutated in Danon disease). Glycogen and fatty acids are substrates for multiprotein complexes located within the mitochondria for the synthesis of ATP. KATP channels composed of an enzyme complex and a potassium pore participate in decoding metabolic signals to maximize cellular functions during stress adaptation. Human mutations (orange text) that cause cardiomyopathies have been identified in the regulatory SUR2A subunit of KATP, the γ2 subunit of AMPK, mitochondrial proteins, α-galactosidase A, and LAMP2.
Nuclear-encoded metabolic mutations. Nuclear gene mutations affecting key regulators of cardiac metabolism are emerging as recognized causes of hypertrophic cardiac remodeling and heart failure (Figure 4). Mutations in genes encoding the γ2 subunit of AMP-activated protein kinase (PRKAG2), α-galactosidase A (GLA), and lysosome-associated membrane protein-2 (LAMP2) can cause profound myocardial hypertrophy in association with electrophysiologic defects (52). AMP-activated protein kinase functions as a metabolic-stress sensor in all cells. This heterotrimeric enzyme complex becomes activated during energy-deficiency states (low ATP, high ADP) and modulates (by phosphorylation) a large number of proteins involved in cell metabolism and energy (53). Most GLA mutations can cause multisystem classic Fabry disease (angiokeratoma, corneal dystrophy, renal insufficiency, acroparesthesia, and cardiac hypertrophy), but some defects produce primarily cardiomyopathy. LAMP2 mutations can also produce either multisystem Danon disease (with skeletal muscle, neurologic, and hepatic manifestations) or a more restricted cardiac phenotype.
Cardiac histopathology reveals that, unlike sarcomere gene mutations, which cause hypertrophic remodeling, the mutations in PRKAG2, LAMP2, and GLA accumulate glycogen in complexes with protein and/or lipids, thereby defining these pathologies as storage cardiomyopathies. Progression from hypertrophy to heart failure is particularly common and occurs earlier with LAMP2 mutations than with other gene mutations that cause metabolic cardiomyopathies. Since both GLA and LAMP2 are encoded on chromosome X, disease expression is more severe in men, but heterozygous mutations in women are not entirely benign, perhaps due to X-inactivation that equally extinguishes a normal or mutant allele. The cellular and molecular pathways that produce either profound hypertrophy or progression to heart failure from PRKAG2, GLA, or LAMP2 mutations are incompletely understood. While accumulated byproducts are likely to produce toxicity, animal models indicate that mutant proteins cause far more profound consequences by changing cardiac metabolism and altering cell signaling. This is particularly evident in PRKAG2 mutations that increase glucose uptake by stimulating translocation of the glucose transporter GLUT-4 to the plasma membrane, increase hexokinase activity, and alter expression of signaling cascades (54).
The cooccurrence of electrophysiologic defects in metabolic mutations raises the possibility that pathologic cardiac conduction and arrhythmias contribute to cardiac remodeling and heart failure in these gene mutations. One mechanism for electrophysiologic defects appears to be the direct consequence of storage: transgenic mice that express a human PRKAG2 mutation (55) developed ventricular pre-excitation due to pathologic atrioventricular connections by glycogen-filled myocytes that ruptured the annulus fibrosis (the normal anatomic insulator which separates atrial and ventricular myocytes). A second and unknown mechanism may be that these gene defects are particularly deleterious to specialized cells of the conduction system. Little is known about the metabolism of these cells, although historical histopathologic data indicate glycogen to be particularly more abundant in the conduction system than in the working myocardium (56–58).
Ca2+ Cycling
Considerable evidence indicates the presence of abnormalities in myocyte calcium homeostasis to be a prevalent and important mechanism for heart failure. Protein and RNA levels of key calcium modulators are altered in acquired and inherited forms of heart failure, and human mutations in molecules directly involved in calcium cycling have been found in several cardiomyopathies (Figure 5).
Figure 5
Human mutations affecting Ca2+ cycling proteins. Intracellular Ca2+ handling is the central coordinator of cardiac contraction and relaxation. Ca2+ entering through L-type channels (LTCC) triggers Ca2+ release (CICR) from the SR via the RyR2, and sarcomere contraction is initiated. Relaxation occurs with SR Ca2+ reuptake through the SERCA2a. Calstabin2 coordinates excitation and contraction by modulating RyR2 release of Ca2+. PLN, an SR transmembrane inhibitor of SERCA2a modulates Ca2+ reuptake. Dynamic regulation of these molecules is effected by PKA-mediated phosphorylation. Ca2+ may further function as a universal signaling molecule, stimulating Ca2+-calmodulin and other molecular cascades. Human mutations (orange text) in molecules involved in calcium cycling cause cardiac remodeling and heart failure. NCX, sodium/calcium exchanger.
Calcium enters the myocyte through voltage-gated L-type Ca2+ channels; this triggers release of calcium from the sarcoplasmic reticulum (SR) via the RyR2. Emerging data define FK506-binding protein (FKBP12.6; calstabin2) as a critical stabilizer of RyR2 function (59), preventing aberrant calcium release during the relaxation phase of the cardiac cycle (Figure 5). Stimuli that phosphorylate RyR2 (such as exercise) by protein kinase A (PKA) dissociate calstabin2 from the receptor, thereby increasing calcium release and enhancing contractility. At low concentrations of intracellular calcium, troponin I and actin interactions block actomyosin ATPase activity; increasing levels foster calcium binding to troponin C, which releases troponin I inhibition and stimulates contraction. Cardiac relaxation occurs when calcium dissociates from troponin C, and intracellular concentrations decline as calcium reuptake into the SR occurs through the cardiac sarcoplasmic reticulum Ca2+-ATPase pump (SERCA2a). Calcium reuptake into SR is regulated by phospholamban (PLN), an inhibitor of SERCA2a activity that when phosphorylated dissociates from SERCA2a and accelerates ventricular relaxation.
RyR2 mutations.While some mutations in the RyR2 are reported to cause ARVD (47) (see discussion of desmosome mutations), defects in this calcium channel are more often associated with catecholaminergic polymorphic ventricular tachycardia (60, 61), a rare inherited arrhythmic disorder characterized by normal heart structure and sudden cardiac death during physical or emotional stress. Mutations in calsequestrin2, an SR calcium-binding protein that interacts with RyR2, also cause catecholaminergic polymorphic ventricular tachycardia (62, 63). Whether the effect of calsequestrin2 mutations directly or indirectly alters RyR2 function is unknown (Figure 5).
While RyR2 mutations affect residues in multiple functional domains of the calcium channel, those affecting residues involved in calstabin2-binding provide mechanistic insights into the substantial arrhythmias found in affected individuals. Mutations that impair calstabin2-binding may foster calcium leak from the SR and trigger depolarization. Diastolic calcium leak can also affect excitation-contraction coupling and impair systolic contractility.
Studies of mice deficient in FKBP12.6 (64) confirmed the relevance of SR calcium leak from RyR2 to clinically important arrhythmias. RyR2 channel activity in FKBP12.6-null mice was significantly increased compared with that of wild-type mice, consistent with a diastolic Ca2+ leak. Mutant myocytes demonstrated delayed after-depolarizations, and exercise-induced syncope, ventricular arrhythmias, and sudden death were observed in FKBP12.6-null mice.
Calcium dysregulation is also a component of hypertrophic remodeling that occurs in sarcomere gene mutations. Calcium cycling is abnormal early in the pathogenesis of murine HCM (65, 66): SR calcium stores are decreased and calcium-binding proteins and RyR2 levels are diminished. Whether calcium changes contribute to ventricular arrhythmias in mouse and human HCM remains an intriguing question.
Related mechanisms may contribute to ventricular dysfunction and arrhythmias in acquired forms of heart failure, in which chronic phosphorylation of RyR2 reduces calstabin2 levels in the channel macromolecular complex and increases calcium loss from SR stores. These data indicate the potential benefit of therapeutics that improve calstabin2-mediated stabilization of RyR2 (67, 68); such agents may both improve ventricular contractility and suppress arrhythmias in heart failure.
PLN mutations. Rare human PLN mutations cause familial DCM and heart failure (69, 70). The pathogenetic mechanism of one mutation (PLN Arg9Cys) was elucidated through biochemical studies, which indicated unusual PKA interactions that inhibited phosphorylation of mutant and wild-type PLN. The functional consequence of the mutation was predicted to be constitutive inhibition of SERCA2a, a result confirmed in transgenic mice expressing mutant, but not wild-type, PLN protein. In mutant transgenic mice, calcium transients were markedly prolonged, myocyte relaxation was delayed, and these abnormalities were unresponsive to β-adrenergic stimulation. Profound biventricular cardiac dilation and heart failure developed in mutant mice, providing clear evidence of the detrimental effects of protracted SERCA2a inhibition due to excess PLN activity.
The biophysical consequences accounting for DCM in humans who are homozygous for a PLN null mutation (Leu39stop; ref. 70) are less clear. PLN-deficient mice show increased calcium reuptake into the SR and enhanced basal contractility (71). Indeed, these effects on calcium cycling appear to account for the mechanism by which PLN ablation rescues DCM in MLP-null mice (72). However, normal responsiveness to β-adrenergic stimulation is blunted in PLN-deficient myocytes, and cells are less able to recover from acidosis that accompanies vigorous contraction or pathologic states, such as ischemia (73). The collective lesson from human PLN mutations appears to be that too little or too much PLN activity is bad for long-term heart function.
Acquired causes of heart failure are also characterized by a relative decrease in SERCA2a function due to excessive PLN inhibition. Downregulation of β-adrenergic responsiveness attenuates PLN phosphorylation, which compromises calcium reuptake and depletes SR calcium levels, which may impair contractile force and enhance arrhythmias. Heterozygote SERCA2 null mice are a good model of this phenotype and exhibit impaired restoration of SR calcium with deficits in systolic and diastolic function (74).
Cardiac ATP-sensitive potassium channel mutations. In response to stress such as hypoxia and ischemia, myocardial cells undergo considerable changes in metabolism and membrane excitability. Cardiac ATP-sensitive potassium channels (KATP channels) contain a potassium pore and an enzyme complex that participate in decoding metabolic signals to maximize cellular functions during stress adaptation (Figure 4) (75). KATP channels are multimeric proteins containing the inwardly rectifying potassium channel pore (Kir6.2) and the regulatory SUR2A subunit, an ATPase-harboring, ATP-binding cassette protein. Recently, human mutations in the regulatory SUR2A subunit (encoded by ABCC9) were identified as a cause of DCM and heart failure (76). These mutations reduced ATP hydrolytic activities, rendered the channels insensitive to ADP-induced conformations, and affected channel opening and closure. Since KATP-null mouse hearts have impaired response to stress and are susceptible to calcium overload (75), some of the pathophysiology of human KATP mutations (DCM and arrhythmias) may reflect calcium increases triggered by myocyte stress.
Transcriptional Regulators
Investigation of the molecular controls of cardiac gene transcription has led to the identification of many key molecules that orchestrate physiologic expression of proteins involved in force production and transmission, metabolism, and calcium cycling. Given that mutation in the structural proteins involved in these complex processes is sufficient to cause cardiac remodeling, it is surprising that defects in transcriptional regulation of these same proteins have not also been identified as primary causes of heart failure. Several possible explanations may account for this. Transcription factor gene mutations may be lethal or may at least substantially impair reproductive fitness so as to be rapidly lost. The consequences of transcription factor gene mutations may be so pleiotropic that these cause systemic rather than single-organ disease. Changes in protein function (produced by a structural protein mutation) may be more potent for remodeling than changes in levels of structural protein (produced by transcription factor mutation). While many other explanations may be relevant, the few human defects discovered in transcriptional regulators that cause heart failure provide an important opportunity to understand molecular mechanisms for heart failure.
Nkx2.5 mutations. The homeodomain-containing transcription factor Nkx2.5, a vertebrate homolog of the Drosophila homeobox gene tinman, is one of the earliest markers of mesoderm. When Nkx2.5 is deleted in the fly, cardiac development is lost (77). Targeted disruption of Nkx2.5 in mice (Nkx2.5–/–) causes embryonic lethality due to the arrested looping morphogenesis of the heart tube and growth retardation (78, 79). Multiple human dominant Nkx2.5 mutations have been identified as causing primarily structural malformations (atrial and ventricular septation defects) accompanied by atrioventricular conduction delay, although cardiac hypertrophic remodeling has also been observed (80). Although the mechanism for ventricular hypertrophy in humans with Nkx2.5 mutations is not fully understood, the pathology is unlike that found in HCM, which perhaps indicates that cardiac hypertrophy is a compensatory event. Several human Nkx2.5 mutations have been shown to abrogate DNA binding (81), which suggests that the level of functional transcription factor is the principle determinant of structural phenotypes. Heterozygous Nkx2.5+/– mice exhibit only congenital malformations with atrioventricular conduction delay (82, 83). Remarkably, however, transgenic mice expressing Nkx2.5 mutations develop profound cardiac conduction disease and heart failure (84) and exhibit increased sensitivity to doxorubicin-induced apoptosis (85), which suggests that this transcription factor plays an important role in postnatal heart function and stress response.
Insights into transcriptional regulation from mouse genetics. Dissection of the combinatorial mechanisms that activate or repress cardiac gene transcription has led to the identification of several key molecules that directly or indirectly lead to cardiac remodeling. While human mutations in these genes have not been identified, these molecules are excellent candidates for triggering cell responses to structural protein gene mutations.
Hypertrophic remodeling is associated with reexpression of cardiac fetal genes. Molecules that activate this program may also regulate genes that directly cause hypertrophy. Activation of calcineurin (Ca2+/calmodulin-dependent serine/threonine phosphatase) results in dephosphorylation and nuclear translocation of nuclear factor of activated T cells 3 (NFAT3), which, in association with the zinc finger transcription factor GATA4, induces cardiac fetal gene expression. Transgenic mice that express activated calcineurin or NFAT3 in the heart develop profound hypertrophy and progressive decompensation to heart failure (86), responses that were prevented by pharmacologic inhibition of calcineurin. Although these data implicated NFAT signaling in hypertrophic heart failure, pharmacologic inhibition of this pathway fails to prevent hypertrophy caused by sarcomere gene mutations in mice and even accelerates disease progression to heart failure (65). Mice lacking calsarcin-1, which is localized with calcineurin to the Z-disc, showed an increase in Z-disc width, marked activation of the fetal gene program, and exaggerated hypertrophy in response to calcineurin activation or mechanical stress, which suggests that calsarcin-1 plays a critical role in linking mechanical stretch sensor machinery to the calcineurin-dependent hypertrophic pathway (87).
Histone deacetylases (HDACs) are emerging as important regulators of cardiac gene transcription. Class II HDACs (4/5/7/9) bind to the cardiac gene transcription factor MEF2 and inhibit MEF2-target gene expression. Stress-responsive HDAC kinases continue to be identified but may include an important calcium-responsive cardiac protein, calmodulin kinase. Kinase-induced phosphorylation of class II HDACs causes nuclear exit, thereby releasing MEF2 for association with histone acetyltransferase proteins (p300/CBP) and activation of hypertrophic genes. Mice deficient in HDAC9 are sensitized to hypertrophic signals and exhibit stress-dependent cardiac hypertrophy. The discovery that HDAC kinase is stimulated by calcineurin (88) implicates crosstalk between these hypertrophic signaling pathways.
Recent attention has also been focused on Hop, an atypical homeodomain-only protein that lacks DNA-binding activity. Hop is expressed in the developing heart, downstream of Nkx2-5. While its functions are not fully elucidated, Hop can repress serum response factor–mediated (SRF-mediated) transcription. Mice with Hop gene ablation have complex phenotypes. Approximately half of Hop-null embryos succumb during mid-gestation with poorly developed myocardium; some have myocardial rupture and pericardial effusion. Other Hop-null embryos survive to adulthood with apparently normal heart structure and function. Cardiac transgenic overexpression of epitope-tagged Hop causes hypertrophy, possibly by recruitment of class I HDACs that may inhibit anti-hypertrophic gene expression (89–92).
PPARα plays important roles in transcriptional control of metabolic genes, particularly those involved in cardiac fatty acid uptake and oxidation. Mice with cardiac-restricted overexpression of PPARα replicate the phenotype of diabetic cardiomyopathy: hypertrophy, fetal gene activation, and systolic ventricular dysfunction (93). Heterozygous PPARγ-deficient mice, when subjected to pressure overload, developed greater hypertrophic remodeling than wild-type controls, implicating the PPARγ-pathway as a protective mechanism for hypertrophy and heart failure (94).
Retinoid X receptor α (RXRα) is a retinoid-dependent transcriptional regulator that binds DNA as an RXR/retinoic acid receptor (RXR/RAR) heterodimer. RXRα-null mice die during embryogenesis with hypoplasia of the ventricular myocardium. In contrast, overexpression of RXRα in the heart does not rescue myocardial hypoplasia but causes DCM (95).
Integrating Functional and Molecular Signals
Study of human gene mutations that cause HCM and DCM provides information about functional triggers of cardiac remodeling. In parallel with evolving information about molecular-signaling cascades that influence cardiac gene expression, there is considerable opportunity to define precise pathways that cause the heart to fail. To understand the integration of functional triggers with molecular responses, a comprehensive data set of the transcriptional and proteomic profiles associated with precise gene mutations is needed. Despite the plethora of information associated with such studies, bioinformatic assembly of data and deduction of pathways should be feasible and productive for defining shared or distinct responses to signals that cause cardiac remodeling and heart failure. Accrual of this data set in humans is a desirable goal, although confounding clinical variables and tissue acquisition pose considerable difficulties that can be more readily addressed by study of animal models with heart disease. With more knowledge about the pathways involved in HCM and DCM, strategies may emerge to attenuate hypertrophy, reduce myocyte death, and diminish myocardial fibrosis, processes that ultimately cause the heart to fail.
CardioGenomics. Genomics of Cardiovascular Development, Adaptation, and Remodeling.
Morita, H, et al. Molecular epidemiology of hypertrophic cardiomyopathy. Cold Spring Harb. Symp. Quant. Biol. 2002. 67:383-388.
Richard, P, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003. 107:2227-2232
Palmer, BM, et al. Effect of cardiac myosin binding protein-C on mechanoenergetics in mouse myocardium. Circ. Res. 2004. 94:1615-1622.
Harris, SP, et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ. Res. 2002. 90:594-601.
Kamisago, M, et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N. Engl. J. Med. 2000. 343:1688-1696.
Itoh-Satoh, M, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem. Biophys. Res. Commun. 2002. 291:385-393.
Gerull, B, et al. Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy. Nat. Genet. 2004. 36:1162-1164.
Tiso, N, et al. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum. Mol. Genet. 2001. 10:189-194.
Anan, R, et al. Cardiac involvement in mitochondrial diseases. A study on 17 patients with documented mitochondrial DNA defects. Circulation. 1995. 91:955-961.
Cardiovascular Autonomic Dysfunction and Predicting Outcomes in Diabetes
Autonomic Dysfunction and Risk of a CV Event In patients with CAD and type 2 diabetes, autonomic dysfunction is common, but its prognostic value is unknown.
A.
data a substudy of patients enrolled in the ARTEMIS trial
,530 patients with CAD and diabetes matched with 530 patients with CAD without diabetes. The patients had a mean age of 67, and 69% were males
patients performed a test on an exercise bicycle, which allowed the researchers to determine their heart-rate recovery, defined as the drop in heart rate from the rate at maximal exercise to the rate one minute after stopping the exercise In univariate analysis, among patients with CAD and type 2 diabetes, those who had a blunted heart-rate recovery after exercise–defined as a drop in heart rate of less than 21 beats per minute–had a 1.69-fold greater risk having a cardiovascular event than their peers. Similarly, those with blunted heart-rate turbulence (<3.4 ms/R-R interval) had a 2.08-fold increased risk of an event, and those with low heart-rate variability (<110 ms) had a 1.96-fold greater risk of having a cardiovascular event. After multivariate analysis, C-reactive protein (CRP), but none of the three measures of autonomic function, still predicted an increased risk of having a cardiovascular event during this short follow-up.
During a two-year follow-up, 127 patients (13%) reached the composite end point of a cardiovascular event, which included
cardiovascular death (2%),
acute coronary event (8%),
stroke (3%), or
hospitalization for heart failure (2%).
B. Autonomic Dysfunction and Risk of Severe Hypoglycemia
Dr Seung-Hyun Ko (Catholic University of Korea, Gyeonggi-do, South Korea
data 894 consecutive patients with type 2 diabetes, aged 25 to 75
heart-rate variability measured at three times: during a Valsalva maneuver, deep breathing, and going from lying down to standing. During close to 10 years of follow-up, 77 episodes of severe hypoglycemia occurred among 62 patients (9.9%). About 16% of patients were diagnosed with early autonomic dysfunction and another 15% were diagnosed with definite autonomic dysfunction. Patients with type 2 diabetes and definite autonomic dysfunction were more than twice as likely to have an episode of severe hypoglycemia as those with normal autonomic function (HR 2.43).
patient education concerning hypoglycemia is essential for patients with definite [cardiovascular autonomic neuropathy] to prevent [severe hypoglycemia] and related mortality
Current Risk-Stratification Role for Heart-Rate Turbulence Monitoring Defined
Measurement of heart-rate turbulence (HRT), an ECG phenomenon that reflects hemodynamic responses to premature ventricular contractions (PVCs), can risk-stratify patients in the post-MI setting and may be similarly useful in heart failure or other heart disease, according to a state-of-the-art review in the October 21, 2008 issue of the Journal of the American College of Cardiology [1]. “Several large-scale retrospective and prospective studies have established beyond any doubt that HRT is one of the strongest independent risk predictors after MI. It thus appears that the stage has now been reached when HRT might be used in large prospective intervention studies,” according to the authors, led by Dr Axel Bauer (Deutsches Herzzentrum, Munich, Germany). The group had been asked to write the review by the International Society for Holter and Noninvasive Electrophysiology (ISHNE), it states. HRT, first published as a potential CV risk stratifier in 1999 [2], and other measures of autonomic function aren’t as well established or even studied as much as some other prognostic markers based on electrocardiography, such as T-wave alternans. As the authors note, it’s usually measured from an average of multiple PVCs on 24-hour Holter monitoring.
The strongest support for the parameter’s risk-stratification role comes from “six large-scale studies and from two prospective studies, both of which have been specifically designed to validate the prognostic value of HRT in post-MI patients receiving state-of-the-art treatment,” the report states.
Other evidence suggests a role for HRT evaluation after PCI to assess the strength of perfusion from the treated coronary artery. “Persistent impairment of HRT after PCI in patients with incomplete reperfusion implies prolonged baroreflex impairment and is consistent with poor prognosis,” write Bauer et al. “Thus, early assessment of HRT may be detecting pathological loss of reflex autonomic response due to incomplete reperfusion or severe microvascular dysfunction after PCI. In heart failure, according to the authors, patients “are known to have significantly impaired baroreflex sensitivity as well as reduced heart-rate variability. . . . This may suggest the possibility of guiding pharmacological therapy [according to HRT responses] in heart-failure patients.” They also note that the prognostic power of HRT in heart failure appears limited to patients with ischemic cardiomyopathy.
Bauer A, Malik M, Schmidt G, et al. Heart rate turbulence: Standards of measurement, physiological interpretation, and clinical use. International Society for Holter and Noninvasive Electrophysiology consensus. J Am Coll Cardiol 2008; 52:1353–1365. http://dx.doi.org/10.1016/j.jacc.2008.07.041
Schmidt G, Malik M, Barthel P, et al. Heart-rate turbulence after ventricular premature beats as a predictor of mortality after acute myocardial infarction. Lancet 1999; 353:1390–1396. Abstract http://www.medscape.com/viewarticle/582091
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
This is the Third article of a multipart series covering Ca(2+) signaling and the cytoskeleton, and two on Ca2+ in cardiac contractility governed by the activations involving a ryanodine (RyR2) receptor and a specific calmodulin protein CaKIIδ with B and C splice variants. In all of these discussions, Ca(2+) has a crucial role in many cellular events, not all of which are detailed, and its importance to cardiac function and function disorders is critical. We shall next undertake the difficult examination of Ca(2+) movements in the kidney, which has a special relationship to vitamin D and bone mineral metabolism that is not of interest here. Nor will we go into any depth on the importance of the kidney to maintenance of plasma H+ and K+ balance and metabolic acidosis. Whereas the lung has a large role in pH maintenance by the respiratory rate (under sympathetic control), it maintains the balance through the expiration of CO2, with H+ tied up in water via the carbonic anhydrase reaction.
(Part II) Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
(Part II) Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
(Part IV) The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part V: Heart Failure and Arrhythmia: Potential for Targeted Intervention — The Effects of Ca 2+ -calmodulin (Ca-CaM) phosphorylation/dephosphorylation/hyperphosphorylation
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses in the Human Heart
Basal-lateral-plasma-membrane vesicles and brush-border-membrane vesicles were isolated from rat kidney cortex by differential centrifugation followed by free-flow electrophoresis. Ca2+ uptake into these vesicles was investigated by a rapid filtration method. Both membranes show a considerable binding of Ca2+ to the vesicle interior, making the analysis of passive fluxes in uptake experiments difficult. Only the basal-lateral-plasma-membrane vesicles exhibit an ATP-dependent pump activity which can be distinguished from the activity in mitochondrial and endoplasmic reticulum by virtue of the different distribution during free-flow electrophoresis and its lack of sensitivity to oligomycin. The basal-lateral plasma membranes contain in addition a Na+/Ca2+-exchange system which mediates a probably rheogenic counter-transport of Ca2+ and Na+ across the basal cell border. The latter system is probably involved in the secondary active Na+-dependent and ouabain-inhibitable Ca2+ reabsorption in the proximal tubule, the ATP-driven system is probably more important for the maintenance of a low concentration of intracellular Ca2+.
In recent micropuncture studies using simultaneously tubular and capillary perfusion it could be demonstrated that in the rat kidney proximal tubule Ca2+ reabsorption is dependent on the presence of Na+- ions and sensitive to ouabain (Ullrich et al., 1976). On the other hand cell-fractionation studies on the distribution of plasma-membrane-bound enzymes in rat proximal tubular epithelial cells revealed a contraluminal localization of a Ca2+-stimulated ATPase (Kinne-Saffran & Kinne, 1974). These results suggested that both Na+-driven and ATP-driven Ca2+ transport systems might be involved in proximal tubular transepithelial Ca2+ transport. Considering the low concentration of intracellular Ca2+ one could expect that these active steps in Ca2+ reabsorption are located at the basal cell pole.
To our knowledge there have been two attempts to study the role of ATP in the Ca2+ transport of renal membranes. In one study increase in Ca2+ uptake by rabbit kidney membranes was observed, but this increase was attributed to a phosphorylation of the membranes and a concomitant binding of Ca2+ to the negative charges newly generated at the membrane surface. Moore et al. (1974) observed an ATP-dependent Ca2+ uptake distinct from that of the mitochondria in a crude fraction of renal plasma membranes as well as in rat renal microsomes. The two uptake systems differed in their capacity, their sensitivity to Na+ and their apparent Km values for Mg2+-ATP.
Experiments are described on the Ca2+ transport into brush-border-membrane vesicles and basal-lateral plasma-membrane vesicles isolated from rat renal cortex. The results show that a primary active ATP-driven Ca2+ pump and an Na+/Ca2+-exchange system are present in the basal-lateral plasma membranes, but not in the brush-border membrane.
These findings indicate that trans-epithelial Ca2+ transport in rat proximal tubule can be
primarily active via the ATP-driven system as well as
secondarily active if the Na+/Ca2+ exchange system is involved.
It appears that the Na+/Ca2+ exchange system
is responsible for the bulk flow of Ca2+ across the epithelium, whereas
the ATP-driven system might be involved in the fine regulation of the concentration of intracellular Ca2+.
(Gmaj P, Murer H, and Kinne R. 1979)
The Renal Na+/Ca2+ Exchange System of the Nephron
The movement of Ca2+ across the basolateral plasma membrane was studied from rabbit proximal and distal convoluted tubules and ATP-dependent Ca2+ uptake was found in both. But the activity was higher distal. The distal tubular membranes had a very active Na+/Ca2+ exchange system, which was absent in the proximal segment. The ATP-dependent Ca2+ uptake in the distal tubular membrane preparations was gradually inhibited by Na+ outside the vesicles, and was a function of the imposed Na+ gradient. The results indicate that an active Na+/Ca2+ exchange system is absent in the proximal tubule. Ramachandram & Brunette, 1989). Parathyroid hormone (PTH) and calcitonin increase Ca2+ uptake by purified distal tubular luminal membranes (DTLM), and both hormone stimulate adenylate cyclase and phospholipase C. Therefore, distal tubules were incubated with dibutyryl cAMP (dbcAMP) and the result was that dbcAMP increased the Ca2+ transport by luminal membranes, but phorbol 12-myristate 13 acetate (PMA) had no such effect. But when PMA was added to low concentrations of dbcAMP the uptake significantly increased. Protein kinase C inhibitors prevented the effect. This indicated that in the distal tubule Ca2+ transport required both the combined effect of PK A and C involves both components of the transport kinetics. (Hila, Claveau, Laclerc, Brunette, 1997)
In the rabbit, calcitonin enhances Ca2+ reabsorption in the distal tubule. Tubules were incubated with or in the absence of calcitonin, and the luminal or basolateral membranes were purified and Ca2+ transport was measured through the vesicles. The results were compared with those obtained from proximal tubule membranes, and the results were no effect of calcitonin on Ca2+ uptake in the proximal tubules. In the distal tubules there was the expected uptake, but the presence of Na+ in the suspension decreased the Ca2+ uptake. The uptake was partially restored by preincubation with calcitonin. Recall the experiment demonstrating a requirement for PK A and C in Ca2+ uptake indicating a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. Calcitonin stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Incubation of distal tubule suspensions with dbcAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. The researchers concluded that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms:
the opening of low affinity Ca2+ channels in the luminal membrane and
the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.
(Zuo Q, Claveau D, Hilal G, Leclerc M, Brunette MG. 1997)
Calcium (Ca2+) filtered in the glomerulus is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by hormones requires
binding of these hormones to the basolateral membrane,
interaction with G proteins,
liberation of messengers,
activation of kinases
opening of the channels at the opposite pole of the cells.
It follows that if the Ca2+ entry through the luminal membranes of proximal and distal tubules is a membrane-limited process, then G proteins have a regulatory role. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPγs or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPγs significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPγs increased 0.5 mM Ca2+ uptake in the absence (p < 0.02) and in the presence (p < 0.02) of Na+. The action of GTPγs, when present, was dose dependent. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPγs increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.
(Brunette MG, Hilal G, Mailloux J, Leclerc M. 2000)
We previously reported a dual kinetics of Ca2+ transport by the distal tubule luminal membrane of the kidney, suggesting the presence of several types of channels. We, therefore, examined the effects of specific inhibitors (i.e., diltiazem, an L-type channel; ω-conotoxin MVIIC, a P/Q-type channel; and mibefradil, a T-type channel antagonist) on Ca2+ uptake by rabbit nephron luminal membranes. None of these inhibitors influenced Ca2+ uptake by the proximal tubule membranes. In contrast, in the absence of sodium (Na+), the three channel antagonists decreased Ca2+ transport by the distal membranes, and their action depended on the substrate concentrations: (P < 0.05) without influencing 0.5 mM Ca2+transport, whereas ω-conotoxin MVIIC decreased 0.5 mM Ca2+ (P < 0.02) and 1 µM mibefradil decreased it (P < 0.05); the latter two inhibitors [P/Q type, T-type] left 0.1 mM Ca2+transport unchanged. Diltiazem [L-type] decreased the Vmax of the high-channels, whereas ω-conotoxin MVIIC and mibefradil influenced exclusively the Vmax of the low-affinity channels. These results not only confirm that the distal luminal membrane is the site of Ca2+ channels, but they suggest that these channels belong to the L, P/Q, and T types. (M G Brunette, M Leclerc, D Couchourel, J Mailloux, Y Bourgeois. 2000)
Calcium (Ca2+) transport by the distal tubule (DT) luminal membrane
Calcium (Ca2+) transport by the distal tubule (DT) luminal membrane is regulated by
the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers,
protein kinases, and
ATP as the phosphate donor.
Could ATP itself, when directly applied to the cytosolic surface of the membrane influence the Ca2+ channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca2+ uptake by these vesicles loaded with ATP or the carrier. The presence of 100 μM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca2+ uptake in the absence of Na+ (P < 0.01) and in the presence of 100 mM Na+ (P < 0.01). This effect was dose dependent with an EC50 value of approximately 40 μM. ATP action involved the high-affinity component of Ca2+ transport, decreasing the Km from 0.08 ± 0.01 to 0.04 ± 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPγs abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, they investigated the effect of intravesicular cytochalasin on Ca2+ transport. Cytochalasin B decreased 0.5 mM Ca2+ uptake (P< 0.01). However, when both ATP and cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca2+ uptake by the PT luminal membrane. They conclude from this that the high-affinity Ca2+ channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca2+ channels within this membrane. (MG. Brunette*, J Mailloux, G Hilal. 1999)
Proximal tubular sodium-calcium exchanger
The functional expression of the renal sodium-calcium exchanger has been amply documented in studies on renal cortical basolateral membranes. In perfused renal tubules, other investigators have shown sodium-calcium exchange activity in the
proximal convolution
in the distalconvolution,
the connecting tubule, and
the collecting tubule of the rabbit.
In rat proximal tubules, we found that the sodium-calcium exchanger is an important determinant of cytosolic calcium homeostasis, since
inhibition of sodium-dependent calcium efflux mode caused a large accumulation of tubular calcium.
In membranes from rat proximal tubulessodium-calcium activity was high, and in intact proximal tubules,
the tubular sodium-calcium exchanger exhibited a high affinity for cytosolic calcium
and had a substantial transport capacity, which may be absolute requirements for the maintenance of stable cytosolic calcium in proximal tubules. (Dominguez JH, Juhaszova M, Feister HA. 1992.)
Proximal tubule Na(+)-Ca2+ exchanger protein is same as the cardiac protein
The activity of the Na(+)-Ca2+ exchanger, a membrane transporter that mediates Ca2+ efflux, has been described in amphibian and mammalian renal proximal tubules. However, demonstration of cell-specific
expression of the Na(+)-Ca2+ exchanger in proximal renal tubules has been restricted to functional assays.
In this work, Na(+)-Ca2+ exchanger gene expression in rat proximal tubules was characterized by three additional criteria:
functional assay of transport activity in membrane vesicles derived from proximal tubules, expression of
specific Na(+)-Ca2+ exchanger protein detected on Western blots, and
determination of specific mRNA encoding Na(+)-Ca2+ exchanger protein on Northern blots.
A new transport activity assay showed that proximal tubule membranes
contained the highest Na(+)-Ca2+ exchanger transport activity reported in renal tissues.
In dog renal proximal tubules and sarcolemma, a specific protein of approximately 70 kDa was detected, whereas in rat proximal tubules and sarcolemma, the specific protein approximated 65 kDa and was localized to the basolateral membrane. On Northern blots, a single 7-kb transcript isolated from rat
proximal tubules,
whole kidney, and
heart
hybridized with rat heart cDNA.
These data indicate that Na(+)-Ca2+ exchanger protein expressed in rat proximal tubule is similar, if not identical, to the cardiac protein. We suggest that the tubular Na(+)-Ca2+ exchanger characterized herein represents the Na(+)-Ca2+ exchanger described in functional assays of renal proximal tubules. (Dominguez JH, Juhaszova M, Kleiboeker SB, Hale CC, Feister HA. 1992.)
Calcium reabsorption regulated by the distal tubules
Extracellular calcium homeostasis involves coordinated calcium absorption by
the intestine,
calcium resorption from bone, and
calcium reabsorption by the kidney.
This review addresses the mechanism and regulation of renal calcium transport. Calcium reabsorption occurs throughout the nephron. However, distal tubules are the nephron site at which calcium reabsorption is regulated by
parathyroid hormone,
calcitonin, and
1 alpha,25-dihydroxyvitamin D3 and
where the magnitude of net reabsorption is largely determined. These and related observations underscore the view that distal tubules are highly specialized
to permit fine regulation of calcium excretion in response to
alterations in extracellular calcium levels.
Progress in understanding the mechanism and regulation of calcium transport has emerged from application of
single cell fluorescence,
patch clamp, and
molecular biological approaches.
These techniques permit the examination of
ion transport at the cellular level and
its regulation at subcellular and molecular levels.
This editorial review focuses on recent and emerging observations and attempts to integrate them into models of cellular calcium transport. (Friedman PA , Gesek FA. 1993)
Calcium-Sensing Receptor (CSR)
Renal tubular calcium reabsorption is a critical determinant of extracellular fluid (ECF) calcium concentration; for the need of constancy of ECF calcium concentration,
the renal tubular handling of calcium is tightly controlled
in order to match renal calcium excretion to the net amount of calcium entering the ECF.
Both parathyroid hormone (PTH) and vitamin D metabolites are involved in
the control of renal tubular calcium reabsorption and
ECF calcium concentration [1].
Besides this hormonal control, it has been recognized recently that
ECF calcium is able to regulate its own reabsorption by the mammalian tubule.
Indeed, a large body of evidence supports the view that ECF calcium exerts this action
by activating the calcium/polyvalent cation-sensing receptor (CaSR)
located in the plasma membrane of many tubular cell types.
First, increasing ECF calcium concentration
elicits a marked increase in urinary calcium (and magnesium) excretion [2,3] and
this occurs independently of any change in the calcium-regulating hormones [2,3].
Second, the inhibitory effect of ECF calcium on its own reabsorption is shared by other CaSR agonists, e.g. magnesium [4].
Third, the relationship between ECF calcium and urinary calcium excretion
is altered in patients bearing mutations of the CASR gene: renal tubular calcium reabsorption
is enhanced in patients with inactivating mutations [5,6]
and decreased in patients with activating mutations.
Therefore, there is abundant evidence that renal tubular CaSR plays a role
in the control of divalent cations reabsorption under
both normal and pathological conditions.
Localization of the extracellular CaSR
Transcripts of the CASR gene are expressed in many nephron segments of rat kidney, extending from glomeruli to the inner medullary collecting duct (IMCD) [7]. The CaSR protein is expressed in
the proximal tubule,
medullary and cortical thick ascending limb (TAL) segments,
macula densa cells,
distal convoluted tubule (DCT) and
type-A intercalated cells in the distal tubule and cortical collecting duct [8]
and in inner medullary collecting duct cells [9].
The polarity of expression varies from segment to segment, the protein being expressed in
the apical membrane of proximal tubule and
IMCD cells and
in the basolateral membrane of TAL and DCT cells [8,9].
Interestingly, the highest density of protein expression has been observed in the cortical TAL (cTAL),
known to reabsorb calcium and magnesium in a regulated manner.
CaSR under physiological conditions
Consistent with its polarized plasma membrane localization,
CaSR has been shown to be involved in the control of thick ascending limb (TAL) calcium and magnesium reabsorption.
In the mouse and rat TAL,
both calcium and magnesium are reabsorbed selectively in the cortical portion (cTAL) [10]
and this reabsorption is passive along an electrical gradient
through the paracellular pathway [10,11]. The electrical gradient is related to
transcellular NaCl reabsorption.
The first step is NaCl entry into the cell via
the electroneutral apical Na- K-2Cl co-transporter BSC1 (NKCC2).
Subsequently, most of the potassium recycles back to the lumen, through an apical potassium channel,
necessary to maintain NaCl absorption via BSC1 (NKCC2).
In the absence of recycling, NaCl absorption is inhibited because of
the low availability of potassium in luminal fluid.
In addition, potassium recycling hyperpolarizes the apical membrane.
Chloride exits the cell
across the basolateral membrane
mainly via the CLC-Kb channel,
which depolarizes the basolateral membrane.
The overall consequence is a lumen-positive transepithelial voltage that
drives calcium, magnesium and also sodium through the paracellular pathway.
The pathway permeability for calcium and magnesium requires the presence of a specific protein,
paracellin-1 (also known as claudin-16),
co-expressed with occludin
in the tight junctions of thick ascending limb (TAL) [12].
Inactivating mutations of the paracellin-1 gene cause a specific
decrease in cTAL calcium and magnesium reabsorption and
renal loss of both cations without renal sodium loss,
which is the landmark of an inherited disease referred to as hypercalciuric hypomagnesaemia with nephrocalcinosis [4].
Calcium and magnesium reabsorption in the cTAL is tightly regulated. Micropuncture studies have shown that peptide hormones, such as
PTH,
arginine vasopressin,
calcitonin and
glucagon,
stimulate NaCl as well as calcium and magnesium reabsorption in the loop of Henle and decrease their excretion in final urine. PTH, the most important peptide hormone for the stimulation of renal calcium transport, elicits an increase in calcium and magnesium reabsorption cTAL.
Wittner et al. [14] demonstrated that PTH stimulation of calcium and magnesium transport
involves an increase in paracellular pathway permeability.
The activation of CaSR also affects a number of intracellular events in TAL cells and
modulates transport processes along the cTAL epithelium.
Activating CaSR increases intracellular free calcium concentration in
cTAL,
DCT and
cortical as well as
outer medullary collecting duct.
This also decreases hormone-dependent cAMP accumulation in cTAL by
inhibition of type-6 adenylyl cyclase [20],
increases inositol phosphate formation [21] and
elicits an increase in phospholipase A2 activity and
in intracellular cellular production of 20-hydroxyeicosatetraenoic acid [22]. ….
In conclusion, a large body of evidence supports the view that CaSR is
a major regulator of calcium and magnesium reabsorption in the cTAL and,
of overall tubular divalent cation handling.
However, several issues remain unresolved. It is still unclear whether CaSR activation in the cTAL decreases NaCl reabsorption in this segment or not. The mechanism through which CaSR activation could alter the function of paracellin-1 and the paracellular pathway permeability also remains unsettled. Finally, the role of CaSR in the medullary part of TAL should be investigated: a CaSR-dependent inhibition of NaCl reabsorption could explain at least part of the polyuria that accompanies hypercalcaemic states. (P Houillier and M Paillar. 2003)
Alpha-Kloth and FGf23
Recent advances that have given rise to marked progress in clarifying actions of alpha(α)-Klotho (alpha-Kl) and FGf23 can be summarized as follows ;
(i) α-Kl binds to Na(+), K(+)-ATPase, and Na(+), K(+)-ATPase is recruited to the plasma membrane by a novel α-Kl dependent pathway in correlation with cleavage and secretion of α-Kl in response to extracellular Ca(2+) fluctuation.
(ii) The increased Na(+) gradient created by Na(+), K(+)-ATPase activity drives the transepithelial transport of Ca(2+) in the choroid plexus and the kidney, this is defective in α-kl(-/-) mice.
(iii) The regulated PTH secretion in the parathyroid glands is triggered via recruitment of Na(+), K(+)-ATPase to the cell surface in response to extracellular Ca(2+) concentrations.
(iv) α-Kl, in combination with FGF23, regulates the production of 1,25 (OH) (2)D in the kidney. In this pathway, α-Kl binds to FGF23, and α-Kl converts the canonical FGF receptor 1c to a specific receptor for FGF23, enabling the high affinity binding of FGF23 to the cell surface of the distal convoluted tubule where α-Kl is expressed.
(v) FGF23 signal down-regulates serum phosphate levels, due to decreased NaPi-IIa abundance in the apical membrane of the kidney proximal tubule cells.
(vi) α-Kl in urine increases TRPV5 channel abundance at the luminal cell surface by hydrolyzing the N-linked extracellular sugar residues of TRPV5, resulting in increased Ca(2+) influx from the lumen.
These findings revealed a comprehensive regulatory scheme of mineral homeostasis that is illustrated by the mutually regulated positive/negative feedback actions of α-Kl, FGF23, PTH and 1,25 (OH) (2)D. In this regard, α-Kl and FGF23 might play pivotal roles in mineral metabolism as regulators that integrate calcium and phosphate homeostasis, although this concept requires further verification in the light of related findings. Here, the unveiling of the molecular functions of α-Klotho and FGF23 has recently given new insight into the field of calcium and phosphate homeostasis. Unveiled molecular functions of α-Kl and FGF23 provided answers for several important questions regarding the mechanisms of calcium and phosphate homeostasis that remained to be solved, such as :
(i) what is the non-hormonal regulatory system that directly responds to the fluctuation of extracellular Ca(2+),
(ii) how is Na(+), K(+)-ATPase activity enhanced in response to low calcium stimuli in the parathyroid glands,
(iii) what is the exact role of FGF23 in calcium and phosphorus metabolism,
(iv) how is Ca(2+) influx through TRPV5 controlled in the DCT nephron, and finally
(v) how is calcium homeostasis regulated in cerebrospinal fluid. However, several critical questions still remain to be solved. So far reported,
α-Kl binds to Na(+),
K(+)-ATPase,
FGF receptors and FGF23, and
α-Kl hydrolyzes the sugar moieties of TRPV5.
The following questions are unresolved:
Does alpha-Kl recognize these proteins directly or indirectly?
Is there any common mechanism?
How can we reconcile such diverse functions of alpha-Kl?What is the Ca(2+) sensor machinery and how can we isolate it?
How do hypervitaminosis D and the subsequently altered mineral-ion balance lead to the multiple phenotypes?
What is the phosphate sensor machinery and how can we isolate it?
How does the Fgf23/α-Kl system regulate phosphorus homeostasis?
How are serum concentrations of Ca(2+) and phosphate mutually regulated?
(Nabeshima Y. 2008)
Cilium and Calcium Signal
We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca2+-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP3-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. (Praetorius HA, Spring KR. 2001)
Fgf23 regulation in chronic renal disease
The mechanism of FGF23 action in calcium/phosphorus metabolism of patients with chronic kidney disease (CKD) was studied using a mathematical model and clinical data in a public domain. We have previously built a physiological model that describes interactions of PTH, calcitriol, and FGF23 in mineral metabolism encompassing organs such as bone, intestine, kidney, and parathyroid glands. Since an elevated FGF23 level in serum is a characteristic symptom of CKD patients, we evaluate herein potential metabolic alterations in response to administration of a neutralizing antibody against FGF23. Using the parameters identified from available clinical data, we observed that a transient decrease in the FGF23 level elevated the serum concentrations of PTH, calcitriol, and phosphorus. The model also predicted that the administration reduced a urinary output of phosphorous. This model-based prediction indicated that the therapeutic reduction of FGF23 by the neutralizing antibody did not reduce phosphorus burden of CKD patients and decreased the urinary phosphorous excretion. Thus, the high FGF23 level in CKD patients was predicted to be a failure of FGF23-mediated phosphorous excretion. The results herein indicate that it is necessary to understand the mechanism in CKD in which the level of FGF23 is elevated without effectively regulating phosphorus.
A traditional, physiological model with PTH and calcitriol needs to be rebuilt in accordance with the emerging role of FGF23 and its interacting molecules. To understand probable interactions among FGF23, PTH and calcitriol, we previously developed a minimum physiological model of calcium/phosphorus metabolism and investigated potential influences of FGF23 on the observable state variables such as the serum concentrations of PTH, calcitriol, calcium (Ca), and phosphorous (P), as well as the urinary excretion of Ca and P.3 In this study, we extended the model and evaluated the mechanism of FGF23-mediated regulation in chronic kidney diseases (CKD).
The FGF23 gene was identified by its mutations associated with autosomal dominant hypophosphatemic rickets (ADHR), which is an inherited phosphate wasting disorder.4 Thereafter, a variety of disorders resulting from gain or loss of FGF23 bioactivity have been reported.5 These disorders, which are caused by mutations in the genes that directly or indirectly interact with FGF23, include hyperphosphatemic familial tumoral calcinosis (HFTC), hereditary hypo-phosphatemic rickets with hypercalciuria (HHRH), autosomal recessive hypophosphatemic rickets (ARHR), and X-linked dominant hypophosphatemic rickets (XLH, HYP). CKD patients who need dialysis have very high levels of FGF23 in serum that are linked with increased rates of death.6
We examined the effect of reduction of FGF23 by neutralizing antibody would modulate phosphorus balance of CKD patients. We evaluated the levels of physiological variables such as the levels of PTH, calcitriol, FGF23, Ca, and P in serum as well as urinary outputs of Ca and P using clinical data. Since a glomerular filtration rate (GFR) is a good indicator of severity of CKD, data were processed as a function of GFR. We then employed the previously developed mathematical model for mineral metabolism, and conducted numerical simulations in response to the modulation of FGF23 by neutralizing antibody.
Estimation of the relationship of the FGF23 level to other physiological variables
The FGF23 concentrations, reported in literature, considerably varied among available datasets, presumably caused by differential baseline levels or sensitivity variations among individual assays. To predict a quantitative relationship among the FGF23 level and other physiological variables, the reported FGF23 level was linearly modified:
[FGF23]AB = {[FGF23]-A}/B (1)
in which [FGF23] = reported FGF23 level, [FGF23]AB = linearly modified FGF23 level, and A and B = two correction factors. Note that these correction factors are constant and they were chosen independently for each of the physiological variables such as the serum level of PTH and the urinary output of P. The “+” and “-” values of the factor B indicate positive and negative correlations to the FGF23 level, respectively. We applied the described modification in analyzing clinical data since the observed FGF23 variation was larger than others. Without this procedure, it was difficult to estimate a quantitative relationship of its concentrations to other variables. [With the significant variation around the linear fit, it might well have been warranted to use the log transform of the modified level, LHB].
Mathematical model and prediction of effects of FGF23 antibody
We previously developed a pair of metabolism models of calcium and phosphorus with and without including the predicted action of FGF23.3,20 In this study we considered an additional state variable, GFRf, as a multiplicative term pertaining to the calcium and phosphorus renal thresholds and the kidney production of calcitriol:
GFRf = (GFR/GFR0)k (2)
in which GFR0 and GFR = glomerular filtration rates in the control state and at any given degree of renal failure, respectively, and a factor k (>0) was chosen so as to fit the clinical data as described previously.7
To predict the effects of intravenous administration of a neutralizing antibody against FGF23, we numerically examined 5 different dosages for i.v. administration at 0.003, 0.01, 0.03, 0.1 and 0.3 mg/kg (dosage levels 1–5). These dosages corresponded to a clinical trial study being proposed for a dose-escalation study of KRN23 (Kyowa Hakko Kirin Pharma Inc.). A primary outcome measure of this Phase I clinical trial is a change in a serum phosphate level, and a single dose by intravenous or subcutaneous administration is planned. The initial target is X-linked hypophosphatemia but no clinical data regarding efficacy and side effects are available. To simulate a probable injection procedure, we assumed a form of a single, smoothed-out pulse. The rise in the antibody concentration was modeled using a Gaussian type diffusion profile with a period dependent on the distribution volume and cardiac output.
Glomerular filtration rate (GFR) as an indicator in cKD patients
We plotted physiological variables of CKD patients as a function of GFR in ml/min/1.73 m2. Figure 1 illustrated the levels of PTH (pg/ml), calcitriol (pg/ml), Ca (mg/dl), and P (mg/dl) in serum as well as urinary outputs of Ca and P expressed as a fraction of the glomerular loads. The numbers in the brackets in Figure 1 were the numbers of patients. The average and SEM values were obtained in each of the sampling bins. As GFR was normal above 90, the levels of PTH and P in serum as well as the fractions of urinary Ca and P outputs were lowered. On the contrary, the level of calcitriol in serum was higher as GFR increased.
Estimation of FGF23 levels in serum in cKD patients
The relationships of the linearly modified FGF23 concentration in serum, [FGF23]AB, to the selected physiological variables in CKD patients were illustrated in Figure 2. First, a strong correlation was observed between log.e(GFR) and a negative form of log.e[FGF23]AB, indicating that the FGF23 level was sharply elevated in CKD patients with reduction in GFR. Second, an increase in [FGF23]AB was correlated to the levels of PTH, calcitriol, P in serum, and the renal threshold for P. Note that a positive correlation (i.e. B > 0) was observed for the levels of PTH and P in serum, while a negative correlation (i.e. B < 0) for the serum level of calcitriol and the renal threshold for P. Note that a majority of data points had the PTH level above 50 pg/ml, indicating a poor balance of mineral metabolism in CKD patients.
Linkage of FGF23 and P levels in serum
In all groups, a positive correction was observed between the level of P and the modified level of FGF23 in serum. Note that CKD data in Figure 2D showed the elevated P level up to 6 mg/dl, while the higher bound of the P level was ∼2 mg/dl (Tumor Induced Osteomalacia), 3.5 ∼4 mg/dl (Fibrous Dysplasia and XLH), and 4.5 mg/dl (healthy populations).
Predicted effects of the antibody specific to FGF23
Although the observed increase of FGF23 in CKD is apparently a physiological response to hyperphos-phatemia, the use of FGF23 antibody is suggested for transplanted hypophosphatemic patients of CKD with a high level of FGF23.21 In response to intravenous administration of the antibody specific to FGF23, we evaluated the predicted changes in the serum levels of PTH, calcitriol, and P as well as a normalized urinary output of P. The results were positive.
(Yokota H, Pires A, Raposa JF, Ferreira HG. 2010.)
Overview of renal Ca2+ handling
About 50% of plasma calcium (ionized and complexed form; ultrafilterable fraction, excluding the protein bound form) is freely filtered through the renal glomerulus, and 99% of the filtered calcium is actually reabsorbed along renal tubules (Table 1- see Fig below on right)). The excreted calcium in the final urine is about 200 mg per day in an adult person with an average diet. Several factors are involved in the regulation of calcium in renal tubules. PTH and activated vitamin D enhance calcium reabsorption in the thick ascending limb (TAL), distal convoluted tubule (DCT) and/or connecting tubule (CNT).
Acidosis contributes to hypercalciuria by reducing calcium reabsorption in the proximal tubule (PT) and DCT, and alkalosis vice versa3). Diuretics like thiazide and furosemide also alter calcium absorption in the renal tubules; thiazide promotes calcium reabsorption and furosemide inhibits it. Plasma calcium itself also controls renal calcium absorption through altered PTH secretion as well as via binding to the calcium sensing receptor (CaSR) in the TAL.
To facilitate Ca2+ reabsorption along renal tubules;
(i) voltage difference between the lumen and blood compartment should be favorable for Ca2+ passage, i.e., a positive voltage in the lumen;
(ii) concentration difference should be favorable for Ca2+ passage with a higher Ca2+ concentration in the lumen;
(iii) an active transporter should exist if the voltage or concentration difference is not favorable for Ca2+ reabsorption. Each renal tubular segment has a different Ca2+ concentration difference or voltage environment for its unique mechanism for calcium re-absorption.
Calcium handling along the tubules
Fifty to sixty percent of filtered calcium is absorbed in parallel with sodium and water in the PT, suggesting that the passive pathway is the main route of Ca2+ absorption in this segment. Claudin-2 is especially concentrated in the tight junction and also expressed in the basolateral membrane of the PT as the candidate for paracellular Ca2+ channel in the PT. There is no evidence that Ca2+ reabsorption occurs in the thin descending and ascending limb. In the TAL, 15% of filtered calcium is absorbed, and the passive absorption through paracellular space is known as the main mechanism (Fig. 1). Paracellin-1 (claudin-16) is exclusively expressed in the tight junction of TAL and has been known as the important magnesium channel in the TAL. Paracellin-1 mutation caused hypercalciuria and nephrocalcinosis in addition to hypomagnesemia. This finding supports that paracellin-1 is not only the main Mg2+ channel, but also works as the paracellular Ca2+ channel in the TAL. There are some evidences that active transport occurs in the TAL, but no specific channel has yet been identified). The CaSR is a member of G protein-coupled receptors and suppresses PTH secretion by sensing high plasma Ca2+ level in the parathyroid glands). In the kidney, the CaSR is most highly expressed in the TAL..
Although only 10-15% of filtered Ca2+ is absorbed in the DCT and CNT, these are the main sites in which the fine regulation of Ca2+ excretion and the major action of PTH and activated vitamin D occur. In the DCT and CNT, the luminal voltage is negative and Ca2+ concentration in the lumen is lower than that of plasma. Thus, active transport mechanism against voltage and concentration gradient should exist in these segments. Several Ca2+ transporting proteins are involved in this active transmembrane transport of Ca2+ in the DCT and CNT. Transcellular Ca2+ re-absorption can occur by three steps;
(i) entry of Ca2+ through the calcium channels (TRPV5, TRPV6) in the apical membrane,
(ii) binding of Ca2+ with calcium-binding protein (calbindin) and diffusion in the cytoplasm (which enables no significant change in the intracellular i[Ca2+], and
(iii) Ca2+ extrusion via an ATP-dependent plasma membrane Ca2+-ATPase (PMCA1b) and an Na2+/Ca2+ exchanger (NCX1) in the basolateral membrane (see Fig below on right).
In the collecting duct (CD), there is no evidence that Ca2+ reabsorption occurs even though calcium channel (TRPV6) was documented to be expressed in CD cells.
Each renal tubule has a unique environment and plays a different role in Ca2+ reabsorption.
The coordinated play of different renal tubules could maintain harmony of renal Ca2+ handling.
Transient receptor potential (TRP) channel is a super-family of ion channels permeable to monovalent and/or divalent cations with six-transmembrane domains. The mammalian TRP family consists of six subfamilies like TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin). TRPV is one of them and consists of six members in mammalians; TRPV1 to TRPV6. TRPV5 (previously known as ECaC1) and TRPV6 (ECaC2), both cloned in 1999, have characteristics distinguished from other TRPV channels; (i) constitutively active at low intracellular Ca2+ concentration, and (ii) exclusively selective for Ca2+ (PCa/PNa >100)9). TRPV5 and TRPV6 have the highest sequence homology (~730 amino acids, amino-terminal ankyrin repeats, TM5 and TM6 each forming the pore-region composed with tetramer, on human chromosome 7q34-35) (Fig. 3a). TRPV5 is exclusively expressed in the DCT and CNT in the kidney10) (Fig. 3b). On the contrary, TRPV6 is more ubiquitously distributed, especially in the intestine, and also found from the DCT to the CD in the kidney11) (Fig. 3b). Both TRPV5 and TRPV6 are located in the apical plasma membrane of the tubular epithelium, and serve as the entrance of Ca2+ from the lumen into the cytoplasm. TRPV5 knockout mice exhibited severe hypercalciuria (more than 6 times of wild type mouse) and low bone densities, but without hypocalcemia due to the compensatory elevation of activated vitamin D, clearly demonstrating that TRPV5 plays a crucial role in renal calcium reabsorption12). TRPV6 knockout mice also showed significant hypercalciuria and bone disease13). Even though TRPV5 and TRPV6 knockout mice showed congenital hypercalciuria, the mutation of the proteins has not been found in the human. Until now, TRPV5 is known
as the main entry of Ca2+ in renal tubular epithelial cells in the DCT and CNT, and TRPV6 is also known to contribute to renal Ca2+ reabsorption in the distal nephron.
Several factors (PTH, 1,25(OH)2D3, calcitonin, estrogen, i[Ca2+], acid-base status, klotho, diuretics, and im-munosuppressive drugs, etc) are involved in the regulation of both TRPV5 and TRPV610) (Table 2). Alteration of TRPV5 and TRPV6 by these factors contributes in disturbance of calcium metabolism: dyscalcemia, hypo- and hypercalciuria. 1,25(OH)2D3-depleted rats showed decreased expression of TRPV5 and calbindin-D28K mRNA and protein, and repletion of the hormone restored the expression of them.
TRPV
Transient receptor potential (TRP) channel is a super-family of ion channels permeable to monovalent and/or divalent cations with six-transmembrane domains. The mammalian TRP family consists of six subfamilies like TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin). TRPV is one of them and consists of six members in mammalians; TRPV1 to TRPV6. TRPV5 (previously known as ECaC1) and TRPV6 (ECaC2), both cloned in 1999, have characteristics distinguished from other TRPV channels;
(i) constitutively active at low intracellular Ca2+ concentration, and
(ii) exclusively selective for Ca2+ (PCa/PNa >100)9). TRPV5 and TRPV6 have the highest sequence homology (~730 amino acids, amino-terminal ankyrin repeats, TM5 and TM6 each forming the pore-region composed with tetramer, on human chromosome 7q34-35). TRPV5 is exclusively expressed in the DCT and CNT in the kidney.
On the contrary, TRPV6 is more ubiquitously distributed, especially in the intestine, and also found from the DCT to the CD in the kidney
Both TRPV5 and TRPV6 are located in the apical plasma membrane of the tubular epithelium, and serve as the entrance of Ca2+ from the lumen into the cytoplasm.
TRPV5 knockout mice exhibited severe hypercalciuria (more than 6 times of wild type mouse) and low bone densities, but without hypocalcemia due to the compensatory elevation of activated vitamin D, clearly demonstrating that TRPV5 plays a crucial role in renal calcium reabsorption. TRPV6 knockout mice also showed significant hypercalciuria and bone disease. Even though TRPV5 and TRPV6 knockout mice showed congenital hypercalciuria, the mutation of the proteins has not been found in the human. Until now, TRPV5 is known as the main entry of Ca2+ in renal tubular epithelial cells in the DCT and CNT, and TRPV6 is also known to contribute to renal Ca2+ reabsorption in the distal nephron.
Several factors (PTH, 1,25(OH)2D3, calcitonin, estrogen, i[Ca2+], acid-base status, klotho, diuretics, and im-munosuppressive drugs, etc) are involved in the regulation of both TRPV5 and TRPV6. Alteration of TRPV5 and TRPV6 by these factors contributes in disturbance of calcium metabolism: dyscalcemia, hypo- and hypercalciuria. 1,25(OH)2D3-depleted rats showed decreased expression of TRPV5 and calbindin-D28K mRNA and protein, and repletion of the hormone restored the expression of them.
Table . The regulation of calcium transporting proteins in the DCT and CNT
Factors
TRPV5 TRPV6 Calbindin- Mechanisms
D28K
PTH
+
NC
+
transcription
Vit D
+
+
+
transcription
Estrogen
+
+
+
transcription
Low Ca2+ diet
+
+
NC
transcription
Acidosis
–
ND
–
transcription
Thiazide
C
ND
C
transcription
Furosemide
+
+
+
transcription
Tacrolimus
–
ND
–
transcription
[Ca2+]
–
–
–
Channel activity
Calbindin-D28K
+
NC
Channel activity
Klotho
+
+
ND
trafficking
FGF23
FGF23, a member of the FGF family (type I trans-membrane phosphotyrosine kinase receptors), is a 30 kDa secreted protein and inactivated by cleavage into two smaller fragments (N-terminal 18 kDa fragment and C-terminal 12 kDa fragment) by a pro-convertase enzyme, furin . It was first cloned as the candidate gene for autosomal dominant hypophosphatemic rickets (ADHR). FGF23 is primarily expressed in the osteoblasts and osteocytes. Because Fgf23 knockout mice showed very similar phenotype to Klotho knockout mice including severe hyperphophatemia and osteoporosis, and gain of function mutation of Fgf23 gene was observed in ADHR patients. The main studies about the role of FGF23 in the kidney have focused on phosphate metabolism rather than calcium metabolism.
It is unknown how the FGF23:klotho complex from the DCT acts in the PT because the main action site of FGF23 in the kidney is the PT, whereas the FGF23:klotho complex is most abundant in the DCT. Both overexpression and deficiency of FGF23 cause several clinical diseases including ADHR and HFTC (hyperphosphatemic familial tumorial calcino-sis). Recently, FGF23 was suggested as a potential bio-marker for management of phosphate balance in chronic kidney disease (CKD) patients because the circulating FGF23 level was higher in CKD patients than healthy controls and the increased FGF23 level was an independent risk factor for higher mortality among dialysis patients26). FGF23 also plays some roles in the parathyroid glands and other organs like the choroid plexus, pituitary gland, and bone. However, further studies are needed to clarify the roles and the mechanisms.
Conclusion
The kidney has been known as the central organ for calcium homeostasis through fine regulation of renal calcium excretion. For the past decade, there has been big progress in the understanding of the roles of the kidney in calcium homeostasis. The identification of calcium transport proteins and the molecular approach to the regulatory mechanisms achieved a major contribution to this progress. TRPV5, TRPV6, calbindin-D28K, NCX1, and PMCA1b have been identified as the main calcium transport proteins in the distal nephron. PTH, vitamin D, i[Ca2+], CaSR, and other various conditions control renal calcium excretion through the regulation of these transport proteins. Klotho and FGF23 emerged as new players in calcium metabolism in the kidney. Thus, the role of the klotho-FGF23 axis in the regulatory mechanisms of calcium transport needs to be addressed.
Disorders of Calcium, Phosphorus and Magnesium Metabolism
Infrequently patients might present in the outpatient settings with non-specific symptoms that might be due to abnormalities of divalent cation (magnesium, calcium) or phosphorous metabolism. Several inherited disorders have been identified that result in renal or intestinal wasting of these elements. Physicians need to have a thorough understanding of the mechanism of calcium, magnesium and phosphorous metabolism and diagnoses disorders due to excess or deficiency of these elements. Prompt identification and treatment of the underlying disorders result in prevention of serious morbidity and mortality.
Maintenance of serum calcium in the extra cellular fluid space (ECF) is tightly regulated. Most calcium (around 99%) is bound and complexed in the bones. Calcium in the ECF is found in three fractions, of which 45% is in biological ionized fraction, 45% is protein bound and not filterable in the kidney and 10% is complexed with anions such as bicarbonate, citrate, phosphate, and lactate (Fig. 1 ). Most of the protein bound calcium is complexed with albumin, and a smaller amount to globulin. Each 1 g/dL of albumin binds 0.8 mg/dL (0.2 mmol/L) calcium. Hence, for each 1g/ dl decrease in serum albumin below normal value of 4.0 g/dl, one needs to add 0.8 mg/ dl to the measured serum calcium. Levels of calcium are also influenced by acid-base status, with acidosis increasing serum calcium while alkalosis decreases serum calcium levels.
Maintenance of normal calcium in ECF is dependent on fluxes of calcium between the intestine, kidneys and bone. The regulation of calcium in serum is regulated by calcium itself, through a calcium sensing receptor (Ca RG) and hormones like parathormone (PTH) and 1, 25-dihydroxyvitamin D3.
Calcium transport across the intestine occurs in two directions, absorption and secretion. The factors that influence calcium absorption in the intestine include daily amount of calcium that is ingested and 1, 25-dihydroxyvitamin D3 that binds to and activates the Vitamin D receptor (VDR) and induces the expression of calcium channel TRPV6, calbindin- D9K, and Ca2+ – ATPase. Other hormones like PTH, estrogen, prolactin and growth hormone may play a minor role in calcium absorption. Conditions that result in decreased intestinal calcium transport include high vegetable fiber and fat content of food, corticosteroid deficiency, estrogen deficiency, advanced age, gastrectomy, intestinal malabsorption, diabetes mellitus, renal failure and low Ca2+ phosphate ratio in the food.
PTH and 1, 25- dihydroxyvitamin D3 stimulate osteoclasts in bones and promote release of calcium in ECF. PTH promotes hydroxylation of 25(OH) D3 to 1, 25(OH) D3 and distal tubular calcium reabsorption.
Hypocalcaemia occurs when the loss of calcium from the ECF via renal excretion is greater than influx of Ca 2+ from intestine or bones. One of the commonest cause of low calcium is hypoalbuminemia, though the level of ionized Ca2+ is normal. The causes of hypocalcaemia is summarized in Table 1 . Acute hypocalcaemia is often seen in acute respiratory alkalosis due to hyperventilation. Idiopathic or acquired (post surgery, radiotherapy) hypoparathyroid states are usually accompanied with elevated phosphate level. Pseudo hypoparathyroidism is characterized by short neck, round face and short metacarpal and results from end-organ resistance to PTH. Chronic kidney disease and massive phosphate administration can result in hypocalcaemia with high serum phosphate levels. Familial hypocalcaemia is linked with activating mutation of Ca RG. Hypocalcaemia with low phosphate levels occur in Vitamin D deficiency, resistance to calcitriol (Type 2 vitamin D- dependent rickets) acute pancreatitis and magnesium deficiency.
Table 1 : Causes of Hypocalcemia
Idiopathic Hypoparathyroidism
Post parathyroidectomy (Hungry bones syndrome)
Pseudo-hypoparathyroidism
Familial hypocalcemia
Rapid correction of severe acidosis with dialysis
Acute respiratory and metabolic alkalosis
Acute pancreatitis
Rhabdomyolysis
Hypomagnesemia
Septic shock
Ethylene glycol toxicity
Vitamin D deficiency
Chronic kidney disease
Massive transfusion- Citrate toxicity
Hypercalcemia occurs when in influx of calcium into the ECF exceeds the efflux of calcium from intestine and kidneys. The normal calcium level ranges from 8.9- 10.1 mg/ dL. The range of serum calcium levels in mild hypercalcemia is (10.1- 12.0 mg/dL), moderate hypercalcemia (12.0 – 14.0 mg/dl) and severe hypercalcemia > 14.0 mg/ dL respectively. The various causes of hypercalcemia is depicted in Table 2. Mutation of the gene for Ca RG results in hypercalcemia in few cases.
Table 2. : Causes of hypercalcemia Parathormone Primary hyperparathyroidism
(PTH) mediated Lithium induced
Familial hypocalciuric hypercalcemia
Tertiary hyperparathyroidism
Cancer Multiple myeloma
PTHrp mediated-Breast, lung,
Exogenous Vitamin D
Dialysis patients (exogenous Vit D)
Other causes Vitamin A toxicity
Thyrotoxicosis
Paget’s disease
Adrenal insufficiency
Thiazide use
Deficiency of calcium, magnesium and phosphorous are common in general practice. A thorough understanding of pathophysiology of these elements, common dietary sources of these elements and pharmacological measures that might be necessary to correct these deficiencies could guide the physician to make an accurate diagnosis, initiate appropriate treatment and prevent future recurrences. (Ghosh AK*, Joshi SR. 2008.)
Renal Disease and the Cardiovascular System
Cardiovascular disease is a leading cause of death among patients with end stage renal failure. Animal models have played a crucial role in teasing apart the complex pathological processes involved. In addition to the anatomical and histological characteristics humans share with other species, human diseases can be reproduced in these species using pharmacological, surgical or genetic manipulation. Experimentation still provides the best evidence for disease causation, and only with this evidence can clinical science proceed to developing treatments. However, experimentation is often not possible or ethical in human subjects, and thus without these animal models the advancement in knowledge of the patho-physiology of disease would come to a standstill.
The way in which kidneys succumb to disease and the development of renal failure involves complex interactions between numerous different systems, mediated by a multitude of chemicals. Current understanding of renal disease is merely the tip of the metaphorical iceberg. The history of renal pathology is plagued by controversy, and nowhere is this more evident than in the development of cardiovascular disease in patients with chronic renal failure. Impairment of renal function increases the risk of cardiac disease to 15-20 times that of individuals with normal renal function. The result is that cardiac disease causes 40% of deaths in patients on dialysis.
This review discusses the principles of using animal models, the history of their use in the study of renal hypertension, the controversies arising from experimental models of non-hypertensive uraemic cardiomyopathy and the lessons learned from these models, and highlights important areas of future research in this field, including de novo cardiomyopathy secondary to renal transplantation.
Myocardial Interstitial Fibrosis, Cardiac Compliance and Vascular Architecture
Using subtotally nephrectomised Sprague-Dawley rats, Mall et al. showed that the increase in total heart weight demonstrated by Rambausek et al. after 21 days of uremia (as well as an increase in both right and left ventricular weight) was secondary to an increase in true interstitial volume, both cellular and non-cellular, with increased deposition of collagen. This was associated with activated interstitial cells, and a reduced capillary cross-sectional area. In 1992, this latter point was confirmed using stereological techniques to analyse perfusion-fixed hearts of subtotally nephrectomised Sprague-Dawley rats. Uremia resulted in increased blood pressure and reduced capillary length per unit myocardial volume, as well as reduced capillary luminal surface density and volume density, compared to control rats. The same group found a blood pressure-independent increase in the wall to lumen ratio of intramyocardial arteries, and in the aorta media thickness of subtotally nephrectomised rats. The intramyocardial arterial wall thickening has been found to be due to hypertrophy rather than hyperplasia, independent of blood pressure. These architectural changes were reported again in 1996. In that experiment, nephrectomised Sprague-Dawley rats were given ramipril, nifedipine or moxonidine to normalise blood pressure; these drugs had differential effects on the above architectural changes, and also acted to prevent these changes. The different changes in interstitial and capillary density in uremic cardiomyopathy have not yet been explained, but the role of growth factors such as basic fibroblast growth factor (BFGF) and vascular endothelial growth factor (VEGF) has been proposed.
Cardiac Function and Energetics in Uremia
The above experiments provided some insight into the structural changes seen in uraemic hearts. They were followed by a study using the subtotal (5/6) nephrectomy model on Wistar rats, in which the authors focused on the mechanical effects of these structural changes in vitro, thereby removing neurohormonal influences on cardiac contractility. Four weeks after surgery, isolated perfusing working heart preparations demonstrated reduced cardiac output. However, blood pressure was not controlled during the four weeks post-operatively, and could have contributed to the effects. An increased susceptibility to ischemic damage was also shown via decreased phosphocreatine content, and an increased release of inosine (a marker of ischaemic damage). These hearts failed in response to increases in calcium; the authors proposed that impaired cytosolic calcium control played a role in the relationship between renal failure and impaired cardiac function.
This in vitro experiment demonstrated the fact that impaired cardiac function was independent of circulating urea and creatinine, as the hearts were perfused with physiological saline, with no effect from the addition of urea and creatinine. The opposite has been shown in spontaneously beating mouse cardiac myocytes, in response to sera from patients on haemodialysis for chronic renal failure. Urea, creatinine, and combinations of the two reduced the cardiac inotropy and resulted in arrhythmias and asynchronies.
These experiments make a good case for uremic cardiomyopathy to be a distinct entity from hypertensive cardiac dysfunction and atherosclerotic cardiac disease secondary to the risk factors common to both heart and kidney disease. The cause of this phenomenon is still controversial, with parathyroid hormone (PTH), angiotensin II, marino-bufagenin (MBG), oxidative stress, and growth hormone.
The Role of Calcium in Uremic Cardiomyopathy
Calcium ions play a crucial role in cardiac physiology, particularly in myocardial excitation-contraction coupling. Therefore, PTH was one of the first culprits to be suspected of playing a role in the pathophysiology of uremic cardiomyopathy; this was as early as 1984. As reviewed by Rostand and Drüeke, there are numerous theories pertaining to the mechanisms whereby PTH could act as an intermediary between renal impairment and cardiomyopathy. These include
direct trophic effects on myocytes
interstitial fibroblasts,
indirect effects via anaemia or large and small vessel changes.
Rostand and Drüeke suggest an increase in blood pressure via hypercalcemia, but the effects on the heart appear to be independent of blood pressure.
Rambausek et al. noted increased cardiac calcium content in experimental rats, and that an increase in heart weight still occurred after parathyroidectomy with calcium supplementation. This was followed in the 1990s by in vitro experiments that demonstrated; an increased cytosolic calcium concentration in isolated rat myocytes in response to PTH, a reduced expression of PTH-related peptide receptor mRNA in rat hearts secondary to hyperparathyroidism due to chronic renal failure, and increased force and frequency of contraction of isolated, beating rat cardiomyocytes.
Subsequent to “chance observations” in the laboratory, Amann et al. argued for the role of PTH in the wall thickening of intramyocardial arterioles and for fibroblast activation and subsequent cardiac fibrosis. Abolishing hyperparathyroidism prevented the cardiac fibrosis and capillary changes normally seen in nephrectomised rats, which was independent of blood pressure.
The Renin-Angiotensin System (RAS) and Endothelin
Many studies have highlighted the importance of the RAS in the development of uremic cardiomyopathy. Tornig et al. showed that in nephrectomised rats, ramipril, an ACE inhibitor, prevented the increased wall thickness of the intramyocardial arterioles, as well as the expansion of nonvascular cardiac interstitial volume and the aortic wall and lumen changes, but not the reduced capillary length density. The same group subsequently repeated these observations, and demonstrated that the beneficial effects of ramipril were prevented by the use of specific bradykinin B2 receptor antagonists, suggesting a role for increased bradykinin as a mediator for the effects of ramipril.
CONCLUSIONS
Experimental models have played a crucial role in the study of the complex interplay between the heart and the kidney in chronic renal disease. In view of the numerous differences in animal and human anatomy, physiology and pathology, the results of these experiments should be interpreted with caution, but in some areas, these studies have led directly to advances in therapeutics.
(RC Grossman. 2010.)
Deficiency of the Calcium-Sensing Receptor
Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)–dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na+-K+-2Cl cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca2+ transport. (Toka HR, Al-Romaih K, Koshy JM, DiBartolo, III S, et al. 2012)
Na(+)-Ca2+ exchanger of rat proximal tubule: gene expression and subcellular localization.
Dominguez JH, Juhaszova M, Kleiboeker SB, Hale CC, Feister HA.
Am J Physiol. 1992 Nov;263(5 Pt 2):F945-50. http://www.ncbi.nlm.nih.gov/pubmed/1443182
Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron.
Zuo Q, Claveau D, Hilal G, Leclerc M, Brunette MG.
Kidney Int. 1997; 51(6):1991-9. http://www.ncbi.nlm.nih.gov/pubmed/9186893
Ca2+ transport by the luminal membrane of the distal nephron: action and interaction of protein kinases A and C.
Hilal G, Claveau D, Leclerc M, Brunette MG.
Biochem J. 1997 Dec 1;328 ( Pt 2):371-5 http://www.ncbi.nlm.nih.gov/pubmed/9371690
Branching points of renal resistance arteries are enriched in L-type calcium channels and initiate vasoconstriction.
M S Goligorsky, D Colflesh, D Gordienko, L C Moore
Am J Physiol 03/1995; 268(2 Pt 2):F251-7. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9186893/
G proteins regulate calcium channels in the luminal membranes of the rabbit nephron
Brunette MG, Hilal G, Mailloux J, Leclerc M.
Nephron. 2000 Jul;85(3):238-47. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10867539/
Characterization of three types of calcium channel in the luminal membrane of the distal nephron
MG Brunette, M Leclerc, D Couchourel, J Mailloux, Y Bourgeois
Can J Phy Pharma 2004; 82(1): 30-37 http://dx.doi.org/10.1139/y03-127
Discovery of alpha-Klotho and FGF23 unveiled new insight into calcium and phosphate homeostasis
Nabeshima Y.
Clin Calcium. 2008;18(7):923-34. http://dx.doi.org/CliCa0807923934
Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormone-independent hypocalciuria
Toka HR, Al-Romaih K, Koshy JM, DiBartolo S, III, Kos, CH, et al.
J Am Soc Nephrol 2012; 23: 1879-1890. http://dx.doi.org/10.1681/ASN.2012030323
The renal Na+/Ca2+ exchange system is located exclusively in the distal tubule
Ramachandram C, Brunette MG.
Biochem J 1989;257:259-264
Calcium ion transport across plasma membranes isolated from rat kidney cortex
Gmaj P, Murer H, Kinne R
Biochem J 1979; 178:549-557
Model-based analysis of Fgf23 regulation in chronic renal disease
Yokota H, Pires A, Raposo JF, Ferreira HG.
Gene Reg and Systems Biol 2010; 4: 53-60
Kidney and Calcium Homeostasis
Un Sil Jeon
Electrolyte & Blood Pressure 2008; 6:68-76
Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
andArticleCurator: Aviva Lev-Ari, PhD, RN
Image created by Adina Hazan 06/30/2021
Voice of Justin Pearlman, MD, PhD, FACC
Catechols refer to the stress hormones that control our response to fright, flight and fight, e.g., epinephrine, also known as adrenaline. Sudden elevation of catechols increases heart rate and also the strength of heart contraction (contractility). In the short term, that provides a boost that supports special demands to run faster, work harder. Like the healthcare system, it is not sustainable in high gear. Excess catechol push causes heart failure (catechol toxicity). Race horses routinely develop pulmonary edema by the end of a race – those pretreated for that with the diuretic LASIX have an L next to their entry in the race ticket. The same issues occur as a whole-body system and at the subcellular level. Catechols increase amount and speed of the release of calcium which in turn triggers heart muscle contraction. However, the failing heart has elevated levels of calcium that impair oxygen utilization. The following discussions address the linkages between catechols and calcium traffic, including both the catechol and calcium stimulation of speed and strength, and their detrimental effects over time.
This article is Part VII in a continuation to the following article series on tightly related topics of the Calcium Release Mechanism.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Introduction to Calcium Release Mechanism in Vascular Smooth Muscle and in Cardiomyocytes
Author: Justin D Pearlman, MD, PhD, FACC PENDING
I. Cellular Contractility Capacity — Actin, Cellular Dynamics and Calcium Efflux: Emergence of the Calcium Release-related Contractile Dysfunction
Author: Justin D Pearlman, MD, PhD, FACC
II. Integration and Interpretation of Research Results in Two Labs: Mark E Anderson’s and Roger Hajjar’s Lab
Author: Justin D Pearlman, MD, PhD, FACC PENDING
Mark Anderson’s Laboratory at the University of Iowa Carver College of Medicine recently summarized the critical roles of calcium in heart failure and arrhythmia in an article in Circulation Research. That laboratory elucidated critical facts, such as the controlling role of phosphorylation of ryanodine receptors among other details of the control and impact of Ca²⁺ homeostatic and structural proteins, ion channels, and enzymes. Their review focuses on the molecular mechanisms of defective Ca²⁺ cycling in heart failure and knowledge of those pathways may translate into new innovative therapies. The highly conserved Ca2+/calmodulin-dependent protein kinase II (CaMKII)plays an essential role in cardiac myocytes. Electrichemical activation of the cariac contraction cycle triggers a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) which activates CaMKII activated through the binding of Ca2+-bound calmodulin (CaM). The activated CaMKII molecules phosphorylate many intracellular target proteins, including the sarcolemmal L-type Ca2+ channel, the ryanodine receptor, and the Ca2+ pump on the sarcoplasmic reticulum. Intersubunit autophosphorylation (positive feedback) promotes accumulation of the active CaMKII. Phosphorylated CaMKII maintains its catalytic activity until it is inactivated by constitutive phosphatase activity.
Roger J. Hajjar MD is the Director of the Cardiovascular Research Center, a cutting-edge translational research laboratory at Mt Sinai Medical Center. He is the Arthur & Janet C. Ross Professor of Medicine, Professor of Gene & Cell Medicine, Director of the Cardiology Fellowship Program, and Co-Director of the Transatlantic Cardiovascular Research Center, which combines Mount Sinai Cardiology Laboratories with those of the Universite de Paris – Madame Curie. He earned a bachelors of science degree in Biomedical Engineering at Johns Hopkins University and a medical degree from Harvard Medical School and the Harvard-MIT Division of Health Sciences and Technology. He completed his fellowship in cardiology at Massachusetts General Hospital in Boston, then became a staff cardiologist in the Heart Failure & Cardiac Transplantation Center, followed by Director of the Cardiovascular Laboratory of Integrative Physiology and Imaging, before moving to Mt. Sinai.
Roger J. Hajjar, MD and his team of investigators translate scientific findings into therapies for cardiovascular diseases. Dr. Hajjar’s team pioneered a potential gene therapy for heart failure, AAV1.SERCA2a, which can revive malfunctioning myocardium. His laboratory has completed Phase 1 and Phase 2 First-in-Man clinical trials of SERCA2a gene transfer in patients with advanced heart failure, and Phase 3 validation began in 2011. His laboratory also studies how to block signaling pathways in cardiac hypertrophy, aging, apoptosis, and diastolic failure.
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
2013
•He BJ, Anderson ME. Aldosterone and Cardiovascular Disease: the heart of the matter. Trends in Endocrinology & Metabolism 24(1):21-30, 2013. [PMID: 23040074]
•Luo M, Anderson ME, Mechanisms of altered Ca2+ handling in heart failure. Circ Res 113(6):690-708. 2013 [PMID: 23989713]
•Anderson ME. Why has it taken so long to learn what we still don’t know? Circ Res 113(7):840-2. 2013 [PMID: 24030016]
•Thomas C, Anderson ME. In memoriam: John B. Stokes, MD. Semin Nephrol. 33(3):207-8, 2013. [PMID: 23953797]
•Gyorke S, Ho HT, Anderson ME, et al. Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides. Cardiovas Res [PMID: Accepted for publication]
•Kline J, Anderson ME, et al, βIV-spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells. Proc Natl Acad Sci. [PMID: Accepted for publication]
•Maier LS, Sag C, Anderson ME, Ionizing Radiation Regulates Cardiac Ca handling via increased ROS and activated CaMKII. Bas Res in Card [PMID: Accepted for publication]
•Chen B, Guo A, Zhang C, Chen R, Zhu Y, Hong J, Kutschke W, Zimmerman K, Weiss RM, Zingman L, Anderson ME, Wehrens XH, Song LS. Critical roles of Junctophilin-2 T-tubule and excitation-contraction coupling maturation during postnatal development. Cardiovas Res 2013 Oct 1; 100(1):54-62. [PMID: 23860812] [PMC3778961]
•Purohit A, Rokita AG, Xiaoqun G, Biyi C, Koval OM, Voigt N, Neef S, Sowa T, Gao Z, Luczak E, Stefansdottir H, Behunin AC, Li N, El-Accaoui RN, Yang B, Swaminathan PD, Weiss RM, Wehrens XH, Song LS, Dobrev D, Maier LS, Anderson ME. Oxidized CaMKII Triggers Atrial Fibrillation. Circulation 2013 Sep 12 [Epub ahead of print] [PMID: 24030498]
•Yoshida-Moriguchi T, Willer T, Anderson ME, Venzke D, Whyte T, Muntoni F, Lee H, Nelson SF, Yu L, Campbell, KP. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function. Science 2013 Aug 23; 341(6148): 896-9. [PMID:23929950]
•Scott JA, Klutho PJ, El Accaoui R, Nguyen E, Venema AN, Xie L, Jiang S, Dibbern M, Scroggins S, Prasad AM, Luczak ED, Davis MK, Li W, Guan X, Backs J, Schlueter AJ, Weiss RM, Miller FJ, Anderson ME, Grumbach IM. The Multifunctional Ca2+/Calmodulin-Dependent Kinase IIδ (CaMKIIδ) Regulates Arteriogenesis in a Mouse Model of Flow-Mediated Remodeling. PLoS One 2013 Aug 8; 8(8):e71550. [PMID: 23951185] [PMC3738514]
•Scholten A, Preisinger C, Corradini E, Bourgonje VJ, Hennrick ML, van Veen TA, Swaminathan PD, Joiner ML, Vos MA, Anderson ME, Heck AJ. A Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin Dependent Protein Kinase II Regulation in the Heart. J Am Heart Assoc 2013 Aug 7; 2(4):e000318. [PMID: 23926118]
•Prasad AM, Nuno DW, Koval OM, Ketsawatsomkron P, Li W, Li H, Shen Y, Joiner ML, Kutschke W, Weiss RM, Sigmund CD, Anderson ME, Lamping KG, Grumbach IM. Differential Control of Calcium Homeostatis and Vascular Reactivity by Ca2+/Calmodulin-Dependent Kinase II. Hypertension 2013 Aug; 62(2):434-41.[PMID:23753415]
•Sanders PN, Koval OM, Jaffer OA, Prasad AM, Businga TR, Scott JA, Hayden PJ, Luczak ED, Dickey DD, Allamargot C, Olivier AK, Meyerholz DK, Robison AJ, Winder DG, Blackwell TS, Dworski R, Sammut D, Wagner BA, Buettner GR, Pope MR, Miller FJ, Dibbern ME, Haitchi HM, Mohler PJ, Howarth PH, Zabner J, Kline JN, Grumbach IM, Anderson ME. CaMKII is Essential for the Proasthmatic Effects of Oxidation. Sci Trans Med 2013 Jul 24; 5(195):195 ra97. [PMID: 23884469] Chosen as a “From the Cover” article in STM and with a commentary in JAMA. 310(9):894. doi: 10.1001/jama.2013.277035
•Wolf RM, Glynn P, Hashemi S, Zarei K, Mitchell CC, Anderson ME, Mohler PJ, Hund TJ. Atrial fibrillation and sinus node dysfunction in human ankyrin-B syndrome: A computational analysis. Am J Physiol Heart and Circ Physiol 2013 May; 304(9):H1253-66. [PMID: 23436330] [PMC3652094]
•Ather S, Wang W, Wang Q, Li N, Anderson ME, Wehrens XH. Inhibition of CaMKII Phosphorylation of RyR2 Prevents Inducible Ventricular Arrhythmias in Mice with Duchenne Muscular Dystrophy. Heart Rhythm 2013 Apr; (10)4:592-9 [PMID: 23246599] [PMC3605194]
•Yang J, Maity B, Huang J, Gao Z, Stewart A, Weiss RM, Anderson ME, Fisher RA. G- protein inactivator RGS6 mediates myocardial cell apoptosis and cardiomyopathy caused by doxorubicin. Cancer Res 2013 Mar 15; 73(6): 1662-7. [PMID: 23338613] [PMC3602152]
•Luo M, Guan X, Luczak ED, Di L, Kutschke W, Gao Z, Yang J, Glynn P , Sossalla S, Swaminathan PD, Weiss RM, Yang B, Rokita AG,5, Maier LS, Efimov I, Hund TJ, Anderson ME. Diabetes increases mortality after myocardial infarction by oxidizing CaMKII. J Clin Invest 2013 Mar 1; 123(3):1262-74. [PMID: 23426181] [ PMC3673230]
•Sierra A, Zhu Z, Sapay N, Sharotri V, Kline CF, Luczak ED, Subbotina E, Sivaprasadarao A, Snyder PM, Mohler PJ, Anderson ME, Vivaudou M, Zingman LV, Hodgson-Zingman DM. Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II. J Biol Chem 2013 Jan 18; 288(3):1568-81. [PMID: 23223335] [PMC3548467]
•Gao Z, Rasmussen TP, Li Y , Kutschke W , Koval OM, Wu Y, Wu Y, Hall DD, Joiner ML, Wu XQ, Swaminathan PD, Purohit A, Zimmerman KA, Weiss RM, Philipson K , Song LS, Hund TJ, Anderson ME. Genetic inhibition of Na+-Ca2+ exchanger current disables fight or flight sinoatrial node activity without affecting resting heart rate. Circ Res 2013 Jan 18;112(2):309-17. [PMID: 23192947][Epub: e157-e179] [PMC3562595]
•Degrande ST, Little S, Nixon DJ, Wright P, Snyder J, Dun W, Murphy N, Kilic A, Higgins R, Binkley PF, Boyden PA, Carnes CA, Anderson ME, Hund TJ, Mohler PJ. Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart. J Biol Chem 2013 Jan 11; 288(2):1032-46. [PMID: 23204520] [PMC3542989]
•He BJ, Anderson ME. Aldosterone and Cardiovascular Disease: the heart of the matter. Trends in Endocrinology & Metabolism 24(1):21-30, 2013. [PMID: 23040074]
• Luo M, Anderson ME, Mechanisms of altered Ca2+ handling in heart failure. Circ Res 113(6):690-708. 2013 [PMID: 23989713]
•Anderson ME. Why has it taken so long to learn what we still don’t know? Circ Res 113(7):840-2. 2013 [PMID: 24030016]
• Thomas C, Anderson ME. In memoriam: John B. Stokes, MD. Semin Nephrol. 33(3):207-8, 2013. [PMID: 23953797]
2012
•Wang Y and Anderson ME. Chapter 22: Intracellular Signaling Pathways in Cardiac Remodeling. Muscle: Fundamental Biology and Mechanisms of Disease. J. Hill and E. Olson (Eds), Elsevier, pp 299-308, 2012.
• Ather S, Wang W, Wang Q, Li N, Anderson ME, Wehrens XH. Inhibition of CaMKII Phosphorylation of RyR2 Prevents Inducible Ventricular Arrhythmiasin Mice with Duchenne Muscular Dystrophy. Heart Rhythm. 2012 Dec 11. doi:pii: S1547-5271(12)01450-6. 10.1016/j.hrthm.2012.12.016. PubMed PMID: 23246599.
• Sierra A, Zhu Z, Sapay N, Sharotri V, Kline CF, Luczak ED, Subbotina E, Sivaprasadarao A, Snyder PM, Mohler PJ, Anderson ME, Vivaudou M, Zingman LV, Hodgson-Zingman DM. Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II. J Biol Chem. 2012 Dec 6. [Epub ahead of print] PubMed PMID: 23223335.
• Degrande S, Nixon D, Koval O, Curran JW, Wright P, Wang Q, Kashef F, Chiang D, Li N, Wehrens XH, Anderson ME, Hund TJ, Mohler PJ. CaMKII inhibition rescues proarrhythmic phenotypes in the model of human ankyrin-B syndrome. Heart Rhythm. 2012 Dec;9(12):2034-41. doi: 10.1016/j.hrthm.2012.08.026. Epub 2012 Aug 28. PubMed PMID: 23059182.
• Degrande ST, Little S, Nixon DJ, Wright P, Snyder J, Dun W, Murphy N, Kilic A, Higgins R, Binkley PF, Boyden PA, Carnes CA, Anderson ME, Hund TJ, Mohler PJ. Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart. J Biol Chem. 2012 Nov 30. [Epub ahead of print] PubMed PMID: 23204520.
• Gao Z, Rasmussen TP, Li Y, Kutschke W, Koval OM, Wu Y, Wu Y, Hall DD, Joiner ML, Wu X, Dominic Swaminathan P, Purohit A, Zimmerman KA, Weiss RM, Philipson K, Song LS, Hund TJ, Anderson ME. Genetic Inhibition of Na+-Ca2+ Exchanger Current Disables Fight or Flight Sinoatrial Node Activity Without Affecting Resting Heart Rate. Circ Res. 2012 Nov 27. PubMed PMID: 23192947
• Joiner ML, Koval OM, Li J, He BJ, Allamargot C, Gao Z, Luczak ED, Hall DD, Fink BD, Chen B, Yang J, Moore SA, Scholz TD, Strack S, Mohler PJ, Sivitz WI, Song LS, Anderson ME. CaMKII determines mitochondrial stress responses in heart. Nature. 2012 Nov 8;491(7423):269-73. doi: 10.1038/nature11444. Epub 2012 Oct 10. PubMed PMID: 23051746; PubMed Central PMCID: PMC3471377.
• Rokita AG, Anderson ME. New therapeutic targets in cardiology: arrhythmias and Ca2+/calmodulin-dependent kinase II (CaMKII). Circulation. 2012 Oct 23;126(17):2125-39. doi: 10.1161/CIRCULATIONAHA.112.124990. Review. PubMed PMID: 23091085; PubMed Central PMCID: PMC3532717.
• Koval OM, Snyder JS, Wolf RM, Pavlovicz RE, Glynn P, Curran J, Leymaster ND, Dun W, Wright PJ, Cardona N, Qian L, Mitchell CC, Boyden PA, Binkley PF, Li C, Anderson ME, Mohler PJ, Hund TJ. Ca2+/calmodulin-dependent protein kinase II-based regulation of voltage-gated Na+ channel in cardiac disease. Circulation. 2012 Oct 23;126(17):2084-94. doi: 10.1161/CIRCULATIONAHA.112.105320. Epub 2012Sep 24. PubMed PMID: 23008441.
• Wagner S, Rokita AG, Anderson ME, Maier LS. Redox Regulation of Sodium and Calcium Handling. Antioxid Redox Signal. 2012 Oct 3. [Epub ahead of print] PubMed PMID: 22900788.
• Wu Y, Luczak ED, Lee EJ, Hidalgo C, Yang J, Gao Z, Li J, Wehrens XH, Granzier H, Anderson ME. CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban. J Mol Cell Cardiol. 2012 Sep;53(3):429-36. doi: 10.1016/j.yjmcc.2012.06.019. Epub 2012 Jul 11. PubMed PMID: 22796260.
• Majumdar S, Anderson ME, Xu CR, Yakovleva TV, Gu LC, Malefyt TR, Siahaan TJ. Methotrexate (MTX)-cIBR conjugate for targeting MTX to leukocytes: conjugate stability and in vivo efficacy in suppressing rheumatoid arthritis. J Pharm Sci. 2012 Sep;101(9):3275-91. doi: 10.1002/jps.23164. Epub 2012 Apr 26. PubMed PMID: 22539217.
• Kashef F, Li J, Wright P, Snyder J, Suliman F, Kilic A, Higgins RS, Anderson ME, Binkley PF, Hund TJ, Mohler PJ. Ankyrin-B protein in heart failure: identification of a new component of metazoan cardioprotection. J Biol Chem. 2012 Aug 31;287(36):30268-81. doi: 10.1074/jbc.M112.368415. Epub 2012 Jul 9. PubMed PMID: 22778271; PubMed Central PMCID: PMC3436279.
• Chen B, Guo A, Gao Z, Wei S, Xie YP, Chen SR, Anderson ME, Song LS. In situ confocal imaging in intact heart reveals stress-induced Ca(2+) release variability in a murine catecholaminergic polymorphic ventricular tachycardia model of type 2 ryanodine receptor(R4496C+/-) mutation. Circ Arrhythm Electrophysiol. 2012 Aug 1;5(4):841-9. doi: 10.1161/CIRCEP.111.969733. Epub 2012 Jun 21. PubMed PMID: 22722659; PubMed Central PMCID: PMC3421047.
• Swaminathan PD, Purohit A, Hund TJ, Anderson ME. Calmodulin-dependent protein kinase II: linking heart failure and arrhythmias. Circ Res. 2012 Jun 8;110(12):1661-77. doi: 10.1161/CIRCRESAHA.111.243956. Review. PubMed PMID: 22679140.
• Chen B, Li Y, Jiang S, Xie YP, Guo A, Kutschke W, Zimmerman K, Weiss RM, Miller FJ, Anderson ME, Song LS. β-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction. FASEB J. 2012 Jun;26(6):2531-7. doi: 10.1096/fj.11-199505. Epub 2012 Feb 28. PubMed PMID: 22375019; PubMed Central PMCID: PMC3360148.
• Scott JA, Xie L, Li H, Li W, He JB, Sanders PN, Carter AB, Backs J, Anderson ME, Grumbach IM. The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9. Am J Physiol Heart Circ Physiol. 2012 May 15;302(10):H1953-64. doi: 10.1152/ajpheart.00978.2011. Epub 2012 Mar 16. PubMed PMID: 22427508; PubMed Central PMCID: PMC3362103.
• Gudmundsson H, Curran J, Kashef F, Snyder JS, Smith SA, Vargas-Pinto P, Bonilla IM, Weiss RM, Anderson ME, Binkley P, Felder RB, Carnes CA, Band H, Hund TJ, Mohler PJ. Differential regulation of EHD3 in human and mammalian heart failure. J Mol Cell Cardiol. 2012 May;52(5):1183-90. doi: 10.1016/j.yjmcc.2012.02.008. Epub 2012 Mar 3. PubMed PMID: 22406195; PubMed Central PMCID: PMC3360944.
• Singh MV, Swaminathan PD, Luczak ED, Kutschke W, Weiss RM, Anderson ME. MyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction. J Mol Cell Cardiol. 2012 May;52(5):1135-44. doi: 10.1016/j.yjmcc.2012.01.021. Epub 2012 Feb 3. PubMed PMID: 22326848; PubMed Central PMCID: PMC3327770.
• Qian H, Matt L, Zhang M, Nguyen M, Patriarchi T, Koval OM, Anderson ME, He K, Lee HK, Hell JW. β2-Adrenergic receptor supports prolonged theta tetanus-induced LTP. J Neurophysiol. 2012 May;107(10):2703-12. doi: 10.1152/jn.00374.2011. Epub 2012 Feb 15. PubMed PMID: 22338020; PubMed Central PMCID: PMC3362273.
2011
• Xie YP, Chen B, Sanders P, Guo A, Li Y, Zimmerman K, Wang LC, Weiss RM, Grumbach IM, Anderson ME, Song LS. Sildenafil Prevents and Reverses Transverse-Tubule Remodeling and Ca2+ Handling Dysfunction in Right Ventricle Failure Induced by Pulmonary Artery Hypertension. Hypertension. 2011 Dec 27.[Epub ahead of print] PubMed PMID: 22203744.
•He BJ, Joiner ML, Singh MV, Luczak ED, Swaminathan PD, Koval OM, Kutschke W, Allamargot C, Yang J, Guan X, Zimmerman K, Grumbach IM, Weiss RM, Spitz DR, Sigmund CD, Blankesteijn WM, Heymans S, Mohler PJ, Anderson ME. Oxidation of CaMKII determines the cardiotoxic effects of aldosterone. Nat Med. 2011 Nov 13;17(12):1610-8. doi: 10.1038/nm.2506. PubMed PMID: 22081025.
• Zhu Z, Burnett CM, Maksymov G, Stepniak E, Sierra A, Subbotina E, Anderson ME, Coetzee WA, Hodgson-Zingman DM, Zingman LV. Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia. Biochem Biophys Res Commun. 2011 Dec 2;415(4):637-41. Epub 2011 Nov 3. PubMed PMID: 22079630; PubMed Central PMCID: PMC3230708.
•Albert CM, Chen PS, Anderson ME, Cain ME, Fishman GI, Narayan SM, Olgin JE, Spooner PM, Stevenson WG, Van Wagoner DR, Packer DL; Heart Rhythm Society Research Task Force. Full report from the first annual Heart Rhythm Society Research Forum: a vision for our research future, “dream, discover, develop, deliver”. Heart Rhythm. 2011 Dec;8(12):e1-12. Epub 2011 Nov 7. PubMed PMID: 22079558.
•Cunha SR, Hund TJ, Hashemi S, Voigt N, Li N, Wright P, Koval O, Li J, Gudmundsson H, Gumina RJ, Karck M, Schott JJ, Probst V, Le Marec H, Anderson ME, Dobrev D, Wehrens XH, Mohler PJ. Defects in ankyrin-based membrane protein targeting pathways underlie atrial fibrillation. Circulation. 2011 Sep 13;124(11):1212-22. Epub 2011 Aug 22. PubMed PMID: 21859974; PubMed Central PMCID: PMC3211046.
•Sag CM, Köhler AC, Anderson ME, Backs J, Maier LS. CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes. J Mol Cell Cardiol. 2011 Nov;51(5):749-59. Epub 2011 Jul 26. PubMed PMID: 21819992; PubMed Central PMCID: PMC3226826.
•Swaminathan PD, Purohit A, Soni S, Voigt N, Singh MV, Glukhov AV, Gao Z, He BJ, Luczak ED, Joiner ML, Kutschke W, Yang J, Donahue JK, Weiss RM, Grumbach IM, Ogawa M, Chen PS, Efimov I, Dobrev D, Mohler PJ, Hund TJ, Anderson ME. Oxidized CaMKII causes cardiac sinus node dysfunction in mice. J Clin Invest. 2011 Aug 1;121(8):3277-88. doi: 10.1172/JCI57833. Epub 2011 Jul 25. PubMed PMID: 21785215; PubMed Central PMCID: PMC3223923.
•Erickson JR, He BJ, Grumbach IM, Anderson ME. CaMKII in the cardiovascular system: sensing redox states. Physiol Rev. 2011 Jul;91(3):889-915. Review. PubMed PMID: 21742790.
•Anderson ME. Pathways for CaMKII activation in disease. Heart Rhythm. 2011 Sep;8(9):1501-3. Epub 2011 May 3. PubMed PMID: 21699838; PubMed Central PMCID: PMC3163819.
•Swaminathan PD, Anderson ME. CaMKII inhibition: breaking the cycle of electrical storm? Circulation. 2011 May 24;123(20):2183-6. Epub 2011 May 9. PubMed PMID: 21555705.
•Schulman H, Anderson ME. Ca/Calmodulin-dependent Protein Kinase II in Heart Failure. Drug Discov Today Dis Mech. 2010 Summer;7(2):e117-e122. PubMed PMID: 21503275; PubMed Central PMCID: PMC3077766.
•Zingman LV, Zhu Z, Sierra A, Stepniak E, Burnett CM, Maksymov G, Anderson ME, Coetzee WA, Hodgson-Zingman DM. Exercise-induced expression of cardiacATP-sensitive potassium channels promotes action potential shortening and energy conservation. J Mol Cell Cardiol. 2011 Jul;51(1):72-81. Epub 2011 Mar 23. PubMed PMID: 21439969; PubMed Central PMCID: PMC3103621.
•Gao Z, Singh MV, Hall DD, Koval OM, Luczak ED, Joiner ML, Chen B, Wu Y, Chaudhary AK, Martins JB, Hund TJ, Mohler PJ, Song LS, Anderson ME. Catecholamine-independent heart rate increases require Ca2+/calmodulin-dependent protein kinase II. Circ Arrhythm Electrophysiol. 2011 Jun 1;4(3):379-87. Epub 2011 Mar 15. PubMed PMID: 21406683; PubMed Central PMCID: PMC3116039.
•Singh MV, Anderson ME. Is CaMKII a link between inflammation and hypertrophy in heart? J Mol Med (Berl). 2011 Jun;89(6):537-43. Epub 2011 Jan 29. Review. PubMed PMID: 21279501.
•Anderson ME, Brown JH, Bers DM. CaMKII in myocardial hypertrophy and heart failure. J Mol Cell Cardiol. 2011 Oct;51(4):468-73. Epub 2011 Jan 27. Review. PubMed PMID: 21276796; PubMed Central PMCID: PMC3158288.
•Wagner S, Ruff HM, Weber SL, Bellmann S, Sowa T, Schulte T, Anderson ME, Grandi E, Bers DM, Backs J, Belardinelli L, Maier LS. Reactive oxygen species-activated Ca/calmodulin kinase IIδ is required for late I(Na) augmentation leading to cellular Na and Ca overload. Circ Res. 2011 Mar 4;108(5):555-65. Epub 2011 Jan 20. PubMed PMID: 21252154; PubMed Central PMCID:PMC3065330.
2010
•Hund TJ, Koval OM, Li J, Wright PJ, Qian L, Snyder JS, Gudmundsson H, Kline CF, Davidson NP, Cardona N, Rasband MN, Anderson ME, Mohler PJ. A β(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice. J Clin Invest. 2010 Oct 1;120(10):3508-19
•Yang J, Huang J, Maity B, Gao Z, Lõrca R, Gudmundsson H, Li J, Stewart A, Swaminathan PD, Ibeawuchi SR, Shepherd A, Chen CK, Kutschke W, Mohler PJ, Mohapatra DP, Anderson ME, Fisher RA. RGS6, a Modulator of Parasympathetic Activation in Heart. Circ Res. 2010 Sep 23. [Epub ahead of print]
•Li J, Kline CF, Hund TJ, Anderson ME, Mohler PJ. Ankyrin-B regulates Kir6.2 membrane expression and function in heart J Biol Chem. 2010 Sep 10;285(37):28723-30.
•Wei S, Guo A, Chen B, Kutschke W, Xie YP, Zimmerman K, Weiss RM, Anderson ME, Cheng H, Song LS. T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res. 2010 Aug 20;107(4):520-31.
•Glukhov AV, Fedorov VV, Anderson ME, Mohler PJ, Efimov IR. Functional anatomy of the murine sinus node: high-resolution optical mapping of ankyrin-B heterozygous mice.Am J Physiol Heart Circ Physiol. 2010 Aug;299(2):H482-91.
•Gudmundsson H, Hund TJ, Wright PJ, Kline CF, Snyder JS, Qian L, Koval OM, Cunha SR, George M, Rainey MA, Kashef FE, Dun W, Boyden PA, Anderson ME, Band H, Mohler PJ. EH domain proteins regulate cardiac membrane protein targeting. Circ Res. 2010 Jul 9;107(1):84-95.
•Gao Z, Chen B, Joiner ML, Wu Y, Guan X, Koval OM, Chaudhary AK, Cunha SR, Mohler PJ, Martins JB, Song LS, Anderson ME .I(f) and SR Ca(2+) release both contribute to pacemaker activity in canine sinoatrial node cells. J Mol Cell Cardiol. 2010 Jul;49(1):33-40.
•Witczak CA, Jessen N, Warro DM, Toyoda T, Fujii N, Anderson ME, Hirshman MF, Goodyear LJ. CaMKII regulates contraction- but not insulin-induced glucose uptake in mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2010 Jun;298(6):E1150-60.
•Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 beta-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4996-5000.
•Li H, Li W, Gupta AK, Mohler PJ, Anderson ME, Grumbach IM. Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy. Am J Physiol Heart Circ Physiol. 2010 Feb;298(2):H688-98.
2009
• Singh, M.V., Kapoun, A., Higgins, L., Kutschke, W., Thurman, J.M., Singh, M., Yang, J., Guan, X., Lowe, J., Weiss, R.M., Zimmerman, K., Zhang, R., Yull, F.E., Blackwell, T.S., Mohler, P.J., Anderson, M.E. Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart. J. Clin. Invest. 119(4):986-996, 2009. (Commentary in Nat Med 15:375, 2009)
• Wu Y, Gao Z, Chen B, Koval O, Singh M, Guan X, Hund T, Kutschke WJ, Sarma S, Grumbach I, Wehrens X, Mohler P, Song L, Anderson M.E. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc. Natl. Acad. Sci. 106:5972-5977, 2009. (Commentary in Sci Signaling, 2:ec130, 2009)
• Chelu M, Sarma S, Sood S, Wang S, Oort V, Jeroen R, Skapura D, Li N, Santonastasi M, Mueller F, Schotten U, Anderson ME, Valderrabano M, Dobrev D, Wehrens XHT. Calmodulin kinase II mediated sarcoplasmic reticulum calcium leak promotes atrial fibrillation. J. Clin. Invest. 119(7): 1940-1951, 2009.
• Timmins J, Ozcan L, Seimon TA, Li G, Malagelada C, Backs J, Backs T, Bassel-Duby R, Olson EN, Anderson ME, and Tabas I. Calcium/calmodulin-dependent protein kinase II links endoplasmic reticulum stress with Fas and mitochondrial apoptosis pathways.J. Clin. Invest. 119(10):2925-2941, 2009.
• Chen B, Wu Y, Mohler PJ, Anderson ME, Song L-S. Local control of Ca2+-induced Ca2+ release in mouse sinoatrial node cells. J. Mol. Cell. Cardiol. 47(5):706-715, 2009.
• Kline CF, Kurata HT, Hund TJ, Cunha SR, Koval OM, Wright PJ, Christensen M, Anderson ME, Nichols CG, Mohler PJ. Dual Role of K ATP channel C-terminal motif in membrane targeting and metabolic regulation. Proc. Natl. Acad. Sci. 106 (39):16669-74, 2009.
• Christensen MD, Dun W, Boyden PA, Anderson ME, Mohler PJ, and Hund TJ. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: A computational analysis. PLoS Comput Biol. 2009. (Accepted).
2008
•Erickson JR, Anderson ME. CaMKII and its role in cardiac arrhythmia. JCardiovasc Electrophysiol. 2008 Dec;19(12):1332-6. Epub 2008 Sep 17. PubMed PMID:18803570.
•Thiel WH, Chen B, Hund TJ, Koval OM, Purohit A, Song LS, Mohler PJ, Anderson ME. Proarrhythmic defects in Timothy syndrome require calmodulin kinase II. Circulation. 2008 Nov 25;118(22):2225-34. Epub 2008 Nov 10. PubMed PMID:19001023.
•Le Scouarnec S, Bhasin N, Vieyres C, Hund TJ, Cunha SR, Koval O, Marionneau C, Chen B, Wu Y, Demolombe S, Song LS, Le Marec H, Probst V, Schott JJ, Anderson ME, Mohler PJ. Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease. Proc Natl Acad Sci U S A. 2008 Oct7;105(40):15617-22. Epub 2008 Oct 1. PubMed PMID: 18832177; PubMed Central PMCID: PMC2563133.
•Couchonnal LF, Anderson ME. The role of calmodulin kinase II in myocardial physiology and disease. Physiology (Bethesda). 2008 Jun;23:151-9. Review. PubMed PMID: 18556468.
•Erickson JR, Joiner ML, Guan X, Kutschke W, Yang J, Oddis CV, Bartlett RK, Lowe JS, O’Donnell SE, Aykin-Burns N, Zimmerman MC, Zimmerman K, Ham AJ, Weiss RM, Spitz DR, Shea MA, Colbran RJ, Mohler PJ, Anderson ME. A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation. Cell. 2008 May 2;133(3):462-74. PubMed PMID: 18455987; PubMed Central PMCID: PMC2435269.
•Werdich AA, Lima EA, Dzhura I, Singh MV, Li J, Anderson ME, Baudenbacher FJ. Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies. Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2352-62. Epub 2008 Mar 21. PubMed PMID: 18359893.
•Mohler PJ, Anderson ME. New insights into genetic causes of sinus node disease and atrial fibrillation. J Cardiovasc Electrophysiol. 2008 May;19(5):516-8. Epub 2008 Feb 21. PubMed PMID: 18298510.
•Grueter CE, Abiria SA, Wu Y, Anderson ME, Colbran RJ. Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits. Biochemistry. 2008 Feb12;47(6):1760-7. Epub 2008 Jan 19.
PubMed PMID: 18205403; PubMed Central PMCID: PMC2814322.
•Khoo MS, Grueter CE, Eren M, Yang J, Zhang R, Bass MA, Lwin ST, Mendes LA, Vaughan DE, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition disrupts cardiomyopathic effects of enhanced green fluorescent protein. J Mol Cell Cardiol. 2008 Feb;44(2):405-10.
Epub 2007 Nov 28. PubMed PMID: 18048055; PubMed Central PMCID: PMC2695824.
•Lowe JS, Palygin O, Bhasin N, Hund TJ, Boyden PA, Shibata E, Anderson ME, Mohler PJ. Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J Cell Biol. 2008 Jan 14;180(1):173-86. Epub 2008 Jan7. PubMed PMID: 18180363; PubMed Central PMCID: PMC2213608.
2007
•Khoo MS, Grueter CE, Eren M, Yang J, Zhang R, Bass MA, Lwin ST, Mendes LA, Vaughan DE, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition disrupts cardiomyopathic effects of enhanced green fluorescent protein. J Mol Cell Cardiol. 2008 Feb;44(2):405-10.
Epub 2007 Nov 28. PubMed PMID: 18048055; PubMed Central PMCID: PMC2695824.
•Li J, Marionneau C, Koval O, Zingman L, Mohler PJ, Nerbonne JM, Anderson ME. Calmodulin kinase II inhibition enhances ischemic preconditioning by augmenting ATP-sensitive K+ current. Channels (Austin). 2007 Sep-Oct;1(5):387-94. Epub 2007 Dec 17. PubMed PMID: 18690039.
•Werdich AA, Baudenbacher F, Dzhura I, Jeyakumar LH, Kannankeril PJ, Fleischer S, LeGrone A, Milatovic D, Aschner M, Strauss AW, Anderson ME, Exil VJ. Polymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice. Am J Physiol Heart Circ Physiol. 2007
May;292(5):H2202-11. Epub 2007 Jan 5. PubMed PMID: 17209005. Anderson ME, Mohler PJ. MicroRNA may have macro effect on sudden death. Nat Med. 2007 Apr;13(4):410-1. PubMed PMID: 17415373.
•Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res. 2007 Mar 1;73(4):657-66. Epub 2006 Dec 12. Review. PubMed PMID: 17254559.
•Grimm M, El-Armouche A, Zhang R, Anderson ME, Eschenhagen T. Reduced contractile response to alpha1-adrenergic stimulation in atria from mice with chronic cardiac calmodulin kinase II inhibition. J Mol Cell Cardiol. 2007 Mar;42(3):643-52. Epub 2006 Dec 28. PubMed PMID: 17292391.
•Grueter CE, Colbran RJ, Anderson ME. CaMKII, an emerging molecular driver for calcium homeostasis, arrhythmias, and cardiac dysfunction. J Mol Med. 2007 Jan;85(1):5-14. Epub 2006 Nov 21. Review. PubMed PMID: 17119905.
2006
• Wu Y, Shintani A, Greuter C, Zhang R, Yang J, Kranias EG, Colbran RJ, Anderson ME. Calmodulin kinase II determines dynamic Ca2+ responses in heart. J Mol Cell Cardiol 2006; 40:213-23.
• Yang Y, Zhu WZ, Joiner M-L, Zhang R, Oddis CV, Hou Y, Yang J, Price EE jr, Gleaves L, Erin M, Ni G, Vaughn DE, Xiao R-P, Anderson ME. Calmodulin kinase inhibition protects against myocardial apoptosis in vivo. Am J Physiol 2006; 291:H3065-H3075.
•Kannankeril PJ, Mitchell BM, Goonasekera SA, Chelu MG, Zhang W, Sood S, Kearney DL, Danila CI, De Biasi M, Pautler RG, Roden DM, Taffet GE, Dirksen RT, Anderson ME, Hamilton SL. Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and mild cardiomyopathy. Proc Natl Acad Sci 2006; 103:12179-12184.
• Khoo MSC, Zhang R, Ni G, Greuter C, Yang Y, Zhang W, Mendes L, Olson EN, Colbran RJ, Anderson ME. Death, cardiac dysfunction and arrhythmias due to up-regulation of calmodulin kinase II in calcineurin-induced cardiomyopathy. Circulation 2006; 114:1352-1359. Published with an accompanying editorial.
• Grueter CE, Abiria SA, Dzhura I, Wu Y, Hamm A-J, Mohler PJ, Anderson ME, Colbran RJ. Molecular basis for facilitation of native Ca2+ channels by CaMKII. Mol Cell 2006; 23:641-650. Selected as a recommended citation by the Faculty of 1000 Biology.
• Li J, Shah V, Hell J, Nerbonne JM, Anderson ME. Calmodulin kinase II inhibition shortens action potential duration by up-regulation of K+ currents. Circ Res 2006; 99:1092-1099. PMID: 17038644. Published with an accompanying editorial.
•Anderson ME, Higgins, LS, Schulman H. Disease mechanisms and emerging therapies: Protein kinases and their inhibitors in myocardial disease. Nature Clin Prac 2006; 3:437-445.
III. Therapeutic Implications of Pharmacological Agents for Cardiac Contractility Dysfunction: “The Fire From Within The Biggest Ca2+ Channel Erupts and Dribbles” by Anderson, ME
Author: Justin D Pearlman, MD, PhD, FACC PENDING –
Therapeutic Implications of these physiological research discoveries
JDP: RECOMMEND SPLIT TO TWO: a. contractility b. arrhythmia
IV. Selective Research Contributions on Calcium Release-related Contractile Dysfunction
Curator: Aviva Lev-Ari, PhD, RN
Summary
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
Author: Larry H Bernstein, MD, FCAP
PENDING
V. Bibliography on Calcium Release Mechanisms in Vascular Smooth Muscle, in Cardiomyocytes and the Role in Heart Failure
Curator: Aviva Lev-Ari, PhD, RN
Anderson ME, General Hospital Iowa City and University of Iowa
Wilson S. Colucci, MD, Heart Failure Lab at BMC
William Gregory Stevenson, M.D.Heart Failure Lab at BWH
Introduction to Calcium Release Mechanism in Vascular Smooth Muscle and in Cardiomyocytes
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
I. Cellular Contractility Capacity — Actin, Cellular Dynamics and Calcium Efflux: Emergence of the Calcium Release-related Contractile Dysfunction
Author: Justin D Pearlman, MD, PhD, FACC
The pumping action of the heart is mediated by repeated cycles of the release and re-uptake of calcium stored within cardiac myocytes. Similar to skeletal muscle function, the protein complex of actinomycin creates mechanical motion when calcium interacts with the threads of the protein strand tropomyosin which are wound around an actin protein filament with the third protein troponin strung out like beads along the string. Calcium (Ca++) released from the storage space (sarcoplasmic reticulum) combines with troponin to actuate a shift in the tropomyosin threads, exposing myosin binding sites to adenosinetriphosphate (ATP, the energy source), which, in turn, consume the high-energy bond of ATP and concommitantly break and make cross-bridges resulting in shifted position (filament sliding, contraction). The spiral layers of these filaments within the heart result in a reduction of chamber size. Normally the two atrial chambers contract first, to boost the load of blood in the ventricles, then the ventricles contract, relying on one-way valves to impose a forward direction to the blood ejected from the heart.
There is barely enough ATP around to complete a single heart beat, so ATP is replenished from a higher energy storage form, phosphocreatine (PCr, aka creatinephosphate), which in turn in reconstituted during the relaxation phase of the heart (low pressure) when oxygenated blood, glucose, and fatty acids are delivered to local mitochondria to restock energy stores. Thus the contraction cycle, unlike a continual pump, provides low pressure respite after each high pressure contraction, which facilitates delivery of oxygenated nutrient blood to the heart muscle to replenish its energy for the action. When switching to a mechanical total heart replacement, it is not necessary to preserve the pulsatile pattern, which primarily serves to facilitate energizing the biologic pump.
The volume of blood ejected by the left ventricle from a single heart beat is called the stroke volume (SV). The amount of blood in the left ventricle just before the heart beat is called the end-diastolic volume (EDV), and just after, the end-systolic volume (ESV), so SV=EDV-ESV. The portion of the filled left ventricle that gets pumped forward through the aortic valve by a single heart beat is called the ejection fraction (EF). Thus EF = SV/EDV, expressed as a percentage. The cardiac output (CO) in liters/minute is simply the product of stroke volume and heart rate (HR): CO = SV x HR.
Heart failure has three clinical forms: high output failure, systolic failure and diastolic heart failure. With high output failure (elevated SV x HR), the demands of the body are elevated beyond the normal capacity of the heart to supply cardiac output. With systolic failure (low EF) the pumping action of the heart is insufficient to meet the needs of fresh blood delivery to the various organs of the body (including in particular the heart, brain, liver, and kidneys). Note that the heart does not draw any significant nutrients or oxygen from the blood in its chambers – rather, it is first in line after the oxygenated blood is pumped out through the aortic valve to tax 10% of the cardiac output via the coronary arteries. In diastolic failure, the LV resists filling (stiff LV) so the back pressure to the lungs is elevated, resulting in pulmonary congestion. Many textbooks incorrectly describe diastolic heart failure as heart failure with a normal EF; however, that would imply that diastolic heart failure (stiff LV) can be “cured” by a myocardial infarction (heart attack) so that the EF drops. Contrary to that mistaken description, the addition of reduced EF to a patient with diastolic heart failure results in combined systolic and diastolic heart failure. Inadequate delivery of blood from low EF has been called “forward failure” and pulmonary congestion from a stiff LV “backward failure” but those terms are not synonymous with systolic and diastolic failure, as low EF also contributes to congestive heart failure, and stiff LV can impede adequate filling, so each has components for forward and backward failure.
One can plot a curve relating stroke volume to the end diastolic volume, called the “Frank-Starling curve” whereby an increase in EDV is generally accommodated by an increase in SV. That adaptive feature is achieved by a stimulation of calcium-mediated increase in contractility (speed and strength of contraction) . In heart failure, the usual amounts of calcium stores are not adequate to meet the demands. Consequently, remodeling occurs, which includes reversion towards a fetal phenotype in which the sarcoplasmic reticulum stores and releases a greater amount of calcium. While this does result in some augmentation of contractility, it occurs at a cost. The higher levels of calcium can interfere with mitochondrial function and reduce the energy efficiency of oxygen replenishment of phosphocreatine and ATP. In research by the author of this section (JDP), the timing of oxygen uptake and utilization is adversely affected by this remodeling, as demonstrated by oxygen uptake sensitive dynamic cardiac MRI.
Thus strategies to genetically re-engineer cardiac function by modifying calcium uptake and release to elevate contractility at a given workload have potentially harmful consequences in terms of lowering the energy efficiency of the heart. If the blood supply of the heart is good (non-ischemic heart failure), one can expect opportunities for benefit. However, if the blood supply to the heart is limited (ischemic heart failure), such changes may be detrimental. Furthermore, the impediments to mitochondrial function may contribute to other adverse effects of remodeling, including in particular activation of fibrosis (adverse remodeling promoting worsened diastolic failure).
II. Integration and Interpretation of Research Results in Two Labs: Mark E Anderson’s and Roger Hajjar’s Lab
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
III. Therapeutic Implications of Pharmacological Agents for Cardiac Contractility Dysfunction: “The Fire From Within The Biggest Ca2+ Channel Erupts and Dribbles” by Anderson, ME
Negative inotropic agents decrease myocardial contractility, and are used to decrease cardiac workload in conditions such as angina. While negative inotropism may precipitate or exacerbate heart failure, certain beta blockers (e.g. carvedilol, bisoprolol and metoprolol) have been believed to reduce morbidity and mortality in congestive heart failure. Quite recently, however, the effectiveness of beta blockers has come under renewed critical scientific scrutiny.
The above list of inotropic agents consists of agents developed to increase the contractile force of the heart and have had a long history of use. Even though they have been proved valid, they are not part of the specific advances that we are seeing that justifies a cardiology specialty in cardiac electrophysiology, the disorders, and the treatments. The developments we now witness were unknown and perhaps unexpected a quarter of a century ago. The methods required to understand the myocardiocyte were not yet developed. Our understanding is now based on a refined knowledge of the Ca(2+) release mechanism between the sarcomere and the myocyte cytoplasm, the Ca(2+) transport, the ion pores, the role of RyR2 and the phosphorylation of the Ca(2+) release mechanism. This and more will lead to far better therapeutic advances in the next few years based on earlier detection of changes preceding heart failure, and the possibility of treatments for potential life-threatening arrhythmias will be averted.
IV. Selective Research Contributions on Calcium Release-related Contractile Dysfunction
Department of Medicine, University Hospital Zurich, Switzerland.
Abstract
In epidemiological surveys and in large-scale therapeutic trials, the prognosis of patients with ischemic heart failure is worse than in patients with a non-ischemic etiology. Even heart transplant candidates may respond better to intensified therapy if they have non-ischemic heart failure. The term ‘non-ischemic heart failure’ includes various subgroups such as hypertensive heart disease, myocarditis, alcoholic cardiomyopathy and cardiac dysfunction due to rapid atrial fibrillation. Some of these causes are reversible. The therapeutic effect of essential drugs such as angiotensin-converting enzyme inhibitors, beta-blockers and diuretics does not, in general, significantly differ between ischemic and non-ischemic heart failure. However, in some trials, response to certain drugs (digoxin, tumor necrosis factor-alpha, inhibition with pentoxifylline, growth hormone and amiodarone) was found to be better in non-ischemic patients. Patients with ischemic heart failure and non-contracting ischemic viable myocardium may, on the other hand, considerably improve following revascularization. In view of prognostic and possible therapeutic differences, the etiology of heart failure should be determined routinely in all patients. http://www.ncbi.nlm.nih.gov/pubmed/12634896
Upregulation of β3-Adrenoceptors and Altered Contractile Response to Inotropic Amines in Human Failing Myocardium
From the Department of Medicine, Unit of Pharmacology and Therapeutics, University of Louvain Medical School (S.M., O.F., J.-L.B.), Brussels, Belgium; INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires (J.-N.T., C.G.) and Faculté des Sciences et Techniques (C.G.), Nantes, France; and Department of Pathology, Brigham and Women’s Hospital, and Physiology Program, Harvard School of Public Health (L.K.), Boston, Mass.
Correspondence to Jean-Luc Balligand, Department of Medicine, Unit of Pharmacology and Therapeutics, FATH 5349, University of Louvain Medical School, 53 avenue Mounier, B1200 Brussels, Belgium, e-mail Balligand@mint.ucl.ac.be; or Chantal Gauthier, INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires, 44093 Nantes, France,
Abstract
Background—Contrary to β1– and β2-adrenoceptors, β3-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of β3-adrenoceptors in nonfailing and failing human hearts.
Methods and Results—We analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6±2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2±3%). β3-Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to β1-adrenoceptor mRNA, Western blot analysis of β3-adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for Gαi-2 proteins that couple β3-adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of β3-preferential agonists was only mildly reduced.
Conclusions—Opposite changes occur in β1– and β3-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.
Increased beta-receptor density and improved hemodynamic response to catecholamine stimulation during long-term metoprolol therapy in heart failure from dilated cardiomyopathy.
Cardiology Division, Stanford University School of Medicine, CA.
Abstract
Severe heart failure is associated with a reduction in myocardial beta-adrenergic receptor density and an impaired contractile response to catecholamine stimulation. Metoprolol was administered during a 6-month period to 14 patients with dilated cardiomyopathy to examine its effects on these abnormalities. The mean daily dose of metoprolol for the group was 105 mg (range, 75-150 mg). Myocardial beta-receptor density, resting hemodynamic output, and peak left ventricular dP/dt response to dobutamine infusions were compared in 9, 14, and 7 patients, respectively, before and after 6 months of metoprolol therapy while the patients were on therapy. The second hemodynamic study was performed 1-2 hours after the morning dose of metoprolol had been given. Myocardial beta-receptor density increased from 39 +/- 7 to 80 +/- 12 fmol/mg (p less than 0.05). Resting hemodynamic output showed a rise in stroke work index from 27 +/- 4 to 43 +/- 3 g/m/m2, p less than 0.05, and ejection fraction rose from 0.26 +/- 0.03 to 0.39 +/- 0.03 after 6 months of metoprolol therapy, p less than 0.05. Before metoprolol therapy, dobutamine caused a 21 +/- 4% increase in peak positive left ventricular dP/dt; during metoprolol therapy, the same dobutamine infusion rate increased peak positive dP/dt by 74 +/- 18% (p less than 0.05). Thus, long-term metoprolol therapy is associated with an increase in myocardial beta-receptor density, significant improvement in resting hemodynamic output, and improved contractile response to catecholamine stimulation. These changes indicate a restoration of beta-adrenergic sensitivity associated with metoprolol therapy, possibly related to the observed up-regulation of beta-adrenergic receptors.
Ryanopathy: causes and manifestations of RyR2 dysfunction in heart failure
Belevych AE, Radwański PB, Carnes CA, Györke S. College of Medicine, The Ohio State University, Columbus, OH. Cardiovasc Res. 2013; 98(2):240-7. doi: 10.1093/cvr/cvt024. Epub 2013 Feb 12. PMID: 23408344 PMCID: PMC3633158 [Available on 2014/5/1] The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodeling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
Circ Res. 2005 Dec 9;97(12):1314-22. Epub 2005 Nov 3.
Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure.
Department of Medicine, University of Illinois at Chicago, IL 60612, USA.
Abstract
Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.
Editorial Comment on the above article abstract made by Anderson, ME
From the University of Iowa, Carver College of Medicine, Iowa City.
Correspondence to Mark E. Anderson, MD, PhD, University of Iowa, Carver College ofMedicine, 200 Hawkins Drive, Room E 315 GH, Iowa City, IA 53342-1081. E-mail mark-e-anderson@uiowa.edu
CaMKII Is a Pluripotent Signaling Molecule in Heart
The multifunctional Ca2+ and calmodulin (CaM)-dependent protein kinase II (CaMKII) is a serine threonine kinase that is abundant in heart where it phosphorylates Ca2+ihomeostatic proteins. It seems likely that CaMKII plays an important role in cardiac physiology because these target proteins significantly overlap with the more extensively studied serine threonine kinase, protein kinase A (PKA), which is a key arbiter of catecholamine responses in heart. However, the physiological functions of CaMKII remain poorly understood, whereas the potential role of CaMKII in signaling myocardial dysfunction and arrhythmias has become an area of intense focus. CaMKII activity and expression are upregulated in failing human hearts and in many animal models of structural heart disease.1 CaMKII inhibitory drugs can prevent cardiac arrhythmias2,3 and suppress afterdepolarizations4 that are a probable proximate focal cause of arrhythmias in heart failure. CaMKII inhibition in mice reduces left ventricular dilation and prevents disordered intracellular Ca2+ (Ca2+i) homeostasis after myocardial infarction.5 CaMKII overexpression in mouse heart causes severe cardiac hypertrophy, dysfunction, and sudden death that is heralded by increased SR Ca2+ leak6; these findings go a long way to making a case for CaMKII as a causative signal in heart disease and arrhythmias but do not identify critical molecular targets or test the potential role of CaMKII in a large non-rodent animal model. The work by Ai et al in this issue of Circulation Research makes an important contribution by demonstrating CaMKII upregulation causes increased Ca2+ leak from ryanodine receptor (RyR) Ca2+ release channels in a clinically-relevant model of structural heart disease.7
Ryanodine Receptors Are Central
Ca2+i release controls cardiac contraction, and most of the Ca2+i for contraction is released from the intracellular sarcoplasmic reticulum (SR) through ryanodine receptors (RyR). RyRs are huge proteins (565 kDa) that assemble with a fourfold symmetry to form a functional Ca2+ release channel. Approximately 90% of the RyR is not directly required to form the pore but instead protrudes into the cytoplasm where it binds numerous proteins, including PKA, CaMKII, CaM, and FK12.6 (calstabin). Cardiac contraction is initiated when Ca2+ current (ICa), through sarcolemmal L-type Ca2+ channels (LTCC), triggers RyR opening by a Ca2+-induced Ca2+ release (CICR) mechanism. LTCCs “face off” with RyRs across a highly ordered cytoplasmic cleft that delineates a kind of Ca2+furnace during each CICR-initiated heart beat (Figure). CICR has an obvious need to function reliably, so it is astounding to consider how this feed forward process is intrinsically unstable. The increased instability of CICR in heart failure is directly relevant to arrhythmias initiated by afterdepolarizations. RyRs partly rely on a collaboration of Ca2+-sensing proteins in the SR lumen to grade their opening probability and the amount of SR Ca2+ release to a given ICa stimulus. Thus the SR Ca2+ content is an important parameter for setting the inotropic state, and heart failure is generally a condition of reduced SR Ca2+ content and diminished myocardial contraction.
Ca2+-induced Ca2+ release (CICR) in health and disease. Each heart beat is initiated by cell membrane depolarization that opens Ca2+channels. The Ca2+ current (ICa) induces ryanodine receptor (RyR) opening that allows release of myofilament activating Ca2+ for contraction. In healthy CICR, RyRs close during diastole while Ca2+ is removed from the cytoplasm by uptake into the sarcoplasmic reticulum (SR). In heart failure the SR has reduced Ca2+ content so that the amount of Ca2+ released to the myofilaments is smaller than in health. RyR hyperphosphorylation by CaMKII promotes repetitive RyR openings leading to a Ca2+ leak in diastole. This leak contributes to the reduction in SR Ca2+ content and can engage the electrogenic Na+-Ca2+ exchanger to trigger afterdepolarizations and arrhythmias.
Kinases Facilitate Communication Between LTCCs and RyRs
LTCCs and RyRs form the protein machinery for initiating contraction in cardiac and skeletal muscle, but in cardiac muscle communication between these proteins occurs without a requirement for physical contact. PKA is preassociated with LTCCs and RyRs, and PKA-dependent phosphorylation increases LTCC8 and RyR9opening. The resultant increase in Ca2+i is an important reason for the positive inotropic response to cathecholamines. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by increased Ca2+I, and so catecholamine stimulation activates CaMKII in addition to PKA.5 In contrast to PKA, which is tightly linked to inotropy, CaMKII inhibition does not cause a reduction in fractional shortening during acute catecholamine stimulation in mice.5 Prolonged catecholamine exposure does reduce contractile function by uncertain mechanisms that require CaMKII.10 CaMKII colocalizes with LTCCs11 and RyRs,12 and CaMKII can also increase LTCC13 and RyR12 opening probability in cardiac myocytes. The ultrastructural environment of LTCCs and RyRs is well-suited for a Ca2+i-responsive kinase to serve as a coordinating signal between LTCCs and RyRs during CICR. The recently identified role of CaMKII in heart failure suggests the possibility that excessive CaMKII activity could cause or contribute to CICR defects present in heart failure
Heart Failure Is a Disease of Disordered Ca2+i Homeostasis
The key clinical phenotypes of contractile dysfunction and electrical instability in heart failure involve problems with Ca2+i homeostasis. Broad changes in Ca2+I-handling proteins can occur in various heart failure models, but in general heart failure is marked by a reduction in the capacity for SR Ca2+ uptake, enhanced activity of the sarcolemmal Na+-Ca2+ exchanger, and reduction in CICR-coordinated SR Ca2+ release. On the other hand, the opening probability of individual LTCCs is increased in human heart failure,14suggesting that posttranslational modifications may also be mechanistically important for understanding these Ca2+i disturbances at Ca2+ homeostatic proteins.
Is Heart Failure a Disease of Enzymatic Over-Activity?
Heart failure is marked by hyper-adrenergic tone, and beta adrenergic receptor antagonist drugs (beta blockers) are a mainstay of therapy for reducing mortality in heart failure patients. The Marks group pioneered the concept that RyRs are hyperphosphorylated by PKA in patients with heart failure and showed that successful therapies, ranging from beta blockers to left ventricular assist devices, reduce RyR phosphorylation in step with improved mechanical function. They have developed a large body of evidence in patients and in animal models that PKA phosphorylation of Ser2809 on cardiac RyRs destabilizes binding of FK12.6 to RyRs and promotes increased RyR opening that causes an insidious Ca2+ leak. This leak is potentially problematic because it can reduce SR Ca2+ content (to depress inotropy), engage pathological Ca2+-dependent transcriptional programs (to promote myocyte hypertrophy), and activate arrhythmia-initiating afterdepolarizations (to cause sudden death). Indeed, RyR hyperphosphorylation can produce arrhythmias as well as mechanical dysfunction, whereas a drug that prevents FK12.6 dissociation from RyR also reduces or prevents arrhythmias.15 Taken together these findings make a strong case that RyR hyperphosphorylation (a result of net excess kinase activity) is a central event in heart failure and sudden death.
Not all findings point to hyperphosphorylation of RyR by PKA and subsequent FK12.6 dissociation as critical determinants of heart failure16 and arrhythmias.17 For example, studies in isolated and permeabilized ventricular myocytes failed to show an increase in RyR openings, called sparks, which are monitored by photoemission of a Ca2+-sensitive fluorescent dye.18 FKBP12.6 dissociation is not universally reported to follow RyR phosphorylation by PKA.19 Furthermore, FKBP12.6 binding to RyR is not affected during catecholamine stimulation that results in arrhythmias in a mouse model of catecholamine-induced ventricular tachycardia,20,21 a genetic disorder of hypersensitive RyR Ca2+release. These findings challenge the PKA hypothesis and make room, conceptually, to consider the role of additional signals for modulating RyR activity in heart disease.
Both PKA and CaMKII may phosphorylate Ser2809, but recently CaMKII was found to exclusively phosphorylate Ser2815 and this phosphorylation caused increased RyR opening.12 However, the PKA and CaMKII responses may be mechanistically distinct because CaMKII evoked increased RyR opening in the absence of FK12.6 dissociation. These findings together with the fact that CaMKII activity is recruited under conditions of increased PKA activity suggest that CaMKII might also be important in regulating RyRs in heart failure.
The article by Ai et al shows that expression of a CaMKII splice variant that is resident in cytoplasm (CaMKIIδc) was increased, and there was enhanced phosphorylation of the recently identified CaMKII site (Ser2815) on RyR. Both Ser2815 and the PKA site (Ser2809) were hyperphosphorylated in failing hearts, but phosphorylation of the CaMKII site was greater than the PKA site. Because both Ser2809 and Ser2815 can increase RyR openings, it seemed likely that PKA and CaMKII would work together to increase Ca2+leak. Surprisingly, CaMKII inhibition but not PKA inhibition suppressed the leak. These experiments were performed with meticulous attention to matching SR Ca2+ load, a technically difficult accomplishment that is not performed by most groups evaluating SR Ca2+ release. Thus, differences in the SR intraluminal Ca2+ could not account for these findings. Although these experiments were carefully controlled, one potential limitation is that the experiments relied exclusively on CaMKII and PKA inhibitor drugs that are notorious for nonspecific actions at ion channel proteins. They also showed that the ratio of inositol tris phosphate receptors (IP3R) to RyRs was increased in failing left ventricular myocytes. IP3R are important for regulating Ca2+i in many cells types, including atrial myocytes, but their role in ventricle remains uncertain. The finding that the IP3R are increased at the expense of RyR suggests that Ca2+i release sites are fundamentally reordered in heart failure but leaves the impact of this change untested. IP3R are also a target for CaMKII, so interesting questions remain about the potential role for this channel and CaMKII in heart failure, at least in this model.
What We Learned and What We Need to Know
CaMKII activity seems to be part and parcel of the adrenergic signaling seen in structural heart disease. This work shows us that CaMKII can contribute directly to increased SR Ca2+ leak in a clinically relevant model of heart failure that is marked by arrhythmias and sudden death.22 Acute experiments with CaMKII inhibitory drugs strongly suggest that SR Ca2+ leak is principally linked to CaMKII rather than PKA activity. Excessive SR Ca2+ release can activate inward (forward mode) Na+-Ca2+ exchanger current to cause delayed afterdepolarizations and arrhythmias and CaMKII inhibition can prevent these inward Na+-Ca2+ exchanger currents.23 An important next step toward translating these findings will be to evaluate the effects of chronic CaMKII inhibition in this model to see whether it reverses cardiac dysfunction, arrhythmias, and whether chronic CaMKII inhibitor therapy can stop the RyR leak to refill the SR. It will be necessary to have improved pharmacological agents with fewer nonspecific effects to convincingly perform these experiments. These future experiments will tell us whether CaMKII inhibition is a potentially viable therapy for structural heart disease and arrhythmias in a non-genetic non-mouse model. We need to know whether CaMKII inhibition is really a highly-specific form of beta blockade that can preserve inotropic responses to catecholamines while preventing the adverse consequences of catecholamines in heart failure.5
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL070250, HL62494, and HL046681). Dr Anderson is an Established Investigator of the American Heart Association.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association
Mazur A, Roden DM, Anderson ME. Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits. Circulation. 1999; 100: 2437–2442.
Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble RW, Roden DM, Passier R, Olson EN, Colbran RJ, Anderson ME. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation. 2002; 106: 1288–1293.
Anderson ME, Braun AP, Wu Y, Lu T, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharm Exp Ther. 1998; 287: 996–1006.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nature Med. 2005; 11:409–417.
Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM Ca2+/-calmodulin-dependent protein kinase modulates cardiac RyR2 phosphorylation and SR Ca2+leak in heart failure. Circ Res. 2005; 97: 1314–1322.
Yue DT, Herzig S, Marban E. Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Nat Acad Sci U S A.1990; 87: 753–757.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): Defective regulation in failing hearts. Cell. 2000; 101:365–376.
Wehrens XH, Lehnart SE, Reiken SR, Marks AR. Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res.2004; 94: e61–e70.
Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L, Schwinger RH, Weil J, Herzig S. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation.1998; 98: 969–976.
Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, Coromilas J, Landry DW, Marks AR. Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2. Science. 2004; 304: 292–296.
Houser SR. Can novel therapies for arrhythmias caused by spontaneous sarcoplasmic reticulum Ca2+ release be developed using mouse models? Circ Res.2005; 96: 1031–1032.
Li Y, Kranias EG, Mignery GA, Bers DM. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes.Circ Res. 2002; 90: 309–316.
Xiao B, Sutherland C, Walsh MP, Chen SR. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res. 2004; 94: 487–495.
Cerrone M, Colombi B, Santoro M, di Barletta MR, Scelsi M, Villani L, Napolitano C, Priori SG. Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor. Circ Res. 2005;96: e77–e82.
Wu Y, Roden DM, Anderson ME. Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current. Circ Res. 1999; 84:906–912.
Clinical Profile Head, Department of Internal Medicine Director, Cardiovascular Research Center Professor of Internal Medicine – Cardiovascular Medicine Professor of Molecular Physiology and Biophysics
Dr. Anderson is clinically trained as a cardiac electrophysiologist. His research is focused on cellular signaling and ionic mechanisms that cause heart failure and sudden cardiac death. The multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII) is upregulated in heart disease and arrhythmias. Work in the Anderson laboratory implicates CaMKII as a signal that drives myocardial hypertrophy, apoptosis, mechanical dysfunction and electrical instability. The laboratory work ranges from molecular structure activity analysis of CaMKII to systems physiology using genetically modified mice to dissect cellular mechanisms of CaMKII signaling in heart. http://www.medicine.uiowa.edu/dept_primary_apr.aspx?appointment=Internal%20Medicine&id=andersonmar
Khoo MS, Li J, Singh MV, Yang Y, Kannankeril P, Wu Y, Grueter CE, Guan X, Oddis CV, Zhang R, Mendes L, Ni G, Madu EC, Yang J, Bass M, Gomez RJ, Wadzinski BE, Olson EN, Colbran RJ, Anderson ME. Circulation. 2006 Sep 26;114(13):1352-9. Epub 2006 Sep 18.
PMID:
16982937 [PubMed – indexed for MEDLINE] Free Article
Yang Y, Zhu WZ, Joiner ML, Zhang R, Oddis CV, Hou Y, Yang J, Price EE, Gleaves L, Eren M, Ni G, Vaughan DE, Xiao RP, Anderson ME. Am J Physiol Heart Circ Physiol. 2006 Dec;291(6):H3065-75. Epub 2006 Jul 21.
PMID:
16861697 [PubMed – indexed for MEDLINE] Free Article
Joiner ML, Koval OM, Li J, He BJ, Allamargot C, Gao Z, Luczak ED, Hall DD, Fink BD, Chen B, Yang J, Moore SA, Scholz TD, Strack S, Mohler PJ, Sivitz WI, Song LS, Anderson ME. Nature. 2012 Nov 8;491(7423):269-73. doi: 10.1038/nature11444. Epub 2012 Oct 10.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE Jr, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Nat Med. 2005 Apr;11(4):409-17. Epub 2005 Mar 27.
Werdich AA, Lima EA, Dzhura I, Singh MV, Li J, Anderson ME, Baudenbacher FJ. Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2352-62. doi: 10.1152/ajpheart.01398.2006. Epub 2008 Mar 21.
PMID:
18359893 [PubMed – indexed for MEDLINE] Free Article
Qian H, Matt L, Zhang M, Nguyen M, Patriarchi T, Koval OM, Anderson ME, He K, Lee HK, Hell JW. J Neurophysiol. 2012 May;107(10):2703-12. doi: 10.1152/jn.00374.2011. Epub 2012 Feb 15.
Publications by Prof. Wilson S. Colucci, MD on Heart Failure
Wilson S. Colucci, MD
Title
Professor
Institution
Boston University School of Medicine
Department
Medicine
Division
Cardiovascular Medicine
Address
75 E. Newton St Boston, MA 02118
Telephone
(617) 638-8706
Title
Chief – Section of Medicine, Cardiovascular Medicine
Institution
Boston University School of Medicine
Department
Medicine
Division
Cardiovascular Medicine
1.
Qin F, Siwik DA, Lancel S, Zhang J, Kuster GM, Luptak I, Wang L, Tong X, Kang YJ, Cohen RA, Colucci WS. Hydrogen Peroxide-Mediated SERCA Cysteine 674 Oxidation Contributes to Impaired Cardiac Myocyte Relaxation in Senescent Mouse Heart. J Am Heart Assoc. 2013; 2(4):e000184.
Gopal DM, Kommineni M, Ayalon N, Koelbl C, Ayalon R, Biolo A, Dember LM, Downing J, Siwik DA, Liang CS, Colucci WS. Relationship of plasma galectin-3 to renal function in patients with heart failure: effects of clinical status, pathophysiology of heart failure, and presence or absence of heart failure. J Am Heart Assoc. 2012 Oct; 1(5):e000760.
Calamaras TD, Lee C, Lan F, Ido Y, Siwik DA, Colucci WS. Post-translational Modification of Serine/Threonine Kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at Lysine Residue 97 Directly Inhibits Kinase Activity. J Biol Chem. 2012 Dec 7; 287(50):42400-6.
Kivikko M, Nieminen MS, Pollesello P, Pohjanjousi P, Colucci WS, Teerlink JR, Mebazaa A. The clinical effects of levosimendan are not attenuated by sulfonylureas. Scand Cardiovasc J. 2012 Dec; 46(6):330-8.
Qin F, Siwik DA, Luptak I, Hou X, Wang L, Higuchi A, Weisbrod RM, Ouchi N, Tu VH, Calamaras TD, Miller EJ, Verbeuren TJ, Walsh K, Cohen RA, Colucci WS. The polyphenols resveratrol and s17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice. Circulation. 2012 Apr 10; 125(14):1757-64.
Mazzini M, Tadros T, Siwik D, Joseph L, Bristow M, Qin F, Cohen R, Monahan K, Klein M, Colucci W. Primary carnitine deficiency and sudden death: in vivo evidence of myocardial lipid peroxidation and sulfonylation of sarcoendoplasmic reticulum calcium ATPase 2. Cardiology. 2011; 120(1):52-8.
Schulze PC, Biolo A, Gopal D, Shahzad K, Balog J, Fish M, Siwik D, Colucci WS. Dynamics in insulin resistance and plasma levels of adipokines in patients with acute decompensated and chronic stable heart failure. J Card Fail. 2011 Dec; 17(12):1004-11.
Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, Zsebo KM, Dittrich H, Hajjar RJ. Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation. 2011 Jul 19; 124(3):304-13.
Kivikko M, Sundberg S, Karlsson MO, Pohjanjousi P, Colucci WS. Acetylation status does not affect levosimendan’s hemodynamic effects in heart failure patients. Scand Cardiovasc J. 2011 Apr; 45(2):86-90.
Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, Shi H, Vincent J, Pocock SJ, Pitt B. Eplerenone in patients with systolic heart failure and mild symptoms. N Engl J Med. 2011 Jan 6; 364(1):11-21.
Qin F, Lennon-Edwards S, Lancel S, Biolo A, Siwik DA, Pimentel DR, Dorn GW, Kang YJ, Colucci WS. Cardiac-specific overexpression of catalase identifies hydrogen peroxide-dependent and -independent phases of myocardial remodeling and prevents the progression to overt heart failure in G(alpha)q-overexpressing transgenic mice. Circ Heart Fail. 2010 Mar; 3(2):306-13.
Dhingra R, Pencina MJ, Schrader P, Wang TJ, Levy D, Pencina K, Siwik DA, Colucci WS, Benjamin EJ, Vasan RS. Relations of matrix remodeling biomarkers to blood pressure progression and incidence of hypertension in the community. Circulation. 2009 Mar 3; 119(8):1101-7.
Biolo A, Greferath R, Siwik DA, Qin F, Valsky E, Fylaktakidou KC, Pothukanuri S, Duarte CD, Schwarz RP, Lehn JM, Nicolau C, Colucci WS. Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate. Proc Natl Acad Sci U S A. 2009 Feb 10; 106(6):1926-9.
Brooks WW, Conrad CH, Robinson KG, Colucci WS, Bing OH. L-arginine fails to prevent ventricular remodeling and heart failure in the spontaneously hypertensive rat. Am J Hypertens. 2009 Feb; 22(2):228-34.
Holubarsch CJ, Colucci WS, Meinertz T, Gaus W, Tendera M. The efficacy and safety of Crataegus extract WS 1442 in patients with heart failure: the SPICE trial. Eur J Heart Fail. 2008 Dec; 10(12):1255-63.
Olshansky B, Sabbah HN, Hauptman PJ, Colucci WS. Parasympathetic nervous system and heart failure: pathophysiology and potential implications for therapy. Circulation. 2008 Aug 19; 118(8):863-71.
Hare JM, Mangal B, Brown J, Fisher C, Freudenberger R, Colucci WS, Mann DL, Liu P, Givertz MM, Schwarz RP. Impact of oxypurinol in patients with symptomatic heart failure. Results of the OPT-CHF study. J Am Coll Cardiol. 2008 Jun 17; 51(24):2301-9.
Fonarow GC, Lukas MA, Robertson M, Colucci WS, Dargie HJ. Effects of carvedilol early after myocardial infarction: analysis of the first 30 days in Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction (CAPRICORN). Am Heart J. 2007 Oct; 154(4):637-44.
Wang TJ, Larson MG, Benjamin EJ, Siwik DA, Safa R, Guo CY, Corey D, Sundstrom J, Sawyer DB, Colucci WS, Vasan RS. Clinical and echocardiographic correlates of plasma procollagen type III amino-terminal peptide levels in the community. Am Heart J. 2007 Aug; 154(2):291-7.
Torre-Amione G, Bourge RC, Colucci WS, Greenberg B, Pratt C, Rouleau JL, Sestier F, Moyé LA, Geddes JA, Nemet AJ, Young JB. A study to assess the effects of a broad-spectrum immune modulatory therapy on mortality and morbidity in patients with chronic heart failure: the ACCLAIM trial rationale and design. Can J Cardiol. 2007 Apr; 23(5):369-76.
Shibata R, Izumiya Y, Sato K, Papanicolaou K, Kihara S, Colucci WS, Sam F, Ouchi N, Walsh K. Adiponectin protects against the development of systolic dysfunction following myocardial infarction. J Mol Cell Cardiol. 2007 Jun; 42(6):1065-74.
Givertz MM, Andreou C, Conrad CH, Colucci WS. Direct myocardial effects of levosimendan in humans with left ventricular dysfunction: alteration of force-frequency and relaxation-frequency relationships. Circulation. 2007 Mar 13; 115(10):1218-24.
Kuster GM, Siwik DA, Pimentel DR, Colucci WS. Role of reversible, thioredoxin-sensitive oxidative protein modifications in cardiac myocytes. Antioxid Redox Signal. 2006 Nov-Dec; 8(11-12):2153-9.
Arnlöv J, Evans JC, Benjamin EJ, Larson MG, Levy D, Sutherland P, Siwik DA, Wang TJ, Colucci WS, Vasan RS. Clinical and echocardiographic correlates of plasma osteopontin in the community: the Framingham Heart Study. Heart. 2006 Oct; 92(10):1514-5.
Gheorghiade M, van Veldhuisen DJ, Colucci WS. Contemporary use of digoxin in the management of cardiovascular disorders. Circulation. 2006 May 30; 113(21):2556-64.
De Luca L, Colucci WS, Nieminen MS, Massie BM, Gheorghiade M. Evidence-based use of levosimendan in different clinical settings. Eur Heart J. 2006 Aug; 27(16):1908-20.
Cohn JN, Colucci W. Cardiovascular effects of aldosterone and post-acute myocardial infarction pathophysiology. Am J Cardiol. 2006 May 22; 97(10A):4F-12F.
Izumiya Y, Shiojima I, Sato K, Sawyer DB, Colucci WS, Walsh K. Vascular endothelial growth factor blockade promotes the transition from compensatory cardiac hypertrophy to failure in response to pressure overload. Hypertension. 2006 May; 47(5):887-93.
Kotlyar E, Vita JA, Winter MR, Awtry EH, Siwik DA, Keaney JF, Sawyer DB, Cupples LA, Colucci WS, Sam F. The relationship between aldosterone, oxidative stress, and inflammation in chronic, stable human heart failure. J Card Fail. 2006 Mar; 12(2):122-7.
Ahmed A, Rich MW, Love TE, Lloyd-Jones DM, Aban IB, Colucci WS, Adams KF, Gheorghiade M. Digoxin and reduction in mortality and hospitalization in heart failure: a comprehensive post hoc analysis of the DIG trial. Eur Heart J. 2006 Jan; 27(2):178-86.
Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, Colucci WS, Walsh K. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest. 2005 Aug; 115(8):2108-18.
Sam F, Kerstetter DL, Pimental DR, Mulukutla S, Tabaee A, Bristow MR, Colucci WS, Sawyer DB. Increased reactive oxygen species production and functional alterations in antioxidant enzymes in human failing myocardium. J Card Fail. 2005 Aug; 11(6):473-80.
Rude MK, Duhaney TA, Kuster GM, Judge S, Heo J, Colucci WS, Siwik DA, Sam F. Aldosterone stimulates matrix metalloproteinases and reactive oxygen species in adult rat ventricular cardiomyocytes. Hypertension. 2005 Sep; 46(3):555-61.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A, Colucci WS, Liao R. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res. 2005 Jul 8; 97(1):52-61.
Communal C, Colucci WS. The control of cardiomyocyte apoptosis via the beta-adrenergic signaling pathways. Arch Mal Coeur Vaiss. 2005 Mar; 98(3):236-41.
Kuster GM, Pimentel DR, Adachi T, Ido Y, Brenner DA, Cohen RA, Liao R, Siwik DA, Colucci WS. Alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes is mediated via thioredoxin-1-sensitive oxidative modification of thiols on Ras. Circulation. 2005 Mar 8; 111(9):1192-8.
McMurray J, Køber L, Robertson M, Dargie H, Colucci W, Lopez-Sendon J, Remme W, Sharpe DN, Ford I. Antiarrhythmic effect of carvedilol after acute myocardial infarction: results of the Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction (CAPRICORN) trial. J Am Coll Cardiol. 2005 Feb 15; 45(4):525-30.
Bianchi P, Pimentel DR, Murphy MP, Colucci WS, Parini A. A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J. 2005 Apr; 19(6):641-3.
Kuster GM, Kotlyar E, Rude MK, Siwik DA, Liao R, Colucci WS, Sam F. Mineralocorticoid receptor inhibition ameliorates the transition to myocardial failure and decreases oxidative stress and inflammation in mice with chronic pressure overload. Circulation. 2005 Feb 1; 111(4):420-7.
Taniyama Y, Ito M, Sato K, Kuester C, Veit K, Tremp G, Liao R, Colucci WS, Ivashchenko Y, Walsh K, Shiojima I. Akt3 overexpression in the heart results in progression from adaptive to maladaptive hypertrophy. J Mol Cell Cardiol. 2005 Feb; 38(2):375-85.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Fourth Edition, Braunwald E (Series Editor). Current Medicine. 2005.
58.
Shibata R, Ouchi N, Ito M, Kihara S, Shiojima I, Pimentel DR, Kumada M, Sato K, Schiekofer S, Ohashi K, Funahashi T, Colucci WS, Walsh K. Adiponectin-mediated modulation of hypertrophic signals in the heart. Nat Med. 2004 Dec; 10(12):1384-9.
Freudenberger RS, Schwarz RP, Brown J, Moore A, Mann D, Givertz MM, Colucci WS, Hare JM. Rationale, design and organisation of an efficacy and safety study of oxypurinol added to standard therapy in patients with NYHA class III – IV congestive heart failure. Expert Opin Investig Drugs. 2004 Nov; 13(11):1509-16.
Sundström J, Evans JC, Benjamin EJ, Levy D, Larson MG, Sawyer DB, Siwik DA, Colucci WS, Wilson PW, Vasan RS. Relations of plasma total TIMP-1 levels to cardiovascular risk factors and echocardiographic measures: the Framingham heart study. Eur Heart J. 2004 Sep; 25(17):1509-16.
Ito M, Adachi T, Pimentel DR, Ido Y, Colucci WS. Statins inhibit beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes via a Rac1-dependent mechanism. Circulation. 2004 Jul 27; 110(4):412-8.
Sundström J, Evans JC, Benjamin EJ, Levy D, Larson MG, Sawyer DB, Siwik DA, Colucci WS, Sutherland P, Wilson PW, Vasan RS. Relations of plasma matrix metalloproteinase-9 to clinical cardiovascular risk factors and echocardiographic left ventricular measures: the Framingham Heart Study. Circulation. 2004 Jun 15; 109(23):2850-6.
Colucci WS. Landmark study: the Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction Study (CAPRICORN). Am J Cardiol. 2004 May 6; 93(9A):13B-6B.
Mann DL, McMurray JJ, Packer M, Swedberg K, Borer JS, Colucci WS, Djian J, Drexler H, Feldman A, Kober L, Krum H, Liu P, Nieminen M, Tavazzi L, van Veldhuisen DJ, Waldenstrom A, Warren M, Westheim A, Zannad F, Fleming T. Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation. 2004 Apr 6; 109(13):1594-602.
Vasan RS, Evans JC, Benjamin EJ, Levy D, Larson MG, Sundstrom J, Murabito JM, Sam F, Colucci WS, Wilson PW. Relations of serum aldosterone to cardiac structure: gender-related differences in the Framingham Heart Study. Hypertension. 2004 May; 43(5):957-62.
Maytin M, Siwik DA, Ito M, Xiao L, Sawyer DB, Liao R, Colucci WS. Pressure overload-induced myocardial hypertrophy in mice does not require gp91phox. Circulation. 2004 Mar 9; 109(9):1168-71.
Sam F, Xie Z, Ooi H, Kerstetter DL, Colucci WS, Singh M, Singh K. Mice lacking osteopontin exhibit increased left ventricular dilation and reduced fibrosis after aldosterone infusion. Am J Hypertens. 2004 Feb; 17(2):188-93.
Giles TD, Chatterjee K, Cohn JN, Colucci WS, Feldman AM, Ferrans VJ, Roberts R. Definition, classification, and staging of the adult cardiomyopathies: a proposal for revision. J Card Fail. 2004 Feb; 10(1):6-8.
Siwik DA, Colucci WS. Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev. 2004 Jan; 9(1):43-51.
Sawyer DB, Colucci WS. Oxidative stress in heart failure; (Chapter 12). In: Mann DL (ed) Heart Failure: A Companion to Braunwald’s Heart Disease. Saunders. 2004; 181-92.
73.
Maytin M, Sawyer DB and Colucci WS. Role of reactive oxygen species in the regulation of cardiac myocyte phenotype. In: Pathophysiology of Cardiovascular Disease. Dhalla NS, Rupp H, Angel A and Pierce GN (eds). 51-7:Kluwer Academic Publishers . 2004.
74.
Kuramochi Y, Lim CC, Guo X, Colucci WS, Liao R, Sawyer DB. Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1beta. Am J Physiol Cell Physiol. 2004 Feb; 286(2):C222-9.
Torre-Amione G, Young JB, Colucci WS, Lewis BS, Pratt C, Cotter G, Stangl K, Elkayam U, Teerlink JR, Frey A, Rainisio M, Kobrin I. Hemodynamic and clinical effects of tezosentan, an intravenous dual endothelin receptor antagonist, in patients hospitalized for acute decompensated heart failure. J Am Coll Cardiol. 2003 Jul 2; 42(1):140-7.
Communal C, Singh M, Menon B, Xie Z, Colucci WS, Singh K. beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of beta-adrenergic receptor-stimulated apoptosis. J Cell Biochem. 2003 May 15; 89(2):381-8.
Sam F, Sawyer DB and Colucci WS. Myocardial nitric oxide in cardiac remodeling. In: Inflammation and Cardiac Diseases. Feuerstein GZ, Libby P and Mann DL (eds). Birkhäuser. 2003; 155-170.
82.
Siwik DA, Pimentel DR, Xiao L, Singh K, Sawyer DB, and Colucci WS. Adrenergic and mechanical regulation of oxidative stress in the myocardium. In: Kukin ML, Fuster V (eds). Oxidative Stress and Cardiac Failure. Armonk, NY:Futura Publishing Co., Inc.. 2003; 153-171.
83.
Ooi H, Colucci WS, Givertz MM. Endothelin mediates increased pulmonary vascular tone in patients with heart failure: demonstration by direct intrapulmonary infusion of sitaxsentan. Circulation. 2002 Sep 24; 106(13):1618-21.
Hare JM, Nguyen GC, Massaro AF, Drazen JM, Stevenson LW, Colucci WS, Fang JC, Johnson W, Givertz MM, Lucas C. Exhaled nitric oxide: a marker of pulmonary hemodynamics in heart failure. J Am Coll Cardiol. 2002 Sep 18; 40(6):1114-9.
Xiao L, Pimentel DR, Wang J, Singh K, Colucci WS, Sawyer DB. Role of reactive oxygen species and NAD(P)H oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am J Physiol Cell Physiol. 2002 Apr; 282(4):C926-34.
Communal C, Colucci WS, Remondino A, Sawyer DB, Port JD, Wichman SE, Bristow MR, Singh K. Reciprocal modulation of mitogen-activated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium. J Card Fail. 2002 Apr; 8(2):86-92.
Sawyer DB, Colucci WS. Molecular and cellular events in myocardial hypertrophy and failure. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
93.
Givertz MM, Colucci WS. Beta-Blockers. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
94.
Givertz MM, Colucci WS. Treatment of heart failure: New approaches. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
95.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Third Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 2002.
Sam F, Sawyer DB, Xie Z, Chang DL, Ngoy S, Brenner DA, Siwik DA, Singh K, Apstein CS, Colucci WS. Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction. Circ Res. 2001 Aug 17; 89(4):351-6.
Loh E, Elkayam U, Cody R, Bristow M, Jaski B, Colucci WS. A randomized multicenter study comparing the efficacy and safety of intravenous milrinone and intravenous nitroglycerin in patients with advanced heart failure. J Card Fail. 2001 Jun; 7(2):114-21.
Givertz MM, Slawsky MT, Moraes DL, McIntyre KM, Colucci WS. Noninvasive determination of pulmonary artery wedge pressure in patients with chronic heart failure. Am J Cardiol. 2001 May 15; 87(10):1213-5; A7.
Yancy CW, Fowler MB, Colucci WS, Gilbert EM, Bristow MR, Cohn JN, Lukas MA, Young ST, Packer M. Race and the response to adrenergic blockade with carvedilol in patients with chronic heart failure. N Engl J Med. 2001 May 3; 344(18):1358-65.
Fowler MB, Vera-Llonch M, Oster G, Bristow MR, Cohn JN, Colucci WS, Gilbert EM, Lukas MA, Lacey MJ, Richner R, Young ST, Packer M. Influence of carvedilol on hospitalizations in heart failure: incidence, resource utilization and costs. U.S. Carvedilol Heart Failure Study Group. J Am Coll Cardiol. 2001 May; 37(6):1692-9.
Givertz MM, Sawyer DB, Colucci WS. Antioxidants and myocardial contractility: illuminating the “Dark Side” of beta-adrenergic receptor activation? Circulation. 2001 Feb 13; 103(6):782-3.
Ooi H and Colucci WS. Pharmacological Treatment of Heart Failure; (Chapter 34). In: Hardman JG, Limbird LE and Gilman AG (eds): Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw Hill. McGraw Hill. 2001; 901-932.
113.
Colucci WS and Braunwald E. Pathophysiology of Heart Failure, (Chapter 16). In: Braunwald E (ed): Heart Disease. 6th Edition. Philadelphia:WB Saunders Co. 2001; 503-533.
114.
Colucci WS and Schoen FJ. Primary Tumors of the Heart; (Chapter 49). In: Braunwald E. (ed): Heart Disease. 6th Edition. Philadelphia:WB Saunders Co. 2001; 1807-22.
115.
Ooi H and Colucci WS. Congestive Heart Failure. In: Rakel & Bope: Conn’s Current Therapy. Philadelphia:WB Saunders Co. 2001; pp. 310-14.
116.
Colucci WS. Heart Failure. In: Essential Atlas of Heart Diseases, Second Edition, Braunwald E (Editor–in-Chief). Philadelphia:Current Medicine. 2001.
117.
Holubarsch CJ, Colucci WS, Meinertz T, Gaus W, Tendera M. Survival and prognosis: investigation of Crataegus extract WS 1442 in congestive heart failure (SPICE)–rationale, study design and study protocol. Eur J Heart Fail. 2000 Dec; 2(4):431-7.
Lim CC, Apstein CS, Colucci WS, Liao R. Impaired cell shortening and relengthening with increased pacing frequency are intrinsic to the senescent mouse cardiomyocyte. J Mol Cell Cardiol. 2000 Nov; 32(11):2075-82.
Slawsky MT, Colucci WS, Gottlieb SS, Greenberg BH, Haeusslein E, Hare J, Hutchins S, Leier CV, LeJemtel TH, Loh E, Nicklas J, Ogilby D, Singh BN, Smith W. Acute hemodynamic and clinical effects of levosimendan in patients with severe heart failure. Study Investigators. Circulation. 2000 Oct 31; 102(18):2222-7.
Moraes DL, Colucci WS, Givertz MM. Secondary pulmonary hypertension in chronic heart failure: the role of the endothelium in pathophysiology and management. Circulation. 2000 Oct 3; 102(14):1718-23.
Singh K, Communal C, Colucci WS. Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation. Basic Res Cardiol. 2000 Oct; 95(5):389-96.
Colucci WS, Elkayam U, Horton DP, Abraham WT, Bourge RC, Johnson AD, Wagoner LE, Givertz MM, Liang CS, Neibaur M, Haught WH, LeJemtel TH. Intravenous nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure. Nesiritide Study Group. N Engl J Med. 2000 Jul 27; 343(4):246-53.
Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, Amin J, Apstein CS, Colucci WS. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol. 2000 Jul; 279(1):H422-8.
Givertz MM, Colucci WS, LeJemtel TH, Gottlieb SS, Hare JM, Slawsky MT, Leier CV, Loh E, Nicklas JM, Lewis BE. Acute endothelin A receptor blockade causes selective pulmonary vasodilation in patients with chronic heart failure. Circulation. 2000 Jun 27; 101(25):2922-7.
Siwik DA, Chang DL, Colucci WS. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro. Circ Res. 2000 Jun 23; 86(12):1259-65.
Cuffe MS, Califf RM, Adams KF, Bourge RC, Colucci W, Massie B, O’Connor CM, Pina I, Quigg R, Silver M, Robinson LA, Leimberger JD, Gheorghiade M. Rationale and design of the OPTIME CHF trial: outcomes of a prospective trial of intravenous milrinone for exacerbations of chronic heart failure. Am Heart J. 2000 Jan; 139(1 Pt 1):15-22.
Sawyer DB, Colucci, WS. Myocardial Nitric Oxide in Heart Failure. In: Loscalzo J and Vita JA, (ed): Contemporary Cardiology: Nitric Oxide and the Cardiovascular System. Totowa, NJ:Humana Press Inc. 2000; pp. 309-19.
137.
Sawyer DB, Colucci WS. Role of oxidative stress, cytokines and apoptosis in myocardial dysfunction. In: Tardiff J-C and Bourassa MG, ed. Antioxidants and Cardiovascular Disease. Dordrecht:Kluwar. 2000.
138.
Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensitive G protein. Circulation. 1999 Nov 30; 100(22):2210-2.
Givertz MM, Colucci WS. Treatment of heart failure: New approaches. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Second Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1999.
143.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Second Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 1999.
144.
Sawyer DB, Colucci WS. Molecular and cellular events in myocardial hypertrophy and failure. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Second Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1999.
145.
Colucci WS. The effects of norepinephrine on myocardial biology: implications for the therapy of heart failure. Clin Cardiol. 1998 Dec; 21(12 Suppl 1):I20-4.
Eberli FR, Sam F, Ngoy S, Apstein CS, Colucci WS. Left-ventricular structural and functional remodeling in the mouse after myocardial infarction: assessment with the isovolumetrically-contracting Langendorff heart. J Mol Cell Cardiol. 1998 Jul; 30(7):1443-7.
Lo MW, Toh J, Emmert SE, Ritter MA, Furtek CI, Lu H, Colucci WS, Uretsky BF, Rucinska E. Pharmacokinetics of intravenous and oral losartan in patients with heart failure. J Clin Pharmacol. 1998 Jun; 38(6):525-32.
Calderone A, Thaik CM, Takahashi N, Chang DL, Colucci WS. Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J Clin Invest. 1998 Feb 15; 101(4):812-8.
Hare JM, Givertz MM, Creager MA, Colucci WS. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta-adrenergic inotropic responsiveness. Circulation. 1998 Jan 20; 97(2):161-6.
Cohn JN, Fowler MB, Bristow MR, Colucci WS, Gilbert EM, Kinhal V, Krueger SK, Lejemtel T, Narahara KA, Packer M, Young ST, Holcslaw TL, Lukas MA. Safety and efficacy of carvedilol in severe heart failure. The U.S. Carvedilol Heart Failure Study Group. J Card Fail. 1997 Sep; 3(3):173-9.
Hare JM, Shernan SK, Body SC, Graydon E, Colucci WS, Couper GS. Influence of inhaled nitric oxide on systemic flow and ventricular filling pressure in patients receiving mechanical circulatory assistance. Circulation. 1997 May 6; 95(9):2250-3.
Cohn JN, Bristow MR, Chien KR, Colucci WS, Frazier OH, Leinwand LA, Lorell BH, Moss AJ, Sonnenblick EH, Walsh RA, Mockrin SC, Reinlib L. Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research. Circulation. 1997 Feb 18; 95(4):766-70.
Colucci WS, Braunwald E. Cardiac tumors, cardiac manifestations of systemic diseases, and traumatic cardiac injury, Chapter 241. In: Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, Kasper DL, Hauser SL, Longo DL, eds. Harrison’s Principles of Internal Medicine, 14th Edition. New York:McGraw-Hill. 1997; pp 1341-4.
161.
Colucci WS, Schoen FJ, Braunwald E. Primary tumors of the heart, Chapter 42. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co. 1997; pp 1464-77.
162.
Colucci WS, Braunwald E. Pathophysiology of heart failure, Chapter 13. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co. 1997; pp 394-420.
163.
Colucci WS. Heart Failure. In: Essential Atlas of Heart Diseases, First Edition, Braunwald E (Editor–in-Chief). Philadelphia:Current Medicine. 1997.
164.
Braunwald E, Colucci WS, Grossman W. Clinical aspects of heart failure, Chapter 15. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co.. 1997; pp 445-70.
165.
Newton GE, Parker AB, Landzberg JS, Colucci WS, Parker JD. Muscarinic receptor modulation of basal and beta-adrenergic stimulated function of the failing human left ventricle. J Clin Invest. 1996 Dec 15; 98(12):2756-63.
Packer M, Colucci WS, Sackner-Bernstein JD, Liang CS, Goldscher DA, Freeman I, Kukin ML, Kinhal V, Udelson JE, Klapholz M, Gottlieb SS, Pearle D, Cody RJ, Gregory JJ, Kantrowitz NE, LeJemtel TH, Young ST, Lukas MA, Shusterman NH. Double-blind, placebo-controlled study of the effects of carvedilol in patients with moderate to severe heart failure. The PRECISE Trial. Prospective Randomized Evaluation of Carvedilol on Symptoms and Exercise. Circulation. 1996 Dec 1; 94(11):2793-9.
Givertz MM, Hare JM, Loh E, Gauthier DF, Colucci WS. Effect of bolus milrinone on hemodynamic variables and pulmonary vascular resistance in patients with severe left ventricular dysfunction: a rapid test for reversibility of pulmonary hypertension. J Am Coll Cardiol. 1996 Dec; 28(7):1775-80.
Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, Shusterman NH. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group. N Engl J Med. 1996 May 23; 334(21):1349-55.
Colucci WS. Pathophysiologic and clinical considerations in the treatment of heart failure: An overview. Chapter 8. In: Cardiovascular Therapeutics, Smith TW (Editor-in-Chief). Philadelphia:WB Saunders. 1996; pp 171-175.
177.
Stevenson LW, Colucci WS. Management of patients hospitalized with heart failure, Chapter 10. In Cardiovascular Therapeutics, Smith TW (Editor-in-Chief). Philadelphia:WB Saunders. 1996; pp 199-209.
178.
Colucci WS. Principles and practice of inotropic therapy, Chapter 126. In: Messerli FH, ed. Cardiovascular Drug Therapy, 2nd Edition. Philadelphia:WB Saunders Co. 1996; pp 1146-1150.
Calderone A, Takahashi N, Izzo NJ, Thaik CM, Colucci WS. Pressure- and volume-induced left ventricular hypertrophies are associated with distinct myocyte phenotypes and differential induction of peptide growth factor mRNAs. Circulation. 1995 Nov 1; 92(9):2385-90.
Hare JM, Loh E, Creager MA, Colucci WS. Nitric oxide inhibits the positive inotropic response to beta-adrenergic stimulation in humans with left ventricular dysfunction. Circulation. 1995 Oct 15; 92(8):2198-203.
Parker JD, Newton GE, Landzberg JS, Floras JS, Colucci WS. Functional significance of presynaptic alpha-adrenergic receptors in failing and nonfailing human left ventricle. Circulation. 1995 Oct 1; 92(7):1793-800.
Thaik CM, Calderone A, Takahashi N, Colucci WS. Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes. J Clin Invest. 1995 Aug; 96(2):1093-9.
Loh E, Barnett JV, Feldman AM, Couper GS, Vatner DE, Colucci WS, Galper JB. Decreased adenylate cyclase activity and expression of Gs alpha in human myocardium after orthotopic cardiac transplantation. Circ Res. 1995 May; 76(5):852-60.
Hare JM, Keaney JF, Balligand JL, Loscalzo J, Smith TW, Colucci WS. Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs. J Clin Invest. 1995 Jan; 95(1):360-6.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, First Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 1995.
189.
Colucci WS. Treatment of stable heart failure: New approaches. In “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1995.
190.
Thaik C, Colucci WS. Molecular and cellular abnormalities in hypertrophied and failing myocardium. In “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1995.
191.
Colucci WS. Secondary molecular alterations in failing human myocardium. In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman ER (ed). London:WB Saunders. 1995.
192.
Loh E, Stamler JS, Hare JM, Loscalzo J, Colucci WS. Cardiovascular effects of inhaled nitric oxide in patients with left ventricular dysfunction. Circulation. 1994 Dec; 90(6):2780-5.
Izzo NJ, Colucci WS. Regulation of alpha 1B-adrenergic receptor half-life: protein synthesis dependence and effect of norepinephrine. Am J Physiol. 1994 Mar; 266(3 Pt 1):C771-5.
Landzberg JS, Parker JD, Gauthier DF, Colucci WS. Effects of intracoronary acetylcholine and atropine on basal and dobutamine-stimulated left ventricular contractility. Circulation. 1994 Jan; 89(1):164-8.
Matoba Y, Colucci WS, Fields BN, Smith TW. The reovirus M1 gene determines the relative capacity of growth of reovirus in cultured bovine aortic endothelial cells. J Clin Invest. 1993 Dec; 92(6):2883-8.
Colucci WS, Sonnenblick EH, Adams KF, Berk M, Brozena SC, Cowley AJ, Grabicki JM, Kubo SA, LeJemtel T, Littler WA, et al. Efficacy of phosphodiesterase inhibition with milrinone in combination with converting enzyme inhibitors in patients with heart failure. The Milrinone Multicenter Trials Investigators. J Am Coll Cardiol. 1993 Oct; 22(4 Suppl A):113A-118A.
Schmidt TA, Allen PD, Colucci WS, Marsh JD, Kjeldsen K. No adaptation to digitalization as evaluated by digitalis receptor (Na,K-ATPase) quantification in explanted hearts from donors without heart disease and from digitalized recipients with end-stage heart failure. Am J Cardiol. 1993 Jan 1; 71(1):110-4.
Packer M, Narahara KA, Elkayam U, Sullivan JM, Pearle DL, Massie BM, Creager MA, and the Principal Investigators of the Reflect Study. Double-blind, placebo-controlled study of the efficacy of flosequinan in patients with chronic heart failure. J Am Coll Cardiol. 1993; 22:65-72.
201.
Colucci WS. In situ assessment of – and -Adrenergic responses in failing human myocardium. Circulation. 1993; 87(Suppl VII):63-7.
202.
Feldman AM, Bristow MR, Parmley WW, Carson PE, Pepine CJ, Gilbert EM, Strobeck JE, Hendrix GH, Powers ER, Bain RP, White BH, for the Vesnarinone Study Group. Effects of vesnarinone on morbidity and mortality in patients with heart failure. N Engl J Med. 1993; 329:149-55.
203.
Bialecki RA, Kulik TJ, Colucci WS. Stretching increases calcium influx and efflux in cultured pulmonary arterial smooth muscle cells. Am J Physiol. 1992 Nov; 263(5 Pt 1):L602-6.
Sen L, Bialecki RA, Smith E, Smith TW, Colucci WS. Cholesterol increases the L-type voltage-sensitive calcium channel current in arterial smooth muscle cells. Circ Res. 1992 Oct; 71(4):1008-14.
Willich SN, Tofler GH, Brezinski DA, Schafer AI, Muller JE, Michel T, Colucci WS. Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability. Eur Heart J. 1992 Apr; 13(4):550-5.
Parker JD, Landzberg JS, Bittl JA, Mirsky I, Colucci WS. Effects of beta-adrenergic stimulation with dobutamine on isovolumic relaxation in the normal and failing human left ventricle. Circulation. 1991 Sep; 84(3):1040-8.
Sen L, Liang BT, Colucci WS, Smith TW. Enhanced alpha 1-adrenergic responsiveness in cardiomyopathic hamster cardiac myocytes. Relation to the expression of pertussis toxin-sensitive G protein and alpha 1-adrenergic receptors. Circ Res. 1990 Nov; 67(5):1182-92.
Colucci WS. In vivo studies of myocardial beta-adrenergic receptor pharmacology in patients with congestive heart failure. Circulation. 1990 Aug; 82(2 Suppl):I44-51.
Arnold JM, Ribeiro JP, Colucci WS. Muscle blood flow during forearm exercise in patients with severe heart failure. Circulation. 1990 Aug; 82(2):465-72.
Treasure CB, Vita JA, Cox DA, Fish RD, Gordon JB, Mudge GH, Colucci WS, Sutton MG, Selwyn AP, Alexander RW, et al. Endothelium-dependent dilation of the coronary microvasculature is impaired in dilated cardiomyopathy. Circulation. 1990 Mar; 81(3):772-9.
Ribeiro JP, White HD, Hartley LH, Colucci WS. Acute increase in exercise capacity with milrinone: lack of correlation with resting hemodynamic responses. Braz J Med Biol Res. 1990; 23(11):1069-78.
Denniss AR, Colucci WS, Allen PD, Marsh JD. Distribution and function of human ventricular beta adrenergic receptors in congestive heart failure. J Mol Cell Cardiol. 1989 Jul; 21(7):651-60.
Denniss AR, Marsh JD, Quigg RJ, Gordon JB, Colucci WS. Beta-adrenergic receptor number and adenylate cyclase function in denervated transplanted and cardiomyopathic human hearts. Circulation. 1989 May; 79(5):1028-34.
Colucci WS. Observations on the intracoronary administration of milrinone and dobutamine to patients with congestive heart failure. Am J Cardiol. 1989 Jan 3; 63(2):17A-22A.
Arai Y, Saul JP, Albrecht P, Hartley LH, Lilly LS, Cohen RJ, Colucci WS. Modulation of cardiac autonomic activity during and immediately after exercise. Am J Physiol. 1989 Jan; 256(1 Pt 2):H132-41.
Colucci WS, Parker JD. Effects of beta-adrenergic agents on systolic and diastolic myocardial function in patients with and without heart failure. J Cardiovasc Pharmacol. 1989; 14 Suppl 5:S28-37.
Leatherman GF, Shook TL, Leatherman SM, Colucci WS. Use of a conductance catheter to detect increased left ventricular inotropic state by end-systolic pressure-volume analysis. Basic Res Cardiol. 1989; 84 Suppl 1:247-56.
Colucci WS, Akers M, Wise GM. Differential effects of norepinephrine and phorbol ester on alpha-1 adrenergic receptor number and surface-accessibility in DDT1 MF-2 cells. Biochem Biophys Res Commun. 1988 Oct 31; 156(2):924-30.
Colucci WS. Do positive inotropic agents adversely affect the survival of patients with chronic congestive heart failure? III. Antagonist’s viewpoint. J Am Coll Cardiol. 1988 Aug; 12(2):566-9.
Colucci WS, Denniss AR, Leatherman GF, Quigg RJ, Ludmer PL, Marsh JD, Gauthier DF. Intracoronary infusion of dobutamine to patients with and without severe congestive heart failure. Dose-response relationships, correlation with circulating catecholamines, and effect of phosphodiesterase inhibition. J Clin Invest. 1988 Apr; 81(4):1103-10.
Givertz MM, Colucci WS. Inotropic and vasoactive agents in the cardiac intensive care unit, Chapter 45. In: Brown DL, ed. Cardiac Intensive Care. Philadelphia:WB Saunders Co. 1988; pp. 545-54.
239.
Colucci WS, Leatherman GF, Ludmer PL, Gauthier DF. Beta-adrenergic inotropic responsiveness of patients with heart failure: studies with intracoronary dobutamine infusion. Circ Res. 1987 Oct; 61(4 Pt 2):I82-6.
Ribeiro JP, Knutzen A, Rocco MB, Hartley LH, Colucci WS. Periodic breathing during exercise in severe heart failure. Reversal with milrinone or cardiac transplantation. Chest. 1987 Sep; 92(3):555-6.
Ribeiro JP, White HD, Arnold JM, Hartley LH, Colucci WS. Exercise responses before and after long-term treatment with oral milrinone in patients with severe heart failure. Am J Med. 1986 Nov; 81(5):759-64.
Arnold JM, Ludmer PL, Wright RF, Ganz P, Braunwald E, Colucci WS. Role of reflex sympathetic withdrawal in the hemodynamic response to an increased inotropic state in patients with severe heart failure. J Am Coll Cardiol. 1986 Aug; 8(2):413-8.
Baim DS, Colucci WS, Monrad ES, Smith HS, Wright RF, Lanoue A, Gauthier DF, Ransil BJ, Grossman W, Braunwald E. Survival of patients with severe congestive heart failure treated with oral milrinone. J Am Coll Cardiol. 1986 Mar; 7(3):661-70.
Colucci WS, Wright RF, Jaski BE, Fifer MA, Braunwald E. Milrinone and dobutamine in severe heart failure: differing hemodynamic effects and individual patient responsiveness. Circulation. 1986 Mar; 73(3 Pt 2):III175-83.
Colucci WS, Alexander RW. Norepinephrine-induced alteration in the coupling of alpha 1-adrenergic receptor occupancy to calcium efflux in rabbit aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1986 Mar; 83(6):1743-6.
Colucci WS, Wright RF, Braunwald E. New positive inotropic agents in the treatment of congestive heart failure. Mechanisms of action and recent clinical developments. 2. N Engl J Med. 1986 Feb 6; 314(6):349-58.
Colucci WS, Wright RF, Braunwald E. New positive inotropic agents in the treatment of congestive heart failure. Mechanisms of action and recent clinical developments. 1. N Engl J Med. 1986 Jan 30; 314(5):290-9.
Ludmer PL, Wright RF, Arnold JM, Ganz P, Braunwald E, Colucci WS. Separation of the direct myocardial and vasodilator actions of milrinone administered by an intracoronary infusion technique. Circulation. 1986 Jan; 73(1):130-7.
Powers RE, Colucci WS. An increase in putative voltage dependent calcium channel number following reserpine treatment. Biochem Biophys Res Commun. 1985 Oct 30; 132(2):844-9.
White HD, Ribeiro JP, Hartley LH, Colucci WS. Immediate effects of milrinone on metabolic and sympathetic responses to exercise in severe congestive heart failure. Am J Cardiol. 1985 Jul 1; 56(1):93-8.
Fifer MA, Colucci WS, Lorell BH, Jaski BE, Barry WH. Inotropic, vascular and neuroendocrine effects of nifedipine in heart failure: comparison with nitroprusside. J Am Coll Cardiol. 1985 Mar; 5(3):731-7.
Jaski BE, Fifer MA, Wright RF, Braunwald E, Colucci WS. Positive inotropic and vasodilator actions of milrinone in patients with severe congestive heart failure. Dose-response relationships and comparison to nitroprusside. J Clin Invest. 1985 Feb; 75(2):643-9.
Colucci WS, Ludmer PL, Wright RF, Arnold JM, Ganz P, Braunwald E. Myocardial and vascular effects of intracoronary versus intravenous milrinone. Trans Assoc Am Physicians. 1985; 98:136-45.
Colucci WS, Brock TA, Atkinson WJ, Alexander RW, Gimbrone MA. Cultured vascular smooth muscle cells: an in vitro system for study of alpha-adrenergic receptor coupling and regulation. J Cardiovasc Pharmacol. 1985; 7 Suppl 6:S79-86.
Monrad ES, McKay RG, Baim DS, Colucci WS, Fifer MA, Heller GV, Royal HD, Grossman W. Improvement in indexes of diastolic performance in patients with congestive heart failure treated with milrinone. Circulation. 1984 Dec; 70(6):1030-7.
Ganz P, Gaspar J, Colucci WS, Barry WH, Mudge GH, Alexander RW. Effects of prostacyclin on coronary hemodynamics at rest and in response to cold pressor testing in patients with angina pectoris. Am J Cardiol. 1984 Jun 1; 53(11):1500-4.
Polak JF, Holman BL, Wynne J, Colucci WS. Right ventricular ejection fraction: an indicator of increased mortality in patients with congestive heart failure associated with coronary artery disease. J Am Coll Cardiol. 1983 Aug; 2(2):217-24.
Colucci WS. New developments in alpha-adrenergic receptor pharmacology: implications for the initial treatment of hypertension. Am J Cardiol. 1983 Feb 24; 51(4):639-43.
Colucci WS, Lorell BH, Schoen FJ, Warhol MJ, Grossman W. Hypertrophic obstructive cardiomyopathy due to Fabry’s disease. N Engl J Med. 1982 Oct 7; 307(15):926-8.
Colucci WS. Alpha-adrenergic receptor blockade with prazosin. Consideration of hypertension, heart failure, and potential new applications. Ann Intern Med. 1982 Jul; 97(1):67-77.
Rude RE, Grossman W, Colucci WS, Benotti JR, Carabello BA, Wynne J, Malacoff R, Braunwald E. Problems in assessment of new pharmacologic agents for the heart failure patient. Am Heart J. 1981 Sep; 102(3 Pt 2):584-90.
Colucci WS, Alexander RW, Mudge GH, Rude RE, Holman BL, Wynne J, Grossman W, Braunwald E. Acute and chronic effects of pirbuterol on left ventricular ejection fraction and clinical status in severe congestive heart failure. Am Heart J. 1981 Sep; 102(3 Pt 2):564-8.
Colucci WS, Williams GH, Braunwald E. Clinical, hemodynamic, and neuroendocrine effects of chronic prazosin therapy for congestive heart failure. Am Heart J. 1981 Sep; 102(3 Pt 2):615-21.
Colucci WS, Alexander RW, Williams GH, Rude RE, Holman BL, Konstam MA, Wynne J, Mudge GH, Braunwald E. Decreased lymphocyte beta-adrenergic-receptor density in patients with heart failure and tolerance to the beta-adrenergic agonist pirbuterol. N Engl J Med. 1981 Jul 23; 305(4):185-90.
Colucci WS, Williams GH, Alexander RW, Braunwald E. Mechanisms and implications of vasodilator tolerance in the treatment of congestive heart failure. Am J Med. 1981 Jul; 71(1):89-99.
Rude RE, Turi Z, Brown EJ, Lorell BH, Colucci WS, Mudge GH, Taylor CR, Grossman W. Acute effects of oral pirbuterol on myocardial oxygen metabolism and systemic hemodynamics in chronic congestive heart failure. Circulation. 1981 Jul; 64(1):139-45.
Dzau VJ, Colucci WS, Hollenberg NK, Williams GH. Relation of the renin-angiotensin-aldosterone system to clinical state in congestive heart failure. Circulation. 1981 Mar; 63(3):645-51.
Colucci WS, Gimbrone MA, Alexander RW. Regulation of the postsynaptic alpha-adrenergic receptor in rat mesenteric artery. Effects of chemical sympathectomy and epinephrine treatment. Circ Res. 1981 Jan; 48(1):104-11.
Colucci WS, Williams GH, Braunwald E. Increased plasma norepinephrine levels during prazosin therapy for severe congestive heart failure. Ann Intern Med. 1980 Sep; 93(3):452-3.
Dzau VJ, Colucci WS, Williams GH, Curfman G, Meggs L, Hollenberg NK. Sustained effectiveness of converting-enzyme inhibition in patients with severe congestive heart failure. N Engl J Med. 1980 Jun 19; 302(25):1373-9.
Colucci WS, Gimbrone MA, Alexander RW. Characterization of postsynaptic alpha-adrenergic receptors by [3H]-dihydroergocryptine binding in muscular arteries from the rat mesentery. Hypertension. 1980 Mar-Apr; 2(2):149-55.
Tokuda M, Kojodjojo P, Tung S, Tedrow UB, Nof E, Inada K, Koplan BA, Michaud GF, John RM, Epstein LM, Stevenson WG. Acute failure of catheter ablation for ventricular tachycardia due to structural heart disease: causes and significance. J Am Heart Assoc. 2013; 2(3):e000072.
Ng J, Barbhaiya C, Chopra N, Reichlin T, Nof E, Tadros T, Stevenson WG, John RM. Automatic external defibrillators-friend or foe? Am J Emerg Med. 2013 Aug; 31(8):1292.e1-2.
Steven D, Sultan A, Reddy V, Luker J, Altenburg M, Hoffmann B, Rostock T, Servatius H, Stevenson WG, Willems S, Michaud GF. Benefit of pulmonary vein isolation guided by loss of pace capture on the ablation line: results from a prospective 2-center randomized trial. J Am Coll Cardiol. 2013 Jul 2; 62(1):44-50.
Nof E, Stevenson WG, Epstein LM, Tedrow UB, Koplan BA. Catheter Ablation of Atrial Arrhythmias After Cardiac Transplantation: Findings at EP Study Utility of 3-D Mapping and Outcomes. J Cardiovasc Electrophysiol. 2013 May; 24(5):498-502.
Tracy CM, Epstein AE, Darbar D, Dimarco JP, Dunbar SB, Mark Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD, Epstein AE, Dimarco JP, Ellenbogen KA, Mark Estes NA, Freedman RA, Gettes LS, Marc Gillinov A, Gregoratos G, Hammill SC, Hayes DL, Hlatky MA, Kristin Newby L, Page RL, Schoenfeld MH, Silka MJ, Warner Stevenson L, Sweeney MO, Anderson JL, Jacobs AK, Halperin JL, Albert NM, Creager MA, Demets D, Ettinger SM, Guyton RA, Hochman JS, Kushner FG, Ohman EM, Stevenson W, Yancy CW. 2012 ACCF/AHA/HRS focused update of the 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Thorac Cardiovasc Surg. 2012 Dec; 144(6):e127-45.
John RM, Tedrow UB, Koplan BA, Albert CM, Epstein LM, Sweeney MO, Miller AL, Michaud GF, Stevenson WG. Ventricular arrhythmias and sudden cardiac death. Lancet. 2012 Oct 27; 380(9852):1520-9.
Tracy CM, Epstein AE, Darbar D, DiMarco JP, Dunbar SB, Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD, Ellenbogen KA, Freedman RA, Gettes LS, Gillinov AM, Gregoratos G, Hayes DL, Page RL, Stevenson LW, Sweeney MO. 2012 ACCF/AHA/HRS focused update of the 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2012 Oct 2; 126(14):1784-800.
Tracy CM, Epstein AE, Darbar D, Dimarco JP, Dunbar SB, Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD. 2012 ACCF/AHA/HRS Focused Update of the 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2012 Oct; 9(10):1737-53.
Wissner E, Stevenson WG, Kuck KH. Catheter ablation of ventricular tachycardia in ischaemic and non-ischaemic cardiomyopathy: where are we today? A clinical review. Eur Heart J. 2012 Jun; 33(12):1440-50.
Vollmann D, Stevenson WG, Lüthje L, Sohns C, John RM, Zabel M, Michaud GF. Misleading long post-pacing interval after entrainment of typical atrial flutter from the cavotricuspid isthmus. J Am Coll Cardiol. 2012 Feb 28; 59(9):819-24.
Albert CM, Chen PS, Anderson ME, Cain ME, Fishman GI, Narayan SM, Olgin JE, Spooner PM, Stevenson WG, Van Wagoner DR, Packer DL. Full report from the first annual Heart Rhythm Society Research Forum: a vision for our research future, “dream, discover, develop, deliver”. Heart Rhythm. 2011 Dec; 8(12):e1-12.
Wijnmaalen AP, Roberts-Thomson KC, Steven D, Klautz RJ, Willems S, Schalij MJ, Stevenson WG, Zeppenfeld K. Catheter ablation of ventricular tachycardia after left ventricular reconstructive surgery for ischemic cardiomyopathy. Heart Rhythm. 2012 Jan; 9(1):10-7.
Stevenson WG, Couper GS. A surgical option for ventricular tachycardia caused by nonischemic cardiomyopathy. Circ Arrhythm Electrophysiol. 2011 Aug; 4(4):429-31.
Bohnen M, Stevenson WG, Tedrow UB, Michaud GF, John RM, Epstein LM, Albert CM, Koplan BA. Incidence and predictors of major complications from contemporary catheter ablation to treat cardiac arrhythmias. Heart Rhythm. 2011 Nov; 8(11):1661-6.
Bohnen M, Shea JB, Michaud GF, John R, Stevenson WG, Epstein LM, Tedrow UB, Albert C, Koplan BA. Quality of life with atrial fibrillation: do the spouses suffer as much as the patients? Pacing Clin Electrophysiol. 2011 Jul; 34(7):804-9.
Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Huezey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Smith SC, Priori SG, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Jacobs AK, Anderson JL, Albert N, Buller CE, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Tarkington LG, Yancy CW. 2011 ACCF/AHA/HRS focused updates incorporated into the ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2011 Mar 15; 123(10):e269-367.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Heuzey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Jacobs AK, Anderson JL, Albert N, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on Dabigatran): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2011 Mar 15; 123(10):1144-50.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on dabigatran): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2011 Mar 15; 57(11):1330-7.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Heuzey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Jacobs AK, Anderson JL, Albert N, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on dabigatran). A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2011 Mar; 8(3):e1-8.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson SB, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann LS, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2011 Jan 11; 57(2):223-42.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson S, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann LS, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2011 Jan; 8(1):157-76.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson S, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann L, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (updating the 2006 guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2011 Jan 4; 123(1):104-23.
Sacher F, Roberts-Thomson K, Maury P, Tedrow U, Nault I, Steven D, Hocini M, Koplan B, Leroux L, Derval N, Seiler J, Wright MJ, Epstein L, Haissaguerre M, Jais P, Stevenson WG. Epicardial ventricular tachycardia ablation a multicenter safety study. J Am Coll Cardiol. 2010 May 25; 55(21):2366-72.
Steven D, Seiler J, Roberts-Thomson KC, Inada K, Stevenson WG. Mapping of atrial tachycardias after catheter ablation for atrial fibrillation: use of bi-atrial activation patterns to facilitate recognition of origin. Heart Rhythm. 2010 May; 7(5):664-72.
Roberts-Thomson KC, Seiler J, Steven D, Inada K, Michaud GF, John RM, Koplan BA, Epstein LM, Stevenson WG, Tedrow UB. Percutaneous access of the epicardial space for mapping ventricular and supraventricular arrhythmias in patients with and without prior cardiac surgery. J Cardiovasc Electrophysiol. 2010 Apr; 21(4):406-11.
Steven D, Reddy VY, Inada K, Roberts-Thomson KC, Seiler J, Stevenson WG, Michaud GF. Loss of pace capture on the ablation line: a new marker for complete radiofrequency lesions to achieve pulmonary vein isolation. Heart Rhythm. 2010 Mar; 7(3):323-30.
Aliot EM, Stevenson WG, Almendral-Garrote JM, Bogun F, Calkins CH, Delacretaz E, Della Bella P, Hindricks G, Jaïs P, Josephson ME, Kautzner J, Kay GN, Kuck KH, Lerman BB, Marchlinski F, Reddy V, Schalij MJ, Schilling R, Soejima K, Wilber D. EHRA/HRS Expert Consensus on Catheter Ablation of Ventricular Arrhythmias: developed in a partnership with the European Heart Rhythm Association (EHRA), a Registered Branch of the European Society of Cardiology (ESC), and the Heart Rhythm Society (HRS); in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA). Heart Rhythm. 2009 Jun; 6(6):886-933.
Aliot EM, Stevenson WG, Almendral-Garrote JM, Bogun F, Calkins CH, Delacretaz E, Bella PD, Hindricks G, Jaïs P, Josephson ME, Kautzner J, Kay GN, Kuck KH, Lerman BB, Marchlinski F, Reddy V, Schalij MJ, Schilling R, Soejima K, Wilber D. EHRA/HRS Expert Consensus on Catheter Ablation of Ventricular Arrhythmias: developed in a partnership with the European Heart Rhythm Association (EHRA), a Registered Branch of the European Society of Cardiology (ESC), and the Heart Rhythm Society (HRS); in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA). Europace. 2009 Jun; 11(6):771-817.
Lee JC, Steven D, Roberts-Thomson KC, Raymond JM, Stevenson WG, Tedrow UB. Atrial tachycardias adjacent to the phrenic nerve: recognition, potential problems, and solutions. Heart Rhythm. 2009 Aug; 6(8):1186-91.
Roberts-Thomson KC, Seiler J, Steven D, Inada K, John R, Michaud G, Stevenson WG. Short AV response to atrial extrastimuli during narrow complex tachycardia: what is the mechanism? J Cardiovasc Electrophysiol. 2009 Aug; 20(8):946-8.
Eckart RE, Field ME, Hruczkowski TW, Forman DE, Dorbala S, Di Carli MF, Albert CE, Maisel WH, Epstein LM, Stevenson WG. Association of electrocardiographic morphology of exercise-induced ventricular arrhythmia with mortality. Ann Intern Med. 2008 Oct 7; 149(7):451-60, W82.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/american College of Cardiology Foundation/heart Rhythm Society scientific statement on noninvasive risk stratification techniques for identifying patients at risk for sudden cardiac death: a scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. Heart Rhythm. 2008 Oct; 5(10):e1-21.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/American College of Cardiology Foundation/Heart Rhythm Society scientific statement on noninvasive risk stratification techniques for identifying patients at risk for sudden cardiac death: a scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. Circulation. 2008 Sep 30; 118(14):1497-1518.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/American College of Cardiology Foundation/Heart Rhythm Society Scientific Statement on Noninvasive Risk Stratification Techniques for Identifying Patients at Risk for Sudden Cardiac Death. A scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. J Am Coll Cardiol. 2008 Sep 30; 52(14):1179-99.
Seiler J, Roberts-Thomson KC, Raymond JM, Vest J, Delacretaz E, Stevenson WG. Steam pops during irrigated radiofrequency ablation: feasibility of impedance monitoring for prevention. Heart Rhythm. 2008 Oct; 5(10):1411-6.
Roy D, Talajic M, Nattel S, Wyse DG, Dorian P, Lee KL, Bourassa MG, Arnold JM, Buxton AE, Camm AJ, Connolly SJ, Dubuc M, Ducharme A, Guerra PG, Hohnloser SH, Lambert J, Le Heuzey JY, O’Hara G, Pedersen OD, Rouleau JL, Singh BN, Stevenson LW, Stevenson WG, Thibault B, Waldo AL. Rhythm control versus rate control for atrial fibrillation and heart failure. N Engl J Med. 2008 Jun 19; 358(25):2667-77.
Roberts-Thomson KC, Seiler J, Raymond JM, Stevenson WG. Exercise induced tachycardia with atrioventricular dissociation: what is the mechanism? Heart Rhythm. 2009 Mar; 6(3):426-8.
Zeppenfeld K, Schalij MJ, Bartelings MM, Tedrow UB, Koplan BA, Soejima K, Stevenson WG. Catheter ablation of ventricular tachycardia after repair of congenital heart disease: electroanatomic identification of the critical right ventricular isthmus. Circulation. 2007 Nov 13; 116(20):2241-52.
Sacher F, Sobieszczyk P, Tedrow U, Eisenhauer AC, Field ME, Selwyn A, Raymond JM, Koplan B, Epstein LM, Stevenson WG. Transcoronary ethanol ventricular tachycardia ablation in the modern electrophysiology era. Heart Rhythm. 2008 Jan; 5(1):62-8.
Sacher F, Jais P, Stephenson K, O’Neill MD, Hocini M, Clementy J, Stevenson WG, Haissaguerre M. Phrenic nerve injury after catheter ablation of atrial fibrillation. Indian Pacing Electrophysiol J. 2007; 7(1):1-6.
Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, Popma JJ, Stevenson W. The cardiovascular disease continuum validated: clinical evidence of improved patient outcomes: part I: Pathophysiology and clinical trial evidence (risk factors through stable coronary artery disease). Circulation. 2006 Dec 19; 114(25):2850-70.
Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, Popma JJ, Stevenson W. The cardiovascular disease continuum validated: clinical evidence of improved patient outcomes: part II: Clinical trial evidence (acute coronary syndromes through renal disease) and future directions. Circulation. 2006 Dec 19; 114(25):2871-91.
Veenhuyzen GD, Hruczkowski T, Dhir SK, Stevenson WG. Another way to prove the presence and participation of an accessory pathway in supraventricular tachycardia? J Cardiovasc Electrophysiol. 2006 Oct; 17(10):1147-9.
Yan AT, Shayne AJ, Brown KA, Gupta SN, Chan CW, Luu TM, Di Carli MF, Reynolds HG, Stevenson WG, Kwong RY. Characterization of the peri-infarct zone by contrast-enhanced cardiac magnetic resonance imaging is a powerful predictor of post-myocardial infarction mortality. Circulation. 2006 Jul 4; 114(1):32-9.
Sapp JL, Cooper JM, Zei P, Stevenson WG. Large radiofrequency ablation lesions can be created with a retractable infusion-needle catheter. J Cardiovasc Electrophysiol. 2006 Jun; 17(6):657-61.
Tedrow UB, Kramer DB, Stevenson LW, Stevenson WG, Baughman KL, Epstein LM, Lewis EF. Relation of right ventricular peak systolic pressure to major adverse events in patients undergoing cardiac resynchronization therapy. Am J Cardiol. 2006 Jun 15; 97(12):1737-40.
Parkash R, Stevenson WG, Epstein LM, Maisel WH. Predicting early mortality after implantable defibrillator implantation: a clinical risk score for optimal patient selection. Am Heart J. 2006 Feb; 151(2):397-403.
Nazarian S, Maisel WH, Miles JS, Tsang S, Stevenson LW, Stevenson WG. Impact of implantable cardioverter defibrillators on survival and recurrent hospitalization in advanced heart failure. Am Heart J. 2005 Nov; 150(5):955-60.
Intini A, Goldstein RN, Jia P, Ramanathan C, Ryu K, Giannattasio B, Gilkeson R, Stambler BS, Brugada P, Stevenson WG, Rudy Y, Waldo AL. Electrocardiographic imaging (ECGI), a novel diagnostic modality used for mapping of focal left ventricular tachycardia in a young athlete. Heart Rhythm. 2005 Nov; 2(11):1250-2.
Parkash R, Maisel WH, Toca FM, Stevenson WG. Atrial fibrillation in heart failure: high mortality risk even if ventricular function is preserved. Am Heart J. 2005 Oct; 150(4):701-6.
Stevenson WG, Chaitman BR, Ellenbogen KA, Epstein AE, Gross WL, Hayes DL, Strickberger SA, Sweeney MO. Clinical assessment and management of patients with implanted cardioverter-defibrillators presenting to nonelectrophysiologists. Circulation. 2004 Dec 21; 110(25):3866-9.
Tedrow U, Maisel WH, Epstein LM, Soejima K, Stevenson WG. Feasibility of adjusting paced left ventricular activation by manipulating stimulus strength. J Am Coll Cardiol. 2004 Dec 7; 44(11):2249-52.
Curtis AB, Abraham WT, Chen PS, Ellenbogen KA, Epstein AE, Friedman PA, Hohnloser SH, Kanter RJ, Stevenson WG. Highlights of Heart Rhythm 2004, the Annual Scientific Sessions of the Heart Rhythm Society: May 19 to 22, 2004, in San Francisco, California. J Am Coll Cardiol. 2004 Oct 19; 44(8):1550-6.
Brunckhorst CB, Delacretaz E, Soejima K, Maisel WH, Friedman PL, Stevenson WG. Identification of the ventricular tachycardia isthmus after infarction by pace mapping. Circulation. 2004 Aug 10; 110(6):652-9.
Sapp JL, Soejima K, Cooper JM, Epstein LM, Stevenson WG. Ablation lesion size correlates with pacing threshold: a physiological basis for use of pacing to assess ablation lesions. Pacing Clin Electrophysiol. 2004 Jul; 27(7):933-7.
Sapp JL, Cooper JM, Soejima K, Sorrell T, Lopera G, Satti SD, Koplan BA, Epstein LM, Edelman E, Rogers C, Stevenson WG. Deep myocardial ablation lesions can be created with a retractable needle-tipped catheter. Pacing Clin Electrophysiol. 2004 May; 27(5):594-9.
Lopera G, Stevenson WG, Soejima K, Maisel WH, Koplan B, Sapp JL, Satti SD, Epstein LM. Identification and ablation of three types of ventricular tachycardia involving the his-purkinje system in patients with heart disease. J Cardiovasc Electrophysiol. 2004 Jan; 15(1):52-8.
Blomström-Lundqvist C, Scheinman MM, Aliot EM, Alpert JS, Calkins H, Camm AJ, Campbell WB, Haines DE, Kuck KH, Lerman BB, Miller DD, Shaeffer CW, Stevenson WG, Tomaselli GF, Antman EM, Smith SC, Alpert JS, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Hiratzka LF, Hunt SA, Jacobs AK, Russell RO, Priori SG, Blanc JJ, Budaj A, Burgos EF, Cowie M, Deckers JW, Garcia MA, Klein WW, Lekakis J, Lindahl B, Mazzotta G, Morais JC, Oto A, Smiseth O, Trappe HJ. ACC/AHA/ESC guidelines for the management of patients with supraventricular arrhythmias–executive summary. a report of the American college of cardiology/American heart association task force on practice guidelines and the European society of cardiology committee for practice guidelines (writing committee to develop guidelines for the management of patients with supraventricular arrhythmias) developed in collaboration with NASPE-Heart Rhythm Society. J Am Coll Cardiol. 2003 Oct 15; 42(8):1493-531.
Blomström-Lundqvist C, Scheinman MM, Aliot EM, Alpert JS, Calkins H, Camm AJ, Campbell WB, Haines DE, Kuck KH, Lerman BB, Miller DD, Shaeffer CW, Stevenson WG, Tomaselli GF, Antman EM, Smith SC, Alpert JS, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Hiratzka LF, Hunt SA, Jacobs AK, Russell RO, Priori SG, Blanc JJ, Budaj A, Burgos EF, Cowie M, Deckers JW, Garcia MA, Klein WW, Lekakis J, Lindahl B, Mazzotta G, Morais JC, Oto A, Smiseth O, Trappe HJ. ACC/AHA/ESC guidelines for the management of patients with supraventricular arrhythmias–executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the European Society of Cardiology Committee for Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Supraventricular Arrhythmias). Circulation. 2003 Oct 14; 108(15):1871-909.
Brunckhorst CB, Stevenson WG, Soejima K, Maisel WH, Delacretaz E, Friedman PL, Ben-Haim SA. Relationship of slow conduction detected by pace-mapping to ventricular tachycardia re-entry circuit sites after infarction. J Am Coll Cardiol. 2003 Mar 5; 41(5):802-9.
Koplan BA, Stevenson WG, Epstein LM, Aranki SF, Maisel WH. Development and validation of a simple risk score to predict the need for permanent pacing after cardiac valve surgery. J Am Coll Cardiol. 2003 Mar 5; 41(5):795-801.
Stevenson WG, Epstein LM. Predicting sudden death risk for heart failure patients in the implantable cardioverter-defibrillator age. Circulation. 2003 Feb 4; 107(4):514-6.
Khan HH, Maisel WH, Ho C, Suzuki M, Soejima K, Solomon S, Stevenson WG. Effect of radiofrequency catheter ablation of ventricular tachycardia on left ventricular function in patients with prior myocardial infarction. J Interv Card Electrophysiol. 2002 Dec; 7(3):243-7.
Fenelon G, Stambler BS, Huvelle E, Brugada P, Stevenson WG. Left ventricular dysfunction is associated with prolonged average ventricular fibrillation cycle length in patients with implantable cardioverter defibrillators. J Interv Card Electrophysiol. 2002 Dec; 7(3):249-54.
Soejima K, Stevenson WG. Ventricular tachycardia associated with myocardial infarct scar: a spectrum of therapies for a single patient. Circulation. 2002 Jul 9; 106(2):176-9.
Friedman RA, Walsh EP, Silka MJ, Calkins H, Stevenson WG, Rhodes LA, Deal BJ, Wolff GS, Demaso DR, Hanisch D, Van Hare GF. NASPE Expert Consensus Conference: Radiofrequency catheter ablation in children with and without congenital heart disease. Report of the writing committee. North American Society of Pacing and Electrophysiology. Pacing Clin Electrophysiol. 2002 Jun; 25(6):1000-17.
Soejima K, Suzuki M, Maisel WH, Brunckhorst CB, Delacretaz E, Blier L, Tung S, Khan H, Stevenson WG. Catheter ablation in patients with multiple and unstable ventricular tachycardias after myocardial infarction: short ablation lines guided by reentry circuit isthmuses and sinus rhythm mapping. Circulation. 2001 Aug 7; 104(6):664-9.
Soejima K, Stevenson WG, Maisel WH, Delacretaz E, Brunckhorst CB, Ellison KE, Friedman PL. The N + 1 difference: a new measure for entrainment mapping. J Am Coll Cardiol. 2001 Apr; 37(5):1386-94.
Delacretaz E, Soejima K, Gottipaty VK, Brunckhorst CB, Friedman PL, Stevenson WG. Single catheter determination of local electrogram prematurity using simultaneous unipolar and bipolar recordings to replace the surface ECG as a timing reference. Pacing Clin Electrophysiol. 2001 Apr; 24(4 Pt 1):441-9.
Soejima K, Stevenson WG, Delacretaz E, Brunckhorst CB, Maisel WH, Friedman PL. Identification of left atrial origin of ectopic tachycardia during right atrial mapping: analysis of double potentials at the posteromedial right atrium. J Cardiovasc Electrophysiol. 2000 Sep; 11(9):975-80.
Maisel WH, Stevenson WG. Sudden death and the electrophysiological effects of angiotensin-converting enzyme inhibitors. J Card Fail. 2000 Jun; 6(2):80-2.
Delacretaz E, Stevenson WG, Ellison KE, Maisel WH, Friedman PL. Mapping and radiofrequency catheter ablation of the three types of sustained monomorphic ventricular tachycardia in nonischemic heart disease. J Cardiovasc Electrophysiol. 2000 Jan; 11(1):11-7.
Delacretaz E, Soejima K, Stevenson WG, Friedman PL. Short ventriculoatrial intervals during orthodromic atrioventricular reciprocating tachycardia: what is the mechanism? J Cardiovasc Electrophysiol. 2000 Jan; 11(1):121-4.
Soejima K, Delacretaz E, Stevenson WG, Friedman PL. DDD-pacing-induced cardiomyopathy following AV node ablation for persistent atrial tachycardia. J Interv Card Electrophysiol. 1999 Dec; 3(4):321-3.
Kocovic DZ, Harada T, Friedman PL, Stevenson WG. Characteristics of electrograms recorded at reentry circuit sites and bystanders during ventricular tachycardia after myocardial infarction. J Am Coll Cardiol. 1999 Aug; 34(2):381-8.
Delacretaz E, Stevenson WG, Winters GL, Mitchell RN, Stewart S, Lynch K, Friedman PL. Ablation of ventricular tachycardia with a saline-cooled radiofrequency catheter: anatomic and histologic characteristics of the lesions in humans. J Cardiovasc Electrophysiol. 1999 Jun; 10(6):860-5.
Ellison KE, Friedman PL, Ganz LI, Stevenson WG. Entrainment mapping and radiofrequency catheter ablation of ventricular tachycardia in right ventricular dysplasia. J Am Coll Cardiol. 1998 Sep; 32(3):724-8.
Lefroy DC, Ellison KE, Friedman PL, Stevenson WG. Arrhythmia of the month: shortening of ventriculoatrial conduction time during radiofrequency catheter ablation of a concealed accessory pathway. J Cardiovasc Electrophysiol. 1998 Apr; 9(4):445-7.
Ganz LI, Couper GS, Friedman PL, Stevenson WG, Ellison K. Use of telemetered permanent pacemaker intracardiac electrograms to diagnose ventricular tachycardia. Am J Cardiol. 1997 Dec 1; 80(11):1511-3.
Ellison KE, Stevenson WG, Couper GS, Friedman PL. Ablation of ventricular tachycardia due to a postinfarct ventricular septal defect: identification and transection of a broad reentry loop. J Cardiovasc Electrophysiol. 1997 Oct; 8(10):1163-6.
Harada T, Stevenson WG, Kocovic DZ, Friedman PL. Catheter ablation of ventricular tachycardia after myocardial infarction: relation of endocardial sinus rhythm late potentials to the reentry circuit. J Am Coll Cardiol. 1997 Oct; 30(4):1015-23.
Maisel WH, Kuntz KM, Reimold SC, Lee TH, Antman EM, Friedman PL, Stevenson WG. Risk of initiating antiarrhythmic drug therapy for atrial fibrillation in patients admitted to a university hospital. Ann Intern Med. 1997 Aug 15; 127(4):281-4.
Hadjis TA, Harada T, Stevenson WG, Friedman PL. Effect of recording site on postpacing interval measurement during catheter mapping and entrainment of postinfarction ventricular tachycardia. J Cardiovasc Electrophysiol. 1997 Apr; 8(4):398-404.
Stevenson WG, Ridker PM. Should survivors of myocardial infarction with low ejection fraction be routinely referred to arrhythmia specialists? JAMA. 1996 Aug 14; 276(6):481-5.
Stevenson WG, Stevenson LW, Middlekauff HR, Fonarow GC, Hamilton MA, Woo MA, Saxon LA, Natterson PD, Steimle A, Walden JA, et al. Improving survival for patients with advanced heart failure: a study of 737 consecutive patients. J Am Coll Cardiol. 1995 Nov 15; 26(6):1417-23.
Stevenson WG, Sager PT, Natterson PD, Saxon LA, Middlekauff HR, Wiener I. Relation of pace mapping QRS configuration and conduction delay to ventricular tachycardia reentry circuits in human infarct scars. J Am Coll Cardiol. 1995 Aug; 26(