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Epilogue: Volume 4 – Translational, Post-Translational and Regenerative Medicine in Cardiology

  • Larry H Bernstein, MD, FCAP, Author and Curator, Volume Four, Co-Editor
  • Justin Pearlman, MD, PhD, FACC, Content Consultant for Series A: Cardiovascular Diseases
  • Aviva Lev-Ari, PhD, RN, Co-Editor of Volume Four and Editor-in-Chief, BioMed e-Series

 

This completes Chapter 4 in two parts on the most dynamic developments in the regulatory pathways guiding cardiovascular dynamics and function in health and disease.  I have covered key features of these in two summaries, so I shall try to look further into important expected future directions and their anticipated implications.

1. Mechanisms of Disease

Signal Transduction: Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

David J. Roberts, Valerie P. Tan-Sah, Jeffery M. Smith and Shigeki Miyamoto
J. Biol. Chem. 2013, 288:23798-23806.  http://dx.doi.org/ 10.1074/jbc.M113.482026

Backgound: Hexokinase II binds to mitochondria and promotes cell survival.
Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.
Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.
Significance: The Akt-HK-II signaling nexus is important in cell survival.

HK-II Phosphorylation

HK-II Phosphorylation

 

 

 

 

 

 

It has been demonstrated that an increased level of HK-II at mitochondria is protective and is increased by protective interventions but decreased under stress.

It   has not  been fully determined   which  molecular  signals  regulate  the    level    of  HK-II at mitochondria.

Thr-473 in HK-II  is phosphorylated by Akt and this phosphorylation  leads to  increases  in  mitochondrial  HK-II binding  through inhibition  of  G-6P-dependent  dissociation, conferring resistance to oxidative stress  (Fig.     7).

Overexpression of  WTHK-II increases mitochondrial HK-II and confers protection against  hydrogen peroxide,  which  is enhanced significantly  in   HK-II   T473D-expressing  cells, whereas  NHK-II, lacking the ability to bind to mitochondria, does not confer protection.   Conversely,  mitochondrial  HK-II from mitochondria (Fig.6, and B) inhibits  the  IGF-1-mediated increase in mitochondrial HK-II and cellular protection.   Similar   dose-dependent  curves were obtained in mitochondrial   HK-II     against stress    (15–25).

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin PhD, Elena V. Dolmatova MD, Michael P. Morley, PhD, Kathryn L. Lunetta PhD, David D. McManus MD, ScM, et al.
Heart Rhythm  2013   http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrialfibrillation (AF). However, the molecular events related to structural and functional changes that
enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic
variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right
atrial tissues.
Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results
implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Long-Term Caspase Inhibition Ameliorates Apoptosis, Reduces Myocardial Troponin-I Cleavage, Protects Left Ventricular Function, and Attenuates Remodeling in Rats With Myocardial Infarction

Y. Chandrashekhar,  Soma Sen, Ruth Anway,  Allan Shuros,  Inder Anand,

J Am Col  Cardiol  2004; 43(2)   http://dx.doi.org/10.1016/j.jacc.2003.09.026

This study was designed to evaluate whether in vivo caspase inhibition can prevent myocardial contractile protein degradation, improve myocardial function, and attenuate ventricular remodeling.
Apoptosis is thought to play an important role in the development and progression of heart failure (HF) after a myocardial infarction (MI). However, it is not known whether inhibiting apoptosis can attenuate left ventricular (LV) remodeling and minimize systolic dysfunction.

A 28-day infusion of caspase inhibitor was administeredimmediately after an anterior MI. In addition, five sham-operated rats given the caspase inhibitor were compared with 17 untreated sham-operated animals to study effects in non-MI rats. Left ventricular function, remodeling parameters, and hemodynamics were studied four weeks later. Myocardial caspase 3 activation and troponin-I contractile protein cleavage were studied in the non-infarct, remote LV myocardium using Western blots. Apoptosis was assessed using immunohistochemistry for activated caspase-positive cells as well as the TUNEL method. Collagen volume was estimated using morphometry.

Caspase inhibition reduced myocardial caspase 3 activation. This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus, and fewer apoptotic cardiomyocytes. Furthermore, caspase inhibition reduced LV-weight-to- body-weight ratio, decreased myocardial interstitial collagen deposition, attenuated LV remodeling, and better preserved LV systolic function after MI.

Caspase inhibition, started soon after MI and continued for four weeks, preserves myocardial contractile proteins, reduces systolic dysfunction, and attenuates ventricular remodeling.

These findings may have important therapeutic implications in post-MI HF. J Am Col Cardiol 2004;43:295–301)

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors

Lincoln T Shenje,  Peter P Rainer , Gun-sik Cho , Dong-ik Lee , Weimin Zhong , Richard P Harvey , David A Kass , Chulan Kwon *,  et al.
eLife 2014.    http://dx.doi.org/10.7554/eLife.02164.001

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, fow cytometric and functional analyses, we fnd that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-defcient CPCs by lineage-specifc mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These fndings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and
that the PA2 serves as a microenvironment for their expansion.

2. Diagnostics and Risk Assessment

Classical and Novel Biomarkers for Cardiovascular Risk Prediction in the United States

Aaron R. Folsom
J Epidemiol 2013;23(3):158-162   http://dx.doi.org/10.2188/jea.JE20120157

Cardiovascular risk prediction models based on classical risk factors identified in epidemiologic cohort studies are useful in primary prevention of cardiovascular disease in individuals. This article briefly reviews aspects of
cardiovascular risk prediction in the United States and efforts to evaluate novel risk factors. Even though many novel risk markers have been found to be associated with cardiovascular disease, few appear to improve risk prediction
beyond the powerful, classical risk factors. A recent US consensus panel concluded that clinical measurement of certain novel markers for risk prediction was reasonable, namely,

  1. hemoglobin A1c (in all adults),
  2. microalbuminuria (in patients with hypertension or diabetes), and
  3. C-reactive protein,
  4. lipoprotein-associated phospholipase,
  5. coronary calcium,
  6. carotid intima-media thickness, and
  7. ankle/brachial index (in patients deemed to be at intermediate cardiovascular risk, based on traditional risk factors).

Diagnostic accuracy of NT-proBNP ratio (BNP-R) for early diagnosis of tachycardia-mediated cardiomyopathy: a pilot study

Amir M. Nia, Natig Gassanov, Kristina M. Dahlem, Evren Caglayan, Martin Hellmich, et al.
Clin Res Cardiol (2011) 100:887–896    http://dx.doi.org/10.1007/s00392-011-0319-y

Tachycardia-mediated cardiomyopathy (TMC) occurs as a consequence of prolonged high heart rate due to ventricular and supraventricular tachycardia. In animal models, rapid pacing induces severe biventricular remodeling with dilation and dysfunction [7]. On a cellular basis, cardiomyocytes exert fundamental morphological and functional roles.

When heart failure and tachycardia occur simultaneously, a useful diagnostic tool for early discrimination of patients with benign tachycardia-mediated  cardiomyopathy (TMC) versus major structural heart disease  (MSHD) is not available. Such a tool is required to prevent unnecessary and wearing diagnostics in patients with reversible TMC. Moreover, it could lead to early additional diagnostics and therapeutic approaches in patients with  MSHD.

A total of 387 consecutive patients with supraventricular arrhythmia underwent assessment.  Of these patients, 40 fulfilled the inclusion criteria
with a resting heart rate C100 bpm and an impaired left ventricular ejection fraction \40%. In all patients, successful electrical cardioversion was performed. At baseline, day 1 and weekly for 4 weeks, levels of NT-proBNP and echocardiographic parameters were evaluated.

NT-proBNP ratio (BNP-R) was calculated as a quotient of baseline NT-proBNP/follow-up NT-proBNP. After 4 weeks, cardiac catheterization was performed to identify patients with a final diagnosis of TMC versus MSHD.

Initial NT-proBNP concentrations were elevated and consecutively decreased after cardioversion in all patients studied. The area under the ROC curve for BNP-R to detect TMC was 0.90 (95% CI 0.79–1.00; p \ 0.001) after 1 week  and 0.995 (95% CI 0.99–1.00; p \ 0.0001) after 4 weeks. One week after cardioversion already, a BNP-R cutoff C2.3 was useful for TMC diagnosis indicated by an accuracy of 90%, sensitivity of 84% and specificity of 95%.

BNP-R was found to be highly accurate for the early diagnosis of TMC.

Omega-3 Index and Cardiovascular Health

Clemens von Schacky
Nutrients 2014; 6: 799-814;  http://dx. doi.org/10.3390/nu602099

Fish, marine oils, and their concentrates all serve as sources of the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as do some products from algae.
To demonstrate an effect of EPA + DHA on heart health, a number of randomized, controlled intervention studies with clinical endpoints like overall mortality or a combination of adverse cardiac events were conducted in populations with elevated cardiovascular risk. One early intervention study with oily fish, rich in EPA + DHA, and some early studies with fish oil or fish oil concentrate or even purified EPA at doses ranging between 0.9 and 1.8 g/day indeed demonstrated effects in terms of fewer sudden cardiac deaths, fatal or non-fatal myocardial infarctions, or a combination of adverse cardiac events.

Recent meta-analyses found no significant benefits on total mortality, cardiovascular mortality, and other adverse cardiac or cardiovascular events [13–18]. This is in contrast to findings in epidemiologic studies, where intake of EPA + DHA had been found to correlate generally with an up to 50% lower incidence of adverse cardiac events [18,19], and in even sharper contrast to epidemiologic studies based on levels of EPA + DHA, demonstrating e.g., a 10-fold lower incidence of sudden cardiac death associated with high levels of the
fatty acids, as compared to low levels.

This seemingly contradictory evidence has led the American Heart Association to recommend “omega-3 fatty acids from fish or fish oil capsules (1 g/day) for cardiovascular disease risk reduction” for secondary prevention, whereas the European Society for Cardiology recommends “Fish at least twice a week, one of which to be oily fish”, but no supplements for cardiovascular prevention.

A similar picture emerges for atrial fibrillation: In epidemiologic studies, consumption of EPA + DHA or higher levels of EPA + DHA were associated with lower risk for developing atrial fibrillation, while intervention studies found no effect. Pertinent guidelines do not mention EPA + DHA. A similar picture also emerges for severe ventricular rhythm disturbances.

Why is it that trial results are at odds with results from epidemiology? What needs to be done to better translate the epidemiologic findings into trial results? The current review will try to shed some light on this  issue, with a special consideration of the Omega-3 Index.

Recent large trials with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the cardiovascular field did not demonstrate a beneficial effect in terms of reductions of clinical endpoints like

  • total mortality,
  • sudden cardiac arrest or
  • other major adverse cardiac events.

Pertinent guidelines do not uniformly recommend EPA + DHA for cardiac patients. In contrast,

  • in epidemiologic findings, higher blood levels of EPA + DHA were consistently associated with a lower risk for the endpoints mentioned.

The following points argue for the use of erythrocytes: erythrocyte fatty acid
composition has a low biological variability, erythrocyte fat consists almost exclusively of phospholipids, erythrocyte fatty acid composition reflects tissue fatty acid composition, pre-analytical stability, and other points.  In 2004, EPA + DHA in erythrocyte fatty acids were defined as the Omega-3 Index and suggested as a risk factor for sudden cardiac death [39]. Integral to the definition was a specific and standardized analytical procedure, conforming the quality management routinely implemented in the field of clinical chemistry.

The laboratories adhering to the HS-Omega-3 Index methodology perform regular proficiency testing, as mandated in routine Clinical Chemistry labs. So far, the HS-Omega-3 Index is the only analytical procedure used in several laboratories. A standardized analytical procedure is a prerequisite to generate the data base necessary to transport a laboratory parameter from research into clinical routine. Moreover, standardization of the analytical procedure is the first important criterion for establishing a new biomarker for cardiovascular risk set forth by the American Heart Association and the US Preventive Services Task Force.

Because of low biological and analytical variability, a standardized analytical procedure, a large database and for other reasons,

  • blood levels of EPA + DHA are frequently assessed in erythrocytes, using the HS-Omega-3 Index methodology.

Table 1. Mean HS-Omega-3 Index values in various populations, Mean (±standard deviation (SD)). Please note that in every population studied, a lower value was found to be associated with a worse condition than a higher value. References are given, if not, unpublished, n = number of individuals measured.

All levels of fatty acids are determined by the balance of substance entering the body and those leaving the body. Neither a recent meal, even if rich in EPA + DHA, nor severe cardiac events altered the HS-Omega-3 Index. However, while long-term intake of EPA + DHA, e.g., as assessed with food questionnaires, was the main predictor of the HS-Omega-3 Index, long-term intake explained only 12%–25% of its variability. A hereditary component of 24% exists. A number of other factors correlated positively (+) or negatively (−), like age (+), body mass index (−), socioeconomic status (+), smoking (−), but no other conventional cardiac risk factors. More factors determining the level of the HS-Omega-3 Index, especially regarding efflux remain to be  defined. Therefore, it is impossible to predict the HS-Omega-3 Index in an individual, as it is impossible to predict the increase in the HS-Omega-3 Index in an individual in response to a given dose of EPA + DHA. In Table 2, current evidence is presented on the relation of the HS-Omega-3 Index to CV events.

The HS-Omega-3 Index has made it possible to reclassify individuals from intermediate cardiovascular risk into the respective high risk and low risk strata, the third criterion for establishing a new biomarker for CV  risk.

A low Omega-3 Index fulfills the current criteria for a novel cardiovascular risk factor.

Increasing the HS-Omega-3 Index by increased intake of EPA + DHA in randomized controlled trials improved a number of surrogate parameters for cardiovascular risk:

  1. heart rate was reduced,
  2. heart rate variability was increased,
  3. blood pressure was reduced,
  4. platelet reactivity was reduced,
  5. triglycerides were reduced,
  6. large buoyant low-density lipoprotein (LDL)-particles were increased and
  7. small dense LDL-particles were reduced,
  8. large buoyant high-density lipoproteins (HDL)2 were increased,
  9. very low-density lipoprotein (VLDL1) + 2 was reduced,
  10. pro-inflammatory cytokines (e.g., tumor necrosis factor alpha, interleukin-1β, interleukins-6,8,10 and monocyte chemoattractant protein-1) were reduced,
  11. anti-inflammatory oxylipins were increased.

Importantly, in a two-year randomized double-blind angiographic intervention trial, increased erythrocyte EPA + DHA

  • reduced progression and increased regression of coronary lesions, an intermediate parameter.

Taken together, increasing the HS-Omega-3 Index improved surrogate and intermediate parameters for cardiovascular events. A large intervention trial with clinical endpoints based on the HS-Omega-3 Index remains to be conducted. Therefore, the fourth criterion, proof of therapeutic consequence of determining the HS-Omega- Index, is only partially fulfilled.

 

Neutral results of intervention trials can be explained by issues of bioavailability and trial design that surfaced after the trials were initiated.

In the future, incorporating the Omega-3 Index into trial designs by

  1. recruiting participants with a low Omega-3 Index and
  2. treating them within a pre-specified target range (e.g., 8%–11%),
  3. will make more efficient trials possible and
    • provide clearer answers to the questions asked than previously possible.

 

3. Stem Cells and Regenerative Biology

Adult Stem Cells Reverse Muscle Atrophy In Elderly Mice   http://www.science20.com/profile/news_staff

Bioengineers at the University of California, Berkeley in a new study published in Nature say they have identified two key regulatory pathways that control how well adult stem cells repair and replace damaged tissue. They then tweaked how those stem cells reacted to those biochemical signals to revive the ability of muscle tissue in old mice to repair itself nearly as well as the muscle in the mice’s much younger counterparts. Irina Conboy, an assistant professor of bioengineering and an investigator at the Berkeley Stem Cell Center and at the California Institute for Quantitative Biosciences (QB3), led the research team conducting this study. Because the findings relate to adult stem cells that reside in existing tissue, this approach to rejuvenating degenerating muscle eliminates the ethical and medical complications associated with transplanting tissues grown from embryonic stem cells. The researchers focused on

  • the interplay of two competing molecular pathways that control the stem cells,

which sit next to the mature, differentiated cells that make up our working body parts. When the mature cells are damaged or wear out, the stem cells are called into action to begin the process of rebuilding.

old muscle tissue is left with

old muscle tissue is left with

 

 

 

 

 

 

 

 

 

 

 

 

“We don’t realize it, but as we grow our bodies are constantly being remodeled,” said Conboy. “We are constantly falling apart, but we don’t notice it much when we’re young because we’re always being restored. As we age, our stem cells are prevented, through chemical signals, from doing their jobs.” The good news, the researchers said, is that

  • the stem cells in old tissue are still ready and able to perform their regenerative function
  • if they receive the appropriate chemical signals.

Studies have shown that when old tissue is placed in an environment of young blood, the stem cells behave as if they are young again. “Conversely, we have found in a study published last year that even young stem cells rapidly age when placed among blood and tissue from old mice,” said Carlson, who will stay on at UC Berkeley to expand his work on stem cell engineering.

  • Adult stem cells have a receptor called Notch that, when activated,
  • tells them that it is time to grow and divide
  • stem cells also have a receptor for the protein TGF-beta
  • that sets off a chain reaction activatingthemoleculepSmad3 and
    • ultimately producing cyclin-dependent kinase (CDK) inhibitors, which regulate the cell’s ability to divide.
  • activated Notch competeswithactivatedpSmad3 for
    • binding to the regulatory regions of the same CDK inhibitors in the stem cell

“We found that Notch is capable of physically kicking off pSmad3 from the promoters for the CDK inhibitors within the stem cell’s nucleus, which tells us that a precise manipulation of the balance of these pathways would allow the ability to control stem cell responses.” Notch and TGF-beta are well known in molecular biology, but Conboy’s lab is the first to connect them to the process of aging, and the first to show that they act in opposition to each other within the nucleus of the adult stem cell. Aging and the inevitable march towards death are, in part, due to the progressive decline of Notch and the increased levels of TGF-beta , producing a one-two punch to the stem cell’s capacity to effectively rebuild the body, the researchers said.

The researchers disabled the “aging pathway” that tells stem cells to stop dividing by using an established method of RNA interference that reduced levels of pSmad3. The researchers then examined the muscle of the different groups of mice one to five days after injury to compare how well the tissue repaired itself. As expected,

  •  muscle tissue in the young mice easily replaced damaged cells with new, healthy cells. In contrast,
  • the areas of damaged muscle in the control group of old mice were characterized by fibroblasts and scar tissue. However,
  • muscles in the old mice whose stem cell “aging pathway”had been dampened showed levels of cellular regeneration that were
    • comparable to their much younger peers, and that were 3 to 4 times greater than those of the group of “untreated” old mice.

Adult Stem Cells To Repair Damaged Heart Muscle

http://www.science20.com/profile/news_staff

In the first trial of its kind in the world, 60 patients who have recently suffered a major heart attack will be injected with selected stem cells from their own bone marrow during routine coronary bypass surgery. The Bristol trial will test

  • whether the stem cells will repair heart muscle cells damaged by the heart attack,
  • by preventing late scar formation and hence impaired heart contraction.

“ Cardiac stem cell therapy aims to repair the damaged heart as it has the potential to replace the damaged tissue.” We have elected to use a very promising stem cell type selected from the patient’s own bone marrow. This approach ensures no risk of rejection or infection. It also gets around the ethical issues that would result from use of stem cells from embryonic or foetal tissue.

In this trial (known as TransACT), all patients will have bone marrow harvested before their heart operation. Then either stem cells from their own bone marrow or a placebo will be injected into the patients’ damaged hearts during routine coronary bypass surgery. The feasibility and safety of this technique has already been demonstrated. As a result of the chosen double blind placebo-controlled design, neither the patients nor the surgeon knows whether the patient is going to be injected with stem cells or placebo. This ensures that results are not biased in any way, and is the most powerful way to prove whether or not the new treatment is effective.

Research of Stem Cells Repair Damaged Heart

By Kelvinlew Minhan | March 26th 2008

Under highly specific growth conditions in laboratory culture dishes, stem cells

  • can be coaxed into developing as new cardiomyocytes and vascular endothelial cells (Kirschstein and Skirboll, 2001).

Discoveries that have triggered the interest in the application of adult stem cells to heart muscle repair in animal models have been made by researchers in the past few years (Kirschstein and Skirboll, 2001). One  study demonstrated that cardiac tissue can be regenerated in the mouse heart attack model through the introduction of adult stem cells from mouse bone marrow (Kirschstein and Skirboll, 2001). These cells were transplanted into the marrow of irradiated mice approximately 10 weeks before the recipient mice were subjected to heart attack thru tying off different major heart blood vessel, the left anterior descending (LAD) coronary artery. The survival rate was 26 percent at two to four weeks after the induced cardiac injury (Kirschstein and Skirboll, 2001). Another study of the region surrounding the damaged tissue in surviving mice showed the presence of donor-derived cardiomyocytes and endothelial cells (Kirschstein and Skirboll, 2001).

  • the mouse hematopoietic stem cells transplanted into the bone marrow had migrated to the border part of the damaged area, and differentiated into several types of tissue for cardiac repair.

Regenerating heart tissue through stem cell therapy

http://www.mayo.edu/research/discoverys-edge/regenerating-heart-tissue-stem-cell-therapy

Summary

A groundbreaking study on repairing damaged heart tissue through stem cell therapy has given patients hope that they may again live active lives. An international team of Mayo Clinic researchers and collaborators has done it by discovering a way to regenerate heart tissue.

“It’s a paradigm shift,” says Andre Terzic, M.D., Ph.D., director of Mayo Clinic’s Center for Regenerative Medicine and senior investigator of the stem cell trial. “We are moving from traditional medicine, which addresses the symptoms of disease to cure disease.” Treating patients with cardiac disease has typically involved managing heart damage with medication.  In collaboration with European researchers, Mayo Clinic researchers have discovered a novel way to repair a damaged heart. In Mayo Clinic’s breakthrough process,
  • stem cells are harvested from a patient’s bone marrow.
  •  undergo a laboratory treatment that guides them into becoming cardiac cells,
  • which are then injected into the patient’s heart in an effort to grow healthy heart tissue.
The study is the first successful demonstration in people of the feasibility and safety of transforming adult stem cells into cardiac cells. Beyond heart failure, the Mayo Clinic research also is a milestone in the emerging field of regenerative medicine, which seeks to fully heal damaged tissue and organs.

Creating a heart repair kit

Process of converting bone marrow cells to heart cells
This image shows the process used in the clinical trials to repair damaged hearts. Cardioprogenitor cells is another term for cardiopoietic cells, those that were transformed into cardiac cells.
Stem cells transforming to cardiac tissue
Transformation: The cardiopoietic cells on the left react to the cardiac environment, cluster together with like cells and form tissue.
 Mayo Clinic researchers pursued this research, inspired by an intriguing discovery. In the early 2000s, they analyzed stem cells from 11 patients undergoing heart bypass surgery. The stem cells from two of the patients had an unusually high expression of certain transcription factors — the proteins that control the flow of genetic information between cells. Clinically, the two patients appeared no different from the others, yet their stem cells seemed to show unique capacity for heart repair.
That observation drove them to  determine how to convert  nonreparative stem cells to become reparative. Doing so required determining precisely how the human heart naturally develops, at a subcellular level. That painstaking work was led by Atta Behfar, M.D., Ph.D., a cardiovascular researcher at Mayo Clinic in Rochester, Minn. With other members of the Terzic research team, Dr. Behfar identified hundreds of proteins involved in the process of heart development (cardiogenesis). The researchers then set out to identify which of these proteins are essential in driving a stem cell to become a cardiac cell. Using computer models,
  • they simulated the effects of eliminating proteins one by one from the process of heart development.
  • That method yielded about 25 proteins.
    • The team then pared that number down to 8 proteins that their data indicated were essential.
The research team was then able to develop the lab procedure that guides stem cells to become heart cells.
The treated stem cells were dubbed cardiopoietic, or heart creative. A proof of principle study about guided cardiopoiesis, whose results were published in the Journal of the American College of Cardiology in 2010, demonstrated that animal models with heart disease that had been injected with caridiopoietic cells had improved heart function compared with animals injected with untreated stem cells. Hailed as “landmark work,” by the journal’s editorial writer, the study showed it was indeed possible to teach stem cells to become cardiac cells. Stem cells from each patient in the cardiopoiesis group were successfully guided to become cardiac cells. The treated cells were injected into the heart wall of each of those patients without apparent complications.
“Ihis newprocessofcardiopoiesiswas achieved in 100 percent of cases, with a very good safety profile,” Dr.Terzic says. “We are enabling the heart toregainitsinitial structure and function,” Dr.Terzic says, “and we will not stop here.” The clinicaltrialfindingsareexpectedto be published in the Journal of the American College of Cardiology in 2013.  Meanwhile, research to improve the injection process and effectiveness is underway.

Stem Cells from Humans Repair Heart Damage in Monkeys

GEN News Highlights  May1, 2014

GPCR Insights Brighten Drug Discovery Outlook

Ken Doyle, Ph.D.

GEN Apr 15, 2014 (Vol. 34, No. 8)

Recent years have seen major advances in understanding the structure-function relationships of G protein-coupled receptors (GPCRs). This large superfamily of transmembrane receptors comprises over 800 members in humans.

GPCRs regulate a wide variety of physiological processes including

  • sensation (vision, taste, and smell),
  • growth,
  • hormone responses, and
  • regulation of the immune and
  • autonomic nervous systems.

Their involvement in multiple disease pathways makes GPCRs attractive targets for drug discovery efforts.

These multifaceted proteins will be the subject of “GPCR Structure, Function and Drug Discovery,” a Global Technology Community conference scheduled to take place May 22–23 in Boston. The conference is expected to cover a broad range of topics including biased signaling, membrane protein structures, GPCR signaling dynamics, computational approaches to disease.

According to Bryan Roth, M.D., Ph.D., Michael Hooker Distinguished Professor at the University of North Carolina, Chapel Hill,

  • drugs that can selectively target various downstream GPCR pathways hold the most promise.

Dr. Roth’s laboratory studies approximately 360 different GPCRs with therapeutic potential using massively parallel screening methods. His research focuses on “functional selectivity,” which he describes as

  • “the ligand-dependent selectivity for certain signal transduction pathways in one and the same receptor.”

Dr. Roth notes that structural data have demonstrated that GPCRs exist in multiple conformations: “The structures of the 5-hydroxytryptamine 2B receptor and the recent high-resolution delta-opioid receptor structure have provided evidence for conformational rearrangements that contribute to functional selectivity.” Drugs that take advantage of this selectivity by preferentially stabilizing certain conformations may have unique therapeutic utility.

“Generally, we look at G protein versus arrestin-based signaling, although it’s also possible to examine how drugs activate one G protein-mediated signaling pathway versus another.

 

fluorescently tagged Arrestin and GPRC of interest

fluorescently tagged Arrestin and GPRC of interest

 

 

 

 

 

 

 

  • β-Arrestins constitute a major class of intracellular scaffolding proteins that regulate GPCR signaling by preventing or enhancing the binding of GPCRs to intracellular signaling molecules. Laura Bohn, Ph.D., associate professor at Scripps Florida,  studies the roles that β-arrestins play in GPCR-mediated signaling.
  • a particular β-arrestin can play multiple, tissue-specific roles—shutting down the signaling of a receptor in one tissue while activating signaling in another.
  • different ligands can direct GPCR signaling to different effectors, which could result in different physiological effects,” comments Dr. Bohn. “Our challenge is in determining what signaling pathways to harness to promote certain effects, while avoiding others.”
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

 

 

 

 

 

 

 

 

 

 

 

Using Designer Proteins

The multifunctional signaling abilities of β-arrestins has prompted large-scale study of their properties. Vsevolod Gurevich, Ph.D., professor of pharmacology at Vanderbilt University, studies

  1. the structure,
  2. function, and
  3. biology of arrestin proteins.

β-arrestins have three main functions.

  1. First, they prevent the coupling of GPCRs to G proteins, thereby blocking further G protein-mediated signaling (a process known as desensitization).
  2. Second, the binding of a GCPR releases the β-arrestin’s carboxy-terminal “tail” and promotes internalization of the receptor.
  3. Third, receptor-bound β-arrestins bind other signaling proteins, resulting in a second wave of arrestin-mediated signaling.

Dr. Gurevich’s laboratory studies β-arrestin biology through the use of three types of specially designed mutants—

  1. enhanced phosphorylation-dependent,
  2. receptor-specific, and
  3. signaling-biased mutants.

an enhanced mutant of visual β-arrestin-1 partially compensates for defects of rhodopsin phosphorylation in vivo,

“Several congenital disorders are caused by mutant GPCRs that cannot be normally phosphorylated because they have lost GPCR kinase (GRK) sites. Enhanced super-active arrestins have the potential to compensate for these defects, bringing the signaling closer to normal.”

  • Dr. Gurevich explains the strategy involved in creating designer β-arrestins: “We identify residues critical for individual β-arrestin functions by mutagenesis, using limited structural information as a guide.
  • We also work on getting more structural information. In collaboration with different crystallographers, we solved the crystal structures of all four vertebrate β-arrestin subtypes in the basal state, as well as the structure of the arrestin-1-rhodopsin complex.”
  • Dr. Gurevich believes that designer β-arrestins “are the next step in research and therapy, moving way beyond what small molecules can achieve.
  • The difference in capabilities between redesigned signaling proteins, including β-arrestins, and conventional small molecule drugs is about the same as that between airplanes and horse-driven carriages.”
  • Dr. Gurevich observes that redesigned signaling proteins face considerable obstacles in terms of gene delivery, but that the efforts are worth it. “Using designer signaling proteins, we can tell the cell what to do in a language it cannot disobey,” asserts Dr. Gurevich.

Synthesis and Antihypertensive Screening of Novel Substituted 1,2- Pyrazoline Sulfonamide Derivatives

Avinash M. Bhagwat , Anilchandra R. Bha , Mahesh S. Palled , Anand P. Khadke , Anuradha M. Patil, et al.

Am. J. PharmTech Res. 2014; 4(2).    http://www.ajptr.com/ 

Angiotensin II receptor antagonists, also known as angiotensin receptor blockers , AT1-receptor antagonists or sartans, are a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system. Their main use is in hypertension, diabetic nephropathy and congestiveheart failure. These substances are AT1-receptor antagonists which

  • block the activationof angiotensin II AT1 receptors.

Blockade of AT1 receptors directly causes

1 vasodilation,

2 reduces secretion of vasopressin,

3 reduces production and secretion of aldosterone, amongst other actions –

4 the combined effect of which is reduction of blood pressure.

Irbesartan is a safe and effectiveangiotensin II receptor antagonist with an affinity for the AT1 receptor that is more than 8,500times greater than its affinity for AT2 receptor. This agent has a higher bioavailability (60-80%) than other drugs in its class . In both Losartan and Irbesartan structures imidazole moiety is being present. A structure analog of losartan and Irbesartan are designed by incorporating the heterocycles like pyrazoline group. We felt it would be interesting to explore the possibilities of 1,2-pyrazoline derivatives for Angiotensin II receptor antagonistic activity.

The Irbesartan structure was a modified Losartan structure, which had all the identity of a Losartan molecule but with groups that would fit the hydrophobic cavity with a tetramethylene group and an alkyl side chain that would fit in the pocket in the AT1 receptor. The hydroxyl methyl group of Losartan being replaced with carbonyl group of Irbesartan. With a view to introduce a hydrogen bonding interaction with AT1 receptor, these structures were further modified with a view of retaining both hydrogen bonding characteristics and as well as lipophilic groups. Losartan and Irbesartan structure contains a diphenyl molecule & imidazole ring.

In Losartan and Irbesartan diphenyl molecule is attached to the nitrogen of the imidazole ring. It is interesting to to see the activity of compounds containing two phenyl rings attached at two different positions namely3,5 position of 1, 2-pyrazoline ring. The sulphonamide derivatives known for its diuretics activity which reduces renal hypertension. We use to synthesize sulphonamide and pyrazoline in one molecule to check its possible Angiotensin II receptor antagonist property. For this reason chalcones were synthesized reacted with hydrazine hydrate to yield the corresponding 1,2-pyrazoline derivatives which further condensed with sulphanilamide and formaldehyde by mannich condensation reaction.

Acute Toxicity Study (LD50)

This study was carried out in order to establish the therapeutic and toxic doses of the newly synthesized 1,2 pyrazoline derivatives. To establish LD50 of these compounds the method described by Miller & Tainter was employed.

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More on the Performance of High Sensitivity Troponin T and with Amino Terminal Pro BNP in Diabetes

Writer and Curator: Larry H. Bernstein, MD, FCAP

This is the final up to date review of the status of hs troponin T (or I) with or without the combined use of the Brain Type Natriuretic Peptide or its Amino Terminal peptide precursor.  In addition, a new identification of the role of the Atrial Natriuretic Peptide has been reported with respect to arrythmogenic activity.  On the one hand, the diagnostic value of the NT-proBNP has been seen as disappointing, in part because of the question of what information is gained by the test in overt known congestive heart failure, and in part because of uncertainty about following the test during a short hospital stay.  At least, this is the view of this reviewer.  However, in the last several years there has been an emphasis on the value this test adds to prediction of adverse outcomes.   In addition, there has been a hidden nvariable that has much to do with the original reference values that were established for age ranges, without any consideration of pathophysiology that might affect the values within those ranges, leading one to consider values in an aging population as normal, that might well be high.  Why is this?  Aging patients are more likely to have hypertension, and also the onset of type-2 diabetes mellitus, with cardiovascular disease consequences.  Type-2 diabetes mellitus (T2DM), for instance, is associated with insulin resistance and also fat gain with generation of adipokines, but the is also a hyalinization of insulin forming beta-cells of the pancreas, and there is hyalinization of glomeruli (glomerulosclerosis) and afferent arteriolonephrosclerosis with expected decline in glomerular filtrattion rate and hypertension as well.   Of course, this is also associated with hepatosteatosis.   Nevertheless, a reference range is established that takes none of this pathophysiology into account.   While a more reasonable approach has been pointed out, there has been no followup in the literature.

On the other hand, there has been much confusion over the restandardization of a high sensitivity troponin I or T test (hs-Tn(I or T).  The reference range declines precipitously, and there is a good identification of patients who are for the most part disease free, but there is no delineation of patients who are at high risk of acute coronary syndrome with plaque rupture, vs a  host of other cardiovascular conditions.  These have no relationship to plaque rupture, but may be serious and require further evaluation.  The question then becomes whether to admit for a hospital stay, to refer to clinic after an evaluation in the ICU without admission, or to do an extensive evaluation in the emergency department overnight before release for followup.  There is still another dimension of this that has to do with prediction of outcomes using hs-Tn(s) with or without the natriuretic peptides.  Another matter that is not for discussion in this article is the underutilization of hs-CRP.  Originally used for a marker of sepsis in the 1970s, it has come to be tied in with identification of an ongoing inflammatory condition.  Therefore, the existence of a known inflammatory condition in the family of autoimmune diseases, with one exception, might make it unnecessary.

The discussion is broken into three parts:

Part 1.   New findings on the troponins.
Part 2.  The use of combined hs-Tn with a natriuretic peptide (NT-proBNP)
Part 3.  Atrial natriuretic peptide

Part 1.    New findings on the troponins.

Troponin: more lessons to learn

C Liebetrau,HM Nef,andCW.Hamm*
KerckhoffHeartandThoraxCenter;DepartmentofCardiology,BadNauheim,
Germany; (GermanCentreforCardiovascularResearch),partnersite
RheinMain,BadNauheim, Germany; and UniversityofGiessen,Medizinische
KlinikI,KardiologieundAngiologie,Giessen,Germany
European Hear tJournal
http://dx.doi.org/10.1093/eurheartj/eht357This editorial refers to ‘Risk stratification in patients with acute chest pain
using three high-sensitivity cardiac troponin assays’,
by P. Haafetal. http://dx.doi.org/10.1093/eurheartj/eht218Cardiac troponin entered our diagnostic armamentarium 20 years ago and –
unlike any other biomarker –

  • is going through constant expansion in its application.

Troponin started out as a marker of risk in unstable angina’, then was used

  • as gold standard for risk stratification and therapy guiding in acute coronary syndrome
  •  served further to redefine myocardial infarction, and
  • has also become a risk factor in apparently healthy subjects.

The recently introduced high-sensitivity cardiac troponin (hs-cTn) assays

  • have not only expanded the potential of troponins, but
  • have also resulted in a certain amount of confusion
    • among unprepared users.

After many years troponins were accepted as the gold standard in

  • patients with chest pain by
  • classifying them into troponin-positive and
    • troponin-negative patients.

The new generation of hs-cTn assays has

  • improved the accuracy at the lower limit of detection and
  • provided incremental diagnostic information especially
    • in the early phase of myocardial infarction.

Moreover, low levels of measurable troponins

  • unrelated to ACS have been associated with
    • an adverse long-term outcome.

Several studies demonstrated that

  • these low levels of cardiac troponin measureable 
    • only by hs-Tn assays
  • are able to predict mortality in patients with ACS
  • as well as patients with assumed
    • stable coronary artery disease.

Furthermore, hs-cTn has the potential

  • to play a role in the care of patients
    • undergoing non-cardiac surgery.

The additional determination of hs-cTn

  • improves risk stratification despite
  • established risk scores providing both diagnosis and
  • for prognosis prediction in chest pain patients.

The daily clinical challenge in using the highly sensitive assays is to 

  • interpret the troponin concentrations, especially
  • in patients with concomitant diseases
    • independently from myocardial ischaemia
  • influencing cardiac troponin concentrations
    (e.g. chronic kidney disease, or stroke). 

The troponin test lost its ‘pregnancy test’ quality with the different users.
Different opinions exist on

  • the change of hs-cTn levels compared to simple ‘positive–negative’ interpretation
  • and thereby makes diagnosis finding more complex than before.

This uncertainty probably has the paradigm that

  • serial measurements of troponins are necessary, and also
    • boosted the number of diagnoses of ACS and
    • invasive diagnostic procedures in some locations.

This is more than understandable, with acute chest pain using

  • three high-sensitivity cardiac troponins with their respective baseline value
    • before the diagnosis of acute myocardial infarction (AMI) can be made.

What is a relevant change in concentrations compatible with acute myocardial necrosis and

  • what is only biological variation for the specific biomarker and assay?

Changes in serial measurements between 20% and 200% have been debated, and
the discussion is ongoing. Furthermore, it has been proposed that

  • absolute changes in cardiac troponin concentrations 
    • have a higher diagnostic accuracy for AMI
  • compared with relative changes, and

it might be helpful in distinguishing AMI from other causes of cardiac troponin elevation.

Do we obtain any helpful directives from experts and guidelines for our daily practice?
Foreseeing this dilemma, the 2011 European Society of Cardiology (ESC) Guidelines

  • on non ST-elevation ACS acted.
  • Minor elevations of  troponins were accepted as hs-cTn values in the ‘grey zone’.

This was and still is the rule, but

  • the ESC provided a general algorithm on how to manage patients with limited data.

The ‘Study Group on Biomarkers in Cardiology’ suggested

  • a rise of 50% from the baseline value at low concentrations.

However, this group of experts could also not find a substitute for the missing data

  • needed to validate the proposed recommendation.

The story is just too complex:

  • different troponin assays with
  • different epitope targets,
  • different patient populations,
  • different sampling protocols,
  • different follow-up lengths, and much more.

Therefore, any study that helps us to see better through the fog is welcome here.

Haaf et al. have now presented the results of their study of

  • different hs-cTn assays
    (hs-cTnT, Roche Diagnostics; hs-cTnI, Beckman-Coulter; and  hs-cTnI, Siemens)

    • with respect to the -outcome of patients with acute chest pain.

The authors examine 1117 consecutive patients presenting with acute chest pain.
[340 patients with ACS (30.5%)] from the Advantageous Predictors of Acute Coronary Syndrome
Evaluation (APACE) study. Blood was collected

  • directly on admission and
  • serially thereafter at 2, 3, and 6h.

Eighty-two patients (7.3%) died during the 2-year follow-up. The main finding of the study is that

  1. hs-cTnT predicts mortality more accurately than the hs-cTnI assays, 
  2. -that a single measurement is sufficient
  3. challenges causes of cardiac troponin elevation.

These results of APACE remain in contrast to recent findings from a GUSTO IV cohortof 1335 patients with ACS (Table1).

Table1 Studies investigating high sensitivity troponins for long-term prognosis

Variable                                                       APACE (n 5 1117)              GUSTO IV (n 5 1335)              PEACE (n 5 3567)

………………………………………………………………………………………………………………………………………………………….

Patient cohort                                                   Unstable                            Unstable                               Stable

Blood sampling                                     On admission,1,2,3,6h                    48h after
study randomization           Before randomization

No. of patients with detection limit             883 (79.1%)                                 UKN                                      UKN

No. of patients with hs-cTnT.
99thpercentile                                        401 (35.9%)                              1015 (76%)                          395 (10.9%)

No. of patients with hs-cTnI (Abbott).
detection limit                                           UKN                                             UKN                              3567 (98.5%)

No.of patients with hs-cTnI (Abbott).
99th percentile                                          UKN                                         988(74%)                           105 (2.9%)

No. of patients with NSTEMI                     170 (15.2%)                              100 (100%)                             0 (0%)

Follow-up                                               24 months                                  12 months                            5.2 years

Non-fatal AMI                                           UKN                                              UKN                               209 (5.9%)

Mortality or primary endpoint                    82 (7.3%)                                 119(8.9%)                           203 (5.7%)

………………………………………………………………………………………………………………………………………………………….

Key findings                                    cTnT better than cTnI                      cTnI ¼cTnT                   cTnI better than cTnT

Single cTn sample sufficient

AMI, acute mycordial infaction; cTn, cardiac tropononin; NSTEMI ,non-ST-elevation myocardial infarction; UKN, unknown

NSTEMI defined in the GUSTO IV trial:
  1. one or more episodes of angina lasting ≥ 5min,
  2. within 24h of admission and
  3. either a positive cardiac TnT or I test
    (above the upper limit of a normal for the local assay; during the years 1999 and 2000)
  4. or ≥ 0.5 mm of transient or persistent ST-segment depression.

the prognostic capacity of four different sensitive cardiac troponin assays were compared

  1. hs-cTnT; Roche Diagnostics,
  2. cTnI and hs-cTnI;
  3. Abbott Diagnostics, and
  4. Acc-cTnI; Beckman-Coulter.

In total, 119 patients (8.9%) died during the 1-year follow-up. Looking at their

  • receiver operating characteristic curve (ROC) analyses,
  • there were only negligible diffferences
    • in the area under the curves between the assays.

Contrasting results have also been reported in patients(n 1/4 3.623)

  • with stable coronary artery disease and preserved systolic left ventricular function

from the PEACE trial (Table1).

During a median follow-up period of 5.2 years,

  • there were 203 (5.6%) cardiovascular deaths or
  • first hospitalization for heart failure.

Concentrations of hs-cTnI (Abbott Diagnostics) at or above

  • the limit of detection of the assay were measured in 3567 patients (98.5%), but
  • concentrations of hs-cTnI at or above the gender-specific 99th percentile
    • were found in only 105 patients (2.9%).

This study revealed that

  • there was a strong and graded association
  • between increasing quartiles of hs-cTnI concentrations and
  • the risk for cardiovascular death or heart failure.

Hs-cTnI provided incremental prognostication information

  • over conventional risk markers and
  • other established cardiovascular biomarkers,
  • including hs-cTnT.

In contrast to the APACE results, only hs-cTnI, but

  • no ths-cTnT, was significantly
  • associated with the risk for AMI.

Is there a real difference between cardiac troponin T and cardiac troponin I

  • in predicting long term prognosis?

The question arises of whether there is a true clinically relevant

  • difference between cTnT and cTnI.

Given the biochemical and analytical differences,the two

  • troponins display rather similar serum profiles during AMI.

While minor biological differences between cTnT and cTnI are

  • apparently not relevant for diagnosis
  • and clinical management in the acute setting of ACS.

This is a provocative theory, but appears premature in our opinion.
Above all, the results of the current study appear

  • too inconsistent to allow such conclusions.

In the present study, hs-cTnT (Roche Diagnostics) outperformed

  • hs-cTnI (Siemens and Beckman-Coulter) in terms of
  • very long term prediction of cardiovascular death and
    • heart failure in stable patients.

We don’t know how hs-cTnI from Abbott Diagnostics

  • performs in the APACE consort.

The number of patients and endpoints provided

  • by the APACE registry are rather low.
  • The results could, therefore, be a chance finding.

It is far too early to favour one high sensitivity assay over the other. The findings need confirmation.

Implications for clinical practice

There is no doubt that high-sensitivity assays

  • are the analytical method of choice
    • in terms of risk stratification in patients with ACS.

What is new?
A single measurement of hs-cTn seems to be adequate

  • for long-term risk stratification in patients without AMI.

However, the question of which troponin might be preferable

  • for long-term risk stratification remains unanswered.

Part 2. ability of high-sensitivity cTnT and NT pro-BNP to predict cardiovascular events and death in patients with T2DM

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; Zoungas S; Poulter N; Mancia G; Williams B; Sattar N; Woodward M
Diabetes Care.  2014; 37(1):295-303 (ISSN: 1935-5548)

OBJECTIVE

Current methods of risk stratification in patients with

  • type 2 diabetes are suboptimal.

The current study assesses the ability of

  • N-terminal pro-B-type natriuretic peptide (NT-proBNP) and
  • high-sensitivity cardiac troponin T (hs-cTnT)

to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease:

Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial.

RESULTS

Seven hundred nine (18%) patients experienced a

  • major cardiovascular event

(composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and

  • 706 (18%) died during a median of 5 years of follow-up.

In Cox regression models, adjusting for all established risk predictors,

  • the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and
  • the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were
    • 1.97 (95% CI 1.73, 2.24) and
    • 1.52 (95% CI 1.37, 1.67), respectively.

The addition of either marker improved 5-year risk classification for cardiovascular events
(net reclassification index in continuous model,

  • 39% for NT-proBNP and 46% for hs-cTnT).

Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted.
The combination of both markers provided optimal risk discrimination.

CONCLUSIONS

NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the

  • risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

PreMedline Identifier: 24089534


Part 3. M-Atrial Natriuretic Peptide

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure:
Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
Journal of the American Heart Association 01/2014; 3(1):e000206. http://dx.doi.org/10.1161/JAHA.113.000206
Source: PubMed

ABSTRACT

Systemic hypertension is a common characteristic in

  • acute heart failure (HF).

This increasingly recognized phenotype

  • is commonly associated with renal dysfunction and
  • there is an unmet need for renal enhancing therapies.

In a canine model of HF and acute vasoconstrictive hypertension

  • we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP),
    a novel particulate guanylyl cyclase (pGC) activator, and
  • nitroglycerin, a soluble guanylyl cyclase (sGC) activator.

HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II
infusion. We characterized the cardiorenal and humoral actions

  • prior to,
  • during, and
  • following intravenous infusions of
  1. M-ANP (n=7),
  2. nitroglycerin (n=7),
  3. and vehicle (n=7) infusion.

Mean arterial pressure (MAP) was reduced by

  1. M-ANP (139±4 to 118±3 mm Hg, P<0.05) and
  2. nitroglycerin (137±3 to 116±4 mm Hg, P<0.05);

similar findings were recorded for

  1. pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05)
  2. and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05).

M-ANP enhanced renal function with significant increases (P<0.05) in

  • glomerular filtration rate (38±4 to 53±5 mL/min),
  • renal blood flow (132±18 to 236±23 mL/min), and
  • natriuresis (11±4 to 689±37 mEq/min) and
  • also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas

nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.

Our results advance

the differential cardiorenal actions of

  • pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation.

These distinct renal and aldosterone modulating actions make

M-ANP an attractive therapeutic for HF with concomitant hypertension, where

  • renal protection is a key therapeutic goal.

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Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)


Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)

Curator:  Larry H. Bernstein, MD, FCAP

This is a multipart article that develops the pathological effects of type-2 diabetes in the progression of a systemic inflammatory disease with a development of neuropathy, and fully developing into cardiovascular disease.  It also identifies a systemic relationship to the development of chronic obstructive pulmonary disease (COPD).

The more we learn about diabetes, we learn about its generalized systemic effects.

This article has the following SIX Parts:

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis of ischemic heart disease in patients with diabetes mellitus

Part 2. A Longitudinal Cohort Study of the Cardiovascular Experience of Individuals at High Risk for Diabetes

Part 3.  Clinical significance of cardiovascular dysmetabolic syndrome

Part 4.   Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Part 5.   How to use C-reactive protein in acute coronary care

Part 6.  Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

INTRODUCTION

Type 2 diabetes mellitus is a common chronic disease which develops insidiously over time, and is associated with obesity, nutritional imbalance (high fructose beverages, high starch and processed foods, carbohydrate excess intake, and an imbalance of proinflammatory to anti-inflammatory polyunsaturated  fatty acids), which makes it an acquired and manageable disease.  The long term effects of T2DM is played out on cardiovascular disease and stroke-risk, obstructive sleep apnea, progressive renal insufficiency, development of neuropathy, congestive heart failure and chronic obstructive pulmonary disease, all of which are occuring related to an systemic inflammatory condition that proceeds for some time prior to the identification of overt diabetes.
A detailed story of a significant part of these associations continues in the SIX Part series.

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis of ischemic heart disease in patients with diabetes mellitus

This article is an abstract only of a related publication of the pathogenesis of autonomic neuropathy in diabetics leading to ischemic heart disease.

Subjects: Medicine (General), Medicine, Medicine (General),
Health Sciences Authors: Popović-Pejičić Snježana, Todorović-Đilas Ljiljana, Pantelinac Pavle
Publisher: Društvo lekara Vojvodine Srpskog lekarskog društva
Publication: Medicinski Pregled 2006; 59(3-4): Pp 118-123 (2006) ISSN(s): 0025-8105  Added to DOAJ: 2010-11-11
http://dx.doi.org/10.2298/MPNS0604118P  http://www.doiserbia.nb.rs/img/doi/0025-8105/2006/0025-81050604118P.pdf

Keywords: diabetes mellitus, autonomic nervous system diseases, heart diseases, myocardial ischemia, comorbidity

Introduction.

Diabetes is strongly associated with macrovascular complications, among which

  • ischemic heart disease is the major cause of mortality.

Autonomic neuropathy increases the risk of complications, which calls for an early diagnosis. The aim of this study was to determine

  • both presence and extent of cardiac autonomic neuropathy,

in regard to the type of diabetes mellitus, as well as

  • its correlation with coronary disease and
  • major cardiovascular risk factors.

Material and methods. We have examined 90 subjects, classified into three groups, with 30 patients each: those with type 1 diabetes, type 2 diabetes and control group of healthy subjects. All patients underwent

  • cardiovascular tests (Valsalva maneuver, deep breathing test, response to standing, blood pressure response to standing sustained, handgrip test),
  • electrocardiogram,
  • treadmill exercise test and
  • filled out a questionnaire referring to major cardiovascular risk factors: smoking, obesity, hypertension, and dyslipidemia.

Results. Our results showed that cardiovascular autonomic neuropathy was

  • more frequent in type 2 diabetes,
  • manifesting as autonomic neuropathy.

In patients with autonomic neuropathy, regardless of the type of diabetes,

  • the treadmill test was positive, i.e. strongly correlating with coronary disease.

In regard to coronary disease risk factors,

  • the most frequent correlation was found for obesity and hypertension.

Discussion

Cardiovascular autonomic neuropathy is considered to be the principal cause of arteriosclerosis and coronary disease. Our results showed that the occurrence of cardiovascular autonomic neuropathy increases the risk of coronary disease due to dysfunction of autonomic nervous system.

Conclusions

Cardiovascular autonomic neuropathy is a common complication of diabetes that significantly correlates with coronary disease. Early diagnosis of cardiovascular autonomic neuropathy points to increased cardiovascular risk, providing a basis for preventive and therapeutic measures.

Part 2. A Longitudinal Cohort Study of the Cardiovascular Experience of Individuals at High Risk for Diabetes

This second part is a description of a longitudinal cohort study of individuals at high-risk for diabetes.  Unlike the SSA study, the study is not focused on protein-energy malnutrition.

Protocol for ADDITION-PRO: a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes recruited from Danish primary care

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Johansen NB, Hansen Anne-Louise S, Jensen TM, Philipsen A, Rasmussen SS, Jørgensen ME, Simmons RK, Lauritzen T, Sandbæk A, Witte DR
Publisher: BioMed Central    Date of publication: 2012 Dec Published in: BMC Public Health 2012; 12(1): 1078    ISSN(s): 1471-2458   Added to DOAJ: 2013-03-12 http://dx.doi.org/10.1186/1471-2458-12-1078       http://www.biomedcentral.com/1471-2458/12/1078

Keywords: Diabetes, Cardiovascular disease, Primary care, Complications, Microvascular, Impaired fasting glucose, Impaired glucose intolerance, Aortic stiffness, Physical activity, Body composition

Background

Screening programmes for type 2 diabetes inevitably find more individuals at high risk for diabetes than people with undiagnosed prevalent disease. While well established guidelines for the treatment of diabetes exist, less is known about treatment or prevention strategies for individuals found at high risk following screening. In order to make better use of the opportunities for primary prevention of diabetes and its complications among this high risk group, it is important to

  • quantify diabetes progression rates and to examine
  • the development of early markers of cardiovascular disease and
  • microvascular diabetic complications.

We also require a better understanding of the

  • mechanisms that underlie and drive early changes in cardiometabolic physiology.

The ADDITION-PRO study was designed to address these issues among individuals at different levels of diabetes risk recruited from Danish primary care.

Methods/Design

ADDITION-PRO is a population-based, longitudinal cohort study of individuals at high risk for diabetes. 16,136 eligible individuals were identified at high risk following participation in a stepwise screening programme in Danish general practice between 2001 and 2006.

  • All individuals with impaired glucose regulation at screening,
  • those who developed diabetes following screening, and
  • a random sub-sample of those at lower levels of diabetes risk

were invited to attend a follow-up health assessment in 2009–2011 (n = 4,188), of whom 2,082 (50%) attended. The health assessment included

  • detailed measurement of anthropometry,
  • body composition,
  • biochemistry,
  • physical activity and
  • cardiovascular risk factors including aortic stiffness and central blood pressure.

All ADDITION-PRO participants are being followed for incident cardiovascular disease and death.

Discussion

The ADDITION-PRO study is designed to increase

  • understanding of cardiovascular risk and
  • its underlying mechanisms among individuals at high risk of diabetes.

Key features of this study include

  • (i) a carefully characterised cohort at different levels of diabetes risk;
  • (ii) detailed measurement of cardiovascular and metabolic risk factors;
  • (iii) objective measurement of physical activity behaviour; and
  • (iv) long-term follow-up of hard clinical outcomes including mortality and cardiovascular disease.

Results will inform policy recommendations concerning cardiovascular risk reduction and treatment among individuals at high risk for diabetes. The detailed phenotyping of this cohort will also allow a number of research questions concerning early changes in cardiometabolic physiology to be addressed.

Part 3.  Clinical significance of cardiovascular dysmetabolic syndrome

This study also addresses the issue of diabetes insulin resistance leading to cardiovascular dysmetabolic syndrome.

Subjects: Diseases of the circulatory (Cardiovascular) system,
Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Deedwania Prakash C Publisher: BioMed Central            Date of publication: 2002 Jan
Published in: Trials 2002; 3: 1(2)   ISSN(s): 1468-6708  Added to DOAJ: 2004-06-03
http://dx.doi.org/10.1186/1468-6708-3-2   http://cvm.controlled-trials.com/content/3/1/2

Keywords: cardiovascular dysmetabolic syndrome, coronary heart disease, diabetes mellitus, hyperinsulinemia, insulin resistance

Although diabetes mellitus is predominantly a metabolic disorder,

  • recent data suggest that it is as much a vascular disorder.
  • Cardiovascular complications are the leading cause
    • of death and disability in patients with diabetes mellitus.

A number of recent reports have emphasized that

  • many patients already have atherosclerosis in progression
  • at the time they are diagnosed with clinical evidence of diabetes mellitus.

The increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to

  • the frequently associated dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, and endothelial dysfunction.

The evolving knowledge regarding the variety of

  • metabolic,
  • hormonal, and
  • hemodynamic abnormalities in patients with diabetes mellitus

has led to efforts designed for early identification of individuals at risk of subsequent disease. It has been suggested that

  • insulin resistance, the key abnormality in type II diabetes,
  • often precedes clinical features of diabetes by 5–6 years.

Careful attention to the criteria described for the cardiovascular dysmetabolic syndrome

  • should help identify those at risk at an early stage.

The application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects

  • in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus
  • by improving insulin sensitivity and related abnormalities.

Early identification and implementation of appropriate therapeutic strategies would be necessary

  • to contain the emerging new epidemic of cardiovascular disease related to diabetes.

Part 4.   Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Siren Reijo, Eriksson Johan G, Vanhanen Hannu
Publisher: BioMed Central      Date of publication: 2012 Aug
Published in: BMC Public Health 2012; 12: 1(631)    ISSN(s): 1471-2458   Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1471-2458-12-631    http://www.biomedcentral.com/1471-2458/12/631

Keywords: Waist circumference, Type 2 diabetes, Cardiovascular disease, Middle-aged men

Background

Abdominal obesity is a more important risk factor than overall obesity in

  • predicting the development of type 2 diabetes and cardiovascular disease.

From a preventive and public health point of view it is crucial that

  • risk factors are identified at an early stage,
  • in order to change and modify behaviour and lifestyle in high risk individuals.

Methods

Data from a community based study was used to assess

  • the risk for type 2 diabetes,
  • cardiovascular disease and
  • prevalence of metabolic syndrome in middle-aged men.

In order to identify those with increased risk for type 2 diabetes and/or cardiovascular disease

  • sensitivity and specificity analysis were performed, including
  • calculation of positive and negative predictive values, and
  • corresponding 95% CI for eleven different cut-off points,
    • with 1 cm intervals (92 to 102 cm), for waist circumference.

Results

A waist circumference ≥94 cm in middle-aged men,

  • identified those with increased risk for type 2 diabetes
  • and/or for cardiovascular disease

with a sensitivity of 84.4% (95% CI 76.4% to 90.0%), and a specificity of 78.2% (95% CI 68.4% to 85.5%). The positive predictive value was 82.9% (95% CI 74.8% to 88.8%), and negative predictive value 80.0% (95% CI 70.3% to 87.1%), respectively .

Conclusions

Measurement of waist circumference in middle-aged men

  • is a reliable test to identify individuals at increased risk for type 2 diabetes and cardiovascular disease.

This measurement should be used more frequently in daily practice in primary care

  • in order to identify individuals at risk and when planning health counselling and interventions.

Part 5.  How to use C-reactive protein in acute coronary care

Luigi M. Biasucci, Wolfgang Koenig, Johannes Mair, Christian Mueller, Mario Plebani, Bertil Lindahl, Nader Rifai,Per Venge,Christian Hamm, and the Study Group on Biomarkers in Cardiology of the Acute Cardiovascular Care Association of the European Society of Cardiology
Department of Cardiology B, Aarhus University Hospital, Tage Hansens Gade2, Aarhus DK-8000,Denmark; Germany, U.K., U.S., Italy
European Heart Journal Advance Access published Nov 7, 2013.  Current Opinion.  http://dx.doi.org/10.1093/eurheartj/eht435

Introduction

 C-reactive protein (CRP) is an acute phase protein and an established marker for detection, risk stratification, and monitoring of infections, and inflammatory and necrotic processes.. Because C-reactive protein is sensitive but not specific, its values must be nterpreted  in the clinical context. Inpatients with acute myocardial infarction (AMI), CRP increases within 4–6h of symptoms, peaks 2–4 days later,and returns to baseline after 7–10 days.

CRP has gained interest recently as a marker for risk stratification in acute coronary syndrome (ACS) when measured by high-sensitivity CRP assays. These assays have greater analytical sensitivity and reliably measure CRP concentrations within the reference range with low imprecision (5–10%). Because of evidence that atherosclerosis is an inflammatory disease, high-sensitivity CRP can be used as a biomarker of risk
in primary prevention and in patients with known cardiovascular disease. The aim of this review is to evaluate the use of CRP in patients with acute coronary disease.

The in-vitro stability of high-sensitivity C-reactive protein is excellent. Specific blood sampling conditions aren’t necessary.  However, retesting may be necessary with some assays if there is marked lipaemia.  Baseline and subsequent measures are in good for agreement for risk stratification despite biological variability of 30–60%.

The upper reference limit is method-dependent but usually 8mg/L for standard assays. The distribution of high-sensitivity CRP concentrations is skewed in both genders with a 50th percentile of_1.5mg/L (excluding women on hormone replacement therapy). Race differences have been reported. Most studies have reported no relationship with age,  but to circadian and seasonal variation. CRP concentrations are increased by smoking, obesity, and hormone replacement therapy and reduced by exercise, moderate alcohol drinking, and statin use. Correction for these factors is essential in reference range studies. CRP assays are not standardized. We recommend  the use of third-generation high-sensitivity CRP assays that combine features of standard and high-sensitivity CRP assays.  Required assay precision should be < 10% in the range of 3 and 10 mg/L.

Biochemical and analytical issues

Critical clinical concepts

(1) CRP concentrations are reported in mg/L
(2) CRP test results are method-dependent

  •  classification of patients into risk categories is usually comparable
(3) Third generation CRP assay are recommended
(4) No specific patient preparation before blood sampling is necessary
(5) The in-vitro stability of CRP is high

This is only a portion of the published concensus document. What is relevant to this discussion is that the hs-CRP is an extremely valuable marker for inflammatory disease.  It is not ordered often enough because of the broad range of values that we have become accustomed to for years, and it is elevated in rheumatologic conditions, but even then, it is widely used in pediatrics because children may present with rapidly emergent sepsis with very minimal sympoms.
The hs-CRP has opened a window to subliminal inflammatory disease that is diabetes, with accompanied arteriolar endothelial inflammation.

Part 6.  Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

Subjects: Diseases of the circulatory (Cardiovascular) system,
Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Mirrakhimov Aibek E
Publisher: BioMed Central      Date of publication: Oct 2012   ISSN(s): 1475-2840
Published in: Cardiovascular Diabetology 2012; 11(1):132   Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1475-2840-11-132      http://www.cardiab.com/content/11/1/132

Keywords: COPD, Dysglycemia, Insulin resistance, Obesity, Metabolic syndrome, Diabetes mellitus endothelial dysfunction, Vasculopathy

Chronic obstructive pulmonary disease, metabolic syndrome and diabetes mellitus

  • are common and underdiagnosed medical conditions.

It was predicted that chronic obstructive pulmonary disease

  • will be the third leading cause of death worldwide by 2020.

The healthcare burden of this disease is even greater

  • if we consider the significant impact of chronic obstructive pulmonary disease on
    • the cardiovascular morbidity and mortality.

Chronic obstructive pulmonary disease

  • may be considered as a novel risk factor for new onset type 2 diabetes mellitus via

multiple pathophysiological alterations such as:

  1. inflammation and oxidative stress,
  2. insulin resistance,
  3. weight gain and
  4. alterations in metabolism of adipokines.

On the other hand, diabetes may act as an independent factor,

  • negatively affecting pulmonary structure and function.

Diabetes is associated with an increased risk of

  1. pulmonary infections,
  2. disease exacerbations and
  3. worsened COPD outcomes.

On the top of that, coexistent OSA

  • may increase the risk for type 2 DM in some individuals.

The current scientific data necessitate a greater outlook on chronic obstructive pulmonary disease and

  • chronic obstructive pulmonary disease may be viewed as a risk factor for
  • the new onset type 2 diabetes mellitus.

Conversely, both types of diabetes mellitus should be viewed as

  • strong contributing factors for the development of obstructive lung disease.

Such approach can potentially improve the outcomes and medical control for both conditions,

  • and, thus, decrease the healthcare burden of these major medical problems.

CONCLUSIONS

This discussion  presents a spectrum of cardiovascular risk associated with type 2 diabetes mellitus, with high risk for CVD, stroke, endothelial dysfunction, and an association with obesity, measured by waist circumference, and an underlying proinflammatory state that can be measured by CRP.

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Notable Contributions to Regenerative Cardiology by Richard T. Lee – Part I

 

Author and Curator: Larry H Bernstein, MD, FCAP

and

Article Commissioner: Aviva Lev-Ari, PhD, RD

 

Introduction

This presentation is a two part discussion of selected articles of a large body of research from Dr. Richard T. Lee, at Harvard Medical School’s Lee Laboratory and Brigham & Womens Hospital.  This work is innovative in the field of stem cell research and myocardial regeneration.  It devolves the complex cellular processes that are involved in the management of a cell transforming from a progenitor to a functional cardiomyocyte.  The cell engineering involves investigating interactions between a cell placed into the layer derived from the interstitial layer between viable cardiomyocytes.  This is only possible from a through actionable knowledge of the mechanism involved in the transformation process, which has occupied the Lee Laboratory for many years.  Part II will cover the cellular mechanisms underlying the conceptual approach to cardiac myocyte regeneration.

The Lee Laboratory uses emerging biotechnologies to discover and design new approaches to cardiovascular diseases. A central theme of the laboratory is that merging bioengineering and molecular biology approaches can yield novel approaches. Thus, the Lee Laboratory works at this interface using a broad variety of techniques in genomics, imaging, nanotechnology, physiology, cell biology, and molecular biology. The approach is to understand problems and design solutions in the laboratory and then demonstrate the effectiveness of these solutions in vivo. Ongoing projects in the laboratory include studies of cardiac regeneration, diabetic vascular disease, wound healing, heart failure, and cardiac hypertrophy.

Richard T. Lee is Professor of Medicine at Harvard Medical School and lecturer in Biological Engineering at the Massachusetts Institute of Technology. He is a 1979 graduate of Harvard College in Biochemical Sciences and received his M.D. from Cornell University Medical College in 1983.  He went on to complete his residency in 1986 and cardiology fellowship in 1989, both at Brigham and Women’s Hospital in Boston, and he obtained post-doctoral training at MIT in Bioengineering.

Dr. Lee is certified by the American Board of Internal Medicine in cardiovascular disease and is a Fellow of the American College of Cardiology. He is Leader of the Cardiovascular Program of the Harvard Stem Cell Institute.  He is a member of the Editorial Boards of the journals Circulation Research, Journal of Clinical Investigation, and Circulation, and has published over 180 peer-reviewed articles based on his research, which combines approaches in biotechnology and molecular biology to discover new avenues to manage and treat heart disease.

Regeneration of the heart

Matthew L. Steinhauser, Richard T. Lee
Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, and (2)             Harvard Stem Cell Institute, Cambridge, MA
EMBO Mol Med 2011; 3: 701–712   http://dx.doi.org/10.1002/emmm.201100175

The death of cardiac myocytes diminishes the heart’s pump function and is a major cause of heart failure. With the exception of heart transplantation and implantation of mechanical ventricular assist devices, current therapies do not address the central problem of decreased pumping capacity owing to a depleted pool of cardiac myocytes. The field is evolving in two important directions. First, although endogenous mammalian cardiac regeneration clearly seems to decline rapidly after birth, it may still persist in adulthood. The careful elucidation of the cellular and molecular mechanisms of endogenous heart regeneration may therefore provide an opportunity for developing therapeutic interventions that amplify this process. Second, recent breakthroughs have enabled reprogramming of cells that were apparently terminally differentiated, either by dedifferentiation into pluripotent stem cells or by trans-differentiation into cardiac myocytes.
The longstanding paradigm held that the mammalian heart is a terminally differentiated organ, incapable of replenishing any myocyte attrition. During the past decade, however, studies revealed not only that mammalian cardiac myocytes retain some capacity for division (Beltrami et al, 2001), but also identified endogenous cardiac progenitor cells in the heart (Beltrami et al, 2003) or bone marrow (Orlic et al, 2001). These cells retain some potential for differentiation into the cellular components of the heart, including endothelial cells, smooth muscle cells and cardiac myocytes.
If progenitor cells residing in the adult are capable of producing new heart cells, the therapeutic delivery of such progenitors might facilitate the generation of de novo functional myocardium. In this context, cell-based therapies for the heart have been rapidly translated into the clinic to treat heart disease, but randomized clinical trials with bone marrow progenitors have shown at best modest improvements in ventricular function (Martin-Rendon et al, 2008). In short, the promise of complete cardiac regeneration has not yet been realized.  Therefore, it is worth revisiting both the foundations of cardiac regeneration and highlight recent advances that may portend future directions in the field.
We will first define the problem, that is elucidating the scope of endogenous mammalian regeneration and, by extension, the scale of the regenerative deficit. We will then summarize current regenerative approaches, including both cell-based therapies and pharmacoregenerative strategies. In this context, we will summarize the many challenges that stand in the way of cardiac regeneration, both endogenous repair processes and exogenous regenerative therapies.
The regenerative deficit of the mammalian heart is obvious when compared with organisms such as zebrafish and newts, which demonstrate a remarkable survival capacity after removal of up to 20% of the heart by transection of the ventricular apex. Pre-existing cardiac myocytes adjacent to the site appear to undergo a process of dedifferentiation, characterized by dissolution of sarcomeric structures. This is followed by incorporation of deoxyribonucleic acid (DNA) synthesis markers (e.g. nucleotide analogues) consistent with proliferation. Ultimately, newly generated cardiac myocytes are functionally integrated with the preexisting myocardium. The heart is left with little residual evidence of the injury, thus providing a natural example of complete myocardial regeneration.

Evidence for heart regeneration in mammals

During embryonic development and the early post-natal period, mice also demonstrate a remarkable regenerative capacity. Embryos heterozygous for a cardiac myocyte-specific null mutation in the x-linked holocytochrome c synthase (Hccs) gene demonstrate cardiac myocyte replacement during foetal development (Drenckhahn et al, 2008): when one of two X-chromosomes is randomly inactivated in each female somatic cell, approximately 50% of the cardiac myocytes are rendered Hccs-null and hence dysfunctional. Proliferative functional Hccs-expressing cardiac myocytes compensate for dysfunctional Hccs-null myocytes, such that, at birth, 90% of the heart is derived from myocytes containing one functional Hccs allele. However, after the first week in post-natal mice, injured myocardium is largely replaced by fibrosis and scarring.  Distinguishing whether the adult mammalian heart is incapable of cardiac myocyte replacement or whether it retains a low-level capacity for repair is therefore fundamentally important. This is the basis for an evolving view of a more plastic mammalian heart.
Arguments against the age old view of the terminally differentiated quiescent cardiac myocyte:

  1. evidence supporting cardiac myocyte plasticity relied on mathematical modelling of the myocyte population based on cytometric indices. (the measured average volume increase of cardiac myocytes was calculated to fall short of the increase predicted by the observed volumetric changes in the whole heart
  • changes in heart volume could not be explained by hypertrophy alone, and that cardiac myocyte hyperplasia contributed to changes in heart mass, but the conclusions relied on a number of assumptions about myocyte size and DNA content.
  • detecting cell cycle markers such as Ki67 or the incorporation of nucleotide analogues (e.g. iododeoxyuridine or 3H-thymidine) into newly synthesized DNA further support the notion that the mammalian heart may generate new myocytes
  • human cardiac myocytes can reenter the cell cycle, but the described rates of this phenomenon differ by more than one order of magnitude
  • experiments, made possible by nuclear arms testing in the middle of the 20th century, provide the most convincing evidence for post-natal human cardiac myocyte turnover.
  • the period of nuclear testing serves as a historical DNA labelling pulse, and the period after the test ban treaty serves as a chase.
  • the genomic DNA of cells generated during either the pulse or the chase reflect the earth’s atmospheric 14C concentration at that point in time, which allows investigators to date the age of cardiac myocytes by measuring the concentration of 14C in their nuclei
  1. Listed are a number of problems in detecting the generation of cardiac myocytes:
  • small errors may magnify projections of absolute yearly or lifetime myocyte turnover
  • mis-identification of cellular components by light microscopy
  • autofluorescence of myocardium, which complicates any method that relies on the detection of a fluorescent signal
  • confounders could also affect the 14C dating method, because it requires the isolation of cardiac myocyte nuclei by digestion and flow cytometric sorting

The heart also presents a unique challenge compared to other organs owing to the propensity of cardiac myocytes to synthesize DNA during S-phase without completing either mitosis and/or cytokinesis (Fig 1).

Figure 1. The majority of post-natal human DNA synthesis in the heart does not lead to new myocyte formation.

Cardiac myocytes can complete S-phase, followed by mitosis and cytokinesis (centre) resulting in myocyte doubling. Cardiac myocytes can also complete mitosis without cytokinesis (left), resulting in a binucleated cell. Cardiac myocytes can also undergo chromosomal replication without completing either mitosis or cytokinesis (right), resulting in polyploidy nuclei. By the completion of post-natal development, the majority of human myocyte nuclei contain ~4n chromosomal copies.

During early post-natal development, for example, the majority of rodent cardiac myocytes and an estimated 25–57% of human cardiac myocytes become binucleated. By adulthood, most cardiac myocyte nuclei have also become polyploid with at least one or two additional rounds of chromosomal replication.

The ploidy state of cardiac myocytes may increase with myocardial hypertrophy or injury, which could be mistaken for myocyte division. Conversely, hearts that have been unloaded by implantation of a ventricular assist device may have a lower percentage of polyploid myocytes, because more 2n cardiac myocytes are being generated. These aspects of cardiac myocyte biology inevitably represent potential confounders that must be considered in any quantification of cardiac myocyte formation.

Defining the cellular source of new cardiac myocytes

The majority of reports suggest some endogenous capacity for cardiac myocyte renewal, which has generated a broad focus on finding the cellular source of newly generated cardiac myocytes.  Newly generated adult mammalian cardiac myocytes may arise from an endogenous pool of progenitor cells after injury. The Lee Laboratory developed a genetic lineage mapping approach to quantify progenitor-dependent cardiac myocyte turnover (Fig 2) (Hsieh et al, 2007). In the bitransgenic MerCreMer/ZEG inducible cardiac myocyte reporter mouse, mature cardiac myocytes undergo an irreversible genetic switch from constitutive 3-galactosidase expression to green fluorescent protein (GFP) expression upon tamoxifen pulse. During a chase period, we evaluated the effect of myocardial injury on the proportion of GFP+ or 3-gal+ cardiac myocytes. Pressure overload or myocardial infarction resulted in a significant reduction in the percentage of GFP+ cardiac myocytes and a corresponding increase in the percentage of B-gal+ cardiac myocytes, consistent with repletion of the myocyte pool by B-gal— expressing progenitors. This approach cannot directly identify the molecular identity or anatomic location of the progenitor pool.
One approach to characterizing the molecular phenotype of cardiac progenitors is to study cardiac embryologic develop-ment, guided by the assumption that developmental paradigms are recapitulated during post-natal repair. When examined through a developmental lens, an increasingly detailed picture emerges of the soluble and transcriptional signals that guide the cardiogenic programme from gastrulation (formation of distinct germ layers) through the ultimate maturation of cardiac myocytes. The induction of mesoderm posterior (MESP)-1 expression by brachyury-expressing primitive mesodermal cells is a proximal require¬ment for the ultimate production of differentiated heart cells. As the developing embryo grows beyond the germ layer phase, its developing heart receives cells from distinct anatomic progeni¬tor sources: the 1st and 2nd heart fields provide the majority of the myocardium, with some contribution from epicardial progenitors.
Also, Certain fields may be preferentially marked by specific transcription factors;

  • the first heart field by T-box transcription factor 5 (Tbx5)
  • the second heart field by
    • Lim-homeodomain protein Islet1 (Isl1)
    • and epicardial progenitors by Wilms tumour-1 (WT1) or
    • T-box transcription factor 18 (Tbx18)
    • identified by embryonic lineage tracing or analysis of gene silencing include
      • homeobox protein nkx2.5
      • myocyte enhancer factor 2C (Mef2c)
      • GATA4
      • there is no consensus yet about the molecular identity of post-natal mammalian cardiac progenitor cells or ‘adult cardiac stem cells’

Figure 2. Lineage-mapping in the adult heart.

Left: Theoretical progenitor lineage-mapping is depicted. Progenitors would be selectively marked by fluorescent protein expression. After injury, the appearance of fluorescently labelled cardiac myocytes would support the concept that these progenitors were contributing to new myocyte formation. Right: Differentiated cell (cardiac myocyte) lineage-mapping. Upon treatment of the MerCreMer-ZEG mouse with OH-tamoxifen, approximately 80% of the cardiac myocytes undergo a permanent switch from I3-galactosidase to GFP expression. The dilution of the GFPþ cardiac myocyte pool after injury is consistent with repletion by I3-galþ progenitors.

A number of laboratories have identified cell populations within the post-natal mouse, which fulfil some criteria of cardiac progenitors:

  • expression of a developmentally important gene (isl-1(Laugwitz et al, 2005))
  • specific cell surface receptor profile (c-kit (Beltrami et al, 2003)
  • or sca-1 (Oh et al, 2003))
  • capacity to actively exclude Hoechst dye (so-called side population cells (Martin et al, 2004)) or based on the outgrowth of typical spherical colonies in tissue culture 

In general, the label of ‘cardiac stem cell’ results from the observation of self-propagation and cardiac myocyte transdifferentiation when exposed to cardiogenic conditions in vitro or when delivered in vivo after injury. However, the field will benefit from careful in vivo lineage tracing studies—without ex vivo culture steps—to study if and how a given cell type contributes to cardiac myocyte replenishment during either normal homeostasis or after injury (Fig 2). The lack of such publications to date owes in part to the lack of specificity of many stem cell markers (Fig 3).

Figure 3. Possible recapitulation of developmental paradigms by endogenous post-natal cardiac stem cells.

Between mesodermal development and the emergence of cardiac myocytes, cardiovascular progenitors express a number of markers that have also been detected in the various post-natal cardiac stem cell (CSC) preparations. Expression as measured by messenger RNA (mRNA) or protein expression is denoted with (þ). Absent expression is denoted by (-). Blank1/4 untested.

Moving towards a regenerative therapy

The therapeutic challenge is considerable: a typical large myocardial infarction that leads to heart failure will kill around 1 billion cardiac myocytes,  roughly a quarter of the heart’s myocytes. A possible therapeutic approach would coax an endogenous stem cell population or an exogenously delivered cell-based therapy to replace lost cardiac myocytes in a coordinated fashion. Amongst the myriad of potential cell-based therapies, no clear winning strategy has so far emerged (Segers & Lee, 2008).

Bone marrow derived progenitors

Conflicting studies sparked excitement and also uncertainty about a possible adult cardiogenic progenitor originating outside of the heart. A post-mortem examination of male heart transplant patients who had received female donor hearts found that approximately 10% of -sarcomeric actin-positive cardiac myocytes had Y-chromosomes, and two cases in which a bone marrow cell population with a higher density of the cell surface receptor c-kit, showed repopulation of murine cardiac myocytes after experimental myocardial infarction. A number of studies that followed failed to demonstrate similar rates of chimerism in transplanted hearts or potency of bone marrow stem cell.  However, some therapeutic effect was observed even in studies with no detectable transdifferentiation.

Figure 4. The challenge of regenerating the heart.

Both exogenously delivered cell therapies and progenitors in the endogenous niche encounter a similar hostile environment after myocardial injury, often including inadequate blood supply (ischemia), inflammation and fibrosis/scarring. Regenerative pathways may be activated by as yet unknown paracrine pathways, responsible for recruiting progenitors from the niche, stimulating proliferation and coaxing differentiation.  Cell-based therapy using autologous bone marrow
progenitors was rapidly translated into the clinic to treat human ischemic heart disease. A number of randomized trials, using bone marrow mononuclear cells have been performed and most studies demonstrated modest cell therapy-mediated improvements in ventricular function.

Pluripotent stem cells

Embryonic stem (ES) cells represent the prototypical stem cells with the hallmarks of clonogenicity, self-renewal and pluripotency. The potency of these cells also represents a real safety concern, given their tendency to form teratomas. One approach to overcoming this prohibitive safety problem has been to generate pluripotent-derived progenitors that have already committed to a cardiogenic pathway. As a proof-of-principle example of such a strategy, cells with an expression profile of Oct4, stage-specific embryonic antigen 1 (SSEA-1) and MESP1 demonstrated some regenerative potency when delivered therapeutically in a primate infarct model, without detectable teratoma formation. One could envision a similar strategy using cardiogenic intermediates that express any of the previously mentioned transcription factors associated with cardiac progenitors or cell surface markers such as the receptor for vascular endothelial growth factor (Flk1/KDR). Yet, such a strategy should still demonstrate both substantial preclinical efficacy without tumorigenicity before human translation. If such criteria are met, ES-derived therapies have the potential of providing ‘off-the-shelf’ cardiac myocytes to treat acute myocardial infarctions or chronic heart failure.
A second approach, which may also obviate the risk of teratomas, is to generate a pure population of ES-derived cardiac myocytes for therapeutic delivery either as a cell suspension or after ex vivo tissue engineering. There has already been enormous progress during the past decade in defining the factors and transcription signals to differentiate cardiac myocytes from ES-cells. As discussed in greater detail, cardiac myocyte development is dictated by the time and dose-dependent exposure to a series of growth factors from the wingless-type MMTV integration site (Wnt), fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and nodal families. Several laboratories have successfully generated ES-derived preparations with more than 50% of functional cardiac myocytes.  The most realistic future for such technical advances may be as an unlimited source of cardiac myocytes for engineering myocardial grafts.
The generation of induced pluripotent stem (iPS) cells may overcome two important limitations of ES cells: ethical concerns about harvesting ES cells from embryos and graft rejection

  • iPS cells can be custom-engineered from a patient’s stromal cells for autologous transplantation.
  • immunogenecity in syngeneically transplanted iPS cells, suggests that the immune system cannot yet be discounted in the development of iPS-based therapies

The initial protocols for iPS cell generation involved retro-viral-mediated expression of four stem-cell genes.
But virally reprogrammed cells may harbour an associated risk of neoplastic conversion. Alternative reprogramming strategies, such as the use of small molecules (Shi et al, 2008) or non-viral gene modifying approaches (Warren et al, 2010) will probably be a necessary component of any future therapeutic strategies. However, the most important lesson from these landmark studies may be the remarkable plasticity of even the most terminally differentiated cells when exposed to the right combination of signals.

Tissue engineering

Historically, the greatest challenge in tissue engineering has been an adequate supply of oxygen and nutrients for metabolically active tissues such as the heart. One approach has been to engineer thin cardiac sheets, which can then be stacked for in vivo delivery. Although these layered sheets demonstrate some degree of electromechanical coordination and neovascularization in vivo, it is not clear yet if such an approach can be optimized to yield full-thickness myocardium with an adequate blood supply. The addition of non-myocyte cellular components, such as fibroblasts and endothelial cells, leads to the formation of primitive vascular structures within engineered grafts, but the electro-mechanical properties are not sufficient for normal functionality.

Circumventing cell-based therapy with pharmacoregeneration?

A short-term goal may be to exploit paracrine signalling to amplify the existent endogenous regenerative response. Recent cell transplantation experiments conducted in our laboratory, using an inducible cre-based genetic lineage mapping approach, tested the hypothesis that cell-based therapies might exert proregenerative effects via a paracrine mechanism (Loffredo et al, 2011) (Fig 5).  Consistent with some prior studies, we found no evidence for transdifferentiation of exogenously delivered bone marrow cells into cardiac myocytes. However, we did find increased generation of cardiac myocytes from endogenous progenitors in mice, which were administered c-kit+ bone marrow cells but not mesenchymal stem cells. This finding suggests paracrine signalling between exogenously delivered cells and endogenous resident progenitors. It provides a rationale for therapeutic interventions aimed at activating progenitors or recruiting them from their niche to the injury site.

Figure 5. Proposed of action for cell-based therapies.

In theory, exogenously delivered cells may directly differentiate into endothelial cells, smooth muscle cells and cardiac myocytes. They may also release paracrine factors which may result in non-regenerative effects, such as immunomodulation, angiogenesis or cardioprotection. Recent work from our laboratory suggests that a dominant mechanism achieved with bone marrow progenitor therapy may be via the activation of endogenous progenitor recruitment (Loffredo et al, 2011).

Controlling the mitotic activity of mononucleated cardiac myocytes may provide an alternative approach to replenishing cardiac myocytes. A major concern with systemic growth factor therapy, however, is the potential for mitogenic effects that may impact other organs. Thus, the future of pharmacologic regeneration may lie in the local delivery of engineered proteins and small molecules that target 

Future directions

In this review, we have described the current status of research on cardiac regeneration, highlighting important recent discoveries and ongoing controversies. The initial hope that a cell progenitor would emerge with the capacity to regenerate the injured mammalian heart in the same manner that bone marrow may be reconstituted has not been realized.
Cardiac myocyte regeneration may lie in the local delivery of engineered proteins and small molecules that target specific survival, growth and differentiation pathways.

Pending issues

Dissect the mechanistic differences between adult mammals with limited regenerative capacity and organisms, such as neonatal mice, zebrafish and newts, that demonstrate unambiguous cardiac myocyte regeneration. Understanding these differences may reveal new pathways that can be therapeutically targeted to achieve more robust regeneration.

Complete molecular and functional characterization of endogenous cardiac myocyte progenitors. Multiple laboratories have isolated progenitors from the heart with different molecular characteristics. What are the in vivo functional roles of these progenitors? Do the observed molecular differences between these isolated cells represent functionally distinct cell types?

Identify paracrine signalling pathways responsible for activation and recruitment of endogenous cardiac myocyte progenitors. This may facilitate a pharmacoregenerative therapy, in which treatment with a protein or small molecule would hold the promise of amplifying endogenous regeneration.

Refine reprogramming strategies to more efficiently produce mature cardiac myocytes, both in vitro and in vivo. The ultimate bioengineering goal is to produce a pure population of mature, fully functional cardiac myocytes for ex vivo tissue engineering (or) to control the proliferation and differentiation of endogenous cell populations or exogenously delivered cell therapies such that scar tissue is replaced by myocardium. These different strategies are unified by an underlying requirement to understand the fundamental pathways involved in cardiac myocyte differentiation and maturation.

There is reason for optimism for a regenerative medicine approach to heart failure, given the intense research efforts and the capacity of higher organisms, including the neonatal mouse, to regenerate myocardium. Perhaps the most important issue in this field is identifying which findings are consistently supported by multiple experimental approaches. Ultimately, the findings that are easily reproduced by most or all laboratories will most likely benefit patients.

Selected references

Hsieh et al, 2007.  Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13: 970¬974
Laugwitz et al, 2005.  Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S, Lin LZ, Cai CL, Lu MM, Reth M et al (2005) Postnatal isl1þ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433: 647-653
Beltrami et al, 2003.  Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K et al (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114: 763¬776
Oh et al, 2003. Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y, Pocius J, Michael LH, Behringer RR, Garry DJ et al (2003) Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 100: 12313-12318
Segers & Lee, 2008.  Segers VF, Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451:937-942
Loffredo et al, 2011.  Loffredo FS, Steinhauser ML, Gannon J, Lee RT (2011) Bone marrow-derived cell therapy stimulates endogenous cardiomyocyte progenitors and promotes cardiac repair. Cell Stem Cell 8: 389-398.
Shi et al, 2008.  Shi Y, Desponts C, Do JT, Hahm HS, Scholer HR, Ding S (2008) Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3: 568-574.
Warren et al, 2010.  Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A et al (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618-630.

Mammalian Heart Renewal by Preexisting Cardiomyocytes

SE Senyo, ML Steinhauser, CL Pizzimenti, VK. Yang, Lei Cai, Mei Wang, …,and Richard T. Lee
Cardiovascular and Genetics Divisions, Brigham and Women’s Hospital and Harvard Medical School,
INSERM, Orsay (Fr), Institut Curie, Laboratoire de Microscopie Ionique, Orsay (Fr), National Resource for Imaging Mass Spectrometry, Harvard Stem Cell Institute
Nature. 2013 January 17; 493(7432): 433–436.  http://dx.doi.org/10.1038/nature11682

Although recent studies have revealed that heart cells are generated in adult mammals, the frequency and source of new heart cells is unclear. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other studies suggest that new cardiomyocytes are born at a very low rate, and that they may be derived from division of pre-existing cardiomyocytes. Thus, the origin of cardiomyocytes in adult mammals remains unknown. Here we combined two different pulse-chase approaches — genetic fate-mapping with stable isotope labeling and Multi-isotope Imaging Mass Spectrometry (MIMS). We show that genesis of cardiomyocytes occurs at a low rate by division of pre-existing cardiomyocytes during normal aging, a process that increases by four-fold adjacent to areas of myocardial injury. Cell cycle activity during normal aging and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleated cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury.

Despite intensive research, fundamental aspects of the mammalian heart’s capacity for self-renewal are actively debated. Estimates of cardiomyocyte turnover range from less than 1% per year to more than 40% per year. Turnover has been reported to either decrease or increase with age, while the source of new cardiomyocytes has been attributed to both division of existing myocytes and to progenitors residing within the heart or in exogenous niches such as bone marrow. Controversy persists regarding the plasticity of the adult heart in part due to methodological challenges associated with studying slowly replenished tissues. Toxicity attributed to radiolabeled thymidine and halogenated nucleotide analogues limits the duration of labeling and may produce direct biological effects. The challenge of measuring cardiomyocyte turnover is further compounded by the faster rate of turnover of cardiac stromal cells relative to cardiomyocytes.

We used Multi-isotope Imaging Mass Spectrometry (MIMS) to study cardiomyocyte turnover and to determine whether new cardiomyocytes are derived from preexisting myocytes or from a progenitor pool (Fig 1a). MIMS uses ion microscopy and mass spectrometry to generate high resolution quantitative mass images and localize stable isotope reporters in domains smaller than one micron cubed15,16,17. MIMS generates 14N quantitative mass images by measuring the atomic composition of the sample surface with a lateral resolution of under 50nm and a depth resolution of a few atomic layers. Cardiomyocyte cell borders and intracellular organelles were easily resolved (Fig 1b). Regions of interest can be analyzed at higher resolution, demonstrating cardiomyocyte-specific subcellular ultrastructure, including sarcomeres (Fig 1c, Supplemental Fig 1a). In all subsequent analyses, cardiomyocyte nuclei were identified by their location within sarcomere-containing cells, distinguishing them from adjacent stromal cells.
An immense advantage of MIMS is the detection of nonradioactive stable isotope tracers. As an integral part of animate and inanimate matter, they do not alter biochemical reactions and are not harmful to the organism18. MIMS localizes stable isotope tracers by simultaneously quantifying multiple masses from each analyzed domain; this enables the generation of a quantitative ratio image of two stable isotopes of the same element15. The incorporation of a tracer tagged with the rare stable isotope of nitrogen (15N) is detectable with high precision by an increase in 15N:14N above the natural ratio (0.37%). Nuclear incorporation of 15N-thymidine is evident in cells having divided during a one-week labeling period, as observed in the small intestinal epithelium, which turns over completely in 3–5 days16 (Fig 1d); in contrast, 15N-thymidine labeled cells are rarely observed in the heart (Fig 1e) after 1 week of labeling. In subsequent studies, small intestine was used as a positive control to confirm label delivery.
To evaluate for an age-related change in cell cycle activity, we administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) (Supplemental Fig 2). We then performed MIMS analysis (Fig 2a, b, Supplemental Fig 3). In the neonatal group, 56% (±3% s.e.m., n=3 mice) of cardiomyocytes demonstrated 15N nuclear labeling, consistent with the well-accepted occurrence of cardiomyocyte DNA synthesis during post-natal development19. We observed a marked decline in the frequency of 15N-labeled cardiomyocyte nuclei (15N+CM) in the young adult (neonate= 1.00%15N+CM/day ±0.05 s.e.m. vs young adult=0.015% 15N+CM/ day ±0.003 s.e.m., n=3 mice/group, p<0.001) (Fig 2a, c; Supplemental Fig 3). We found a further reduction in cardiomyocyte DNA synthesis in old mice (young adult=0.015%15N+CM/day ±0.003 s.e.m. vs. old adult=0.007 %15N+CM/day ±0.002 s.e.m., n=3/group, p<0.05) (Fig 2c). The observed pattern of nuclear 15N-labeling in cardiomyocytes is consistent with the known chromatin distribution pattern in cardiomyocytes20 (Supplemental Fig 1b) and was measured at levels that could not be explained by DNA repair (Supplemental Fig 4). Extrapolating DNA synthesis measured in cardiomyocytes over 8 weeks yields a yearly rate of 5.5% in the young adult and 2.6% in the old mice. Given that cardiomyocytes are known to undergo DNA replication without completing the cell cycle19,21,22, these calculations represent the upper limit of cardiomyocyte generation under normal homeostatic conditions, indicating a low rate of cardiogenesis.
To test whether cell cycle activity occurred in preexisting cardiomyocytes or was dependent on a progenitor pool, we performed 15N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, previously developed for genetic lineage mapping (Fig 3a)23,24. MerCreMer/ZEG cardiomyocytes irreversibly express green fluorescent protein (GFP) after treatment with 4OH-tamoxifen, allowing pulse labeling of existing cardiomyocytes with a reproducible efficiency of approximately 80%. Although some have reported rare GFP expression by non-cardiomyocytes with this approach25, we did not detect GFP expression in interstitial cells isolated from MerCreMer/ZEG hearts nor did we detect GFP expression by Sca1 or ckit-expressing progenitors in histological sections (Supplemental Fig 5). Thus, during a chase period, cardiomyocytes generated from progenitors should be GFP−, whereas cardiomyocytes arising from preexisting cardiomyocytes should express GFP at a frequency similar to the background rate induced by 4OH-tamoxifen. We administered 4OH-tamoxifen for two weeks to 8 wk-old mice (n=4); during a subsequent 10-week chase, mice received 15N-thymidine via osmotic minipump.

We next used MIMS and genetic fate mapping to study myocardial injury. Cardiomyocyte GFP labeling was induced in MerCreMer/ZEG mice with 4OH-tamoxifen. Mice then underwent experimental myocardial infarction or sham surgery followed by continuous labeling with 15N-thymidine for 8wks. The frequency of 15N-labeled cardiomyocytes in sham-operated mice was similar to prior experiments in unoperated mice (yearly projected rates: sham=6.8%; unoperated=4.4%), but increased significantly adjacent to infarcted myocardium (total 15N+ cardiomyocyte nuclei: MI=23.0% vs sham=1.1%, Fig 4a–b, Supplemental Fig 8). We examined GFP expression, nucleation and ploidy status of 15N-labeled cardiomyocytes and surrounding unlabeled cardiomyocytes. We found a significant dilution of the GFP+ cardiomyocyte pool at the border region as previously shown23,24 (67% vs. 79%, p<0.05, Table 2, Supplemental Fig 9); however, 15N+ myocytes demonstrated a similar frequency of GFP expression compared to unlabeled myocytes (71% vs. 67%, Fisher’s exact=n.s.), suggesting that DNA synthesis was primarily occurring in pre-existing cardiomyocytes. Of 15N-labeled cardiomyocytes, approximately 14% were mononucleated and diploid consistent with division of pre-existing cardiomyocytes (Supplemental Fig 6, 7). We observed higher DNA content (>2N) in the remaining cardiomyocytes as expected with compensatory hypertrophy after injury. Thus, in the 8wks after myocardial infarction, approximately 3.2% of the cardiomyocytes adjacent to the infarct had unambiguously undergone division (total 15N+ × mononucleated diploid fraction = 23% × 0.14 = 3.2%). The low rate of cardiomyocyte cell cycle completion is further supported by the absence of detectable Aurora B Kinase, a transiently expressed cytokinesis marker, which was detected in rapidly proliferating small intestinal cells but not in cardiomyocytes (Supplemental Fig 10). We also considered the possibility that a subset of 15N+ myocytes that were multinucleated and/or polyploid resulted from division followed by additional rounds of DNA synthesis without division. However, quantitative analysis of the 15N+ population did not identify a subpopulation that had accumulated additional 15N-label as would be expected in such a scenario (Supplemental Fig 11). Together, these data suggest that adult cardiomyocytes retain some capacity to reenter the cell cycle, but that the majority of DNA synthesis after injury occurs in preexisting cardiomyocytes without completion of cell division.
If dilution of the GFP+ cardiomyocyte pool cannot be attributed to division and differentiation of endogenous progenitors, do these data exclude a role for progenitors in the adult mammalian heart? These data could be explained by preferential loss of GFP+ cardiomyocytes after injury, a process that we have previously considered but for which we have not found supporting evidence23. Such an explanation excludes a role for endogenous progenitors in cardiac repair and would be consistent with data emerging from lower vertebrates8,26 and the neonatal mouse27 in which preexisting cardiomyocytes are the cellular source for cardiomyocyte repletion. A second possibility to explain the dilution of the GFP+ cardiomyocyte pool is that injury stimulates progenitor differentiation without division; inevitably, this would lead to exhaustion of the progenitor pool, which if true could explain the limited regenerative potential of the adult mammalian heart.

In summary, this study demonstrates birth of cardiomyocytes from preexisting cardiomyocytes at a projected rate of approximately 0.76%/year (15N+ annual rate × mononucleated diploid fraction = 4.4% × 0.17) in the young adult mouse under normal homeostatic conditions, a rate that declines with age but increases by approximately four-fold after myocardial injury in the border region. This study shows that cardiac progenitors do not play a significant role in myocardial homeostasis in mammals and suggests that their role after injury is also limited.

Engineering insulin-like growth factor-1 for local delivery

T Tokunou, R Miller, P Patwari, ME Davis, VFM Segers, AJ Grodzinsky, and RT Lee
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA and Biological Engineering, MIT, Cambridge, MA
FASEB J. 2008 June ; 22(6): 1886–1893.   http://dx.doi.org/10.1096/fj.07-100925

Insulin-like growth factor-1 (IGF-1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF-1 has been considered to have therapeutic potential, systemic side effects of IGF-1 are significant, and local delivery of IGF-1 for tissue repair has been a long-standing challenge. In this study, we designed and purified a novel protein, heparin-binding IGF-1 (Xp-HB-IGF-1), which is a fusion protein of native IGF-1 with the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor. Xp-HB-IGF-1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp-HB-IGF-1 activated the IGF-1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activity due to the heparin-binding domain. Because cartilage is a proteoglycan-rich environment and IGF-1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp-HB-IGF-1 in cartilage. Xp-HB-IGF-1 was selectively retained by cartilage explants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF-1. These data show that the strategy of engineering a “long-distance” growth factor like IGF-1 for local delivery may be useful for tissue repair and minimizing systemic effects.

INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) is a growth factor well known as an important mediator of cell growth and differentiation. IGF-1 stimulates several signaling pathways through the tyrosine kinase IGF-1 receptor, including phosphatidylinositol (PI) 3-kinase and mitogen-activated protein kinases (MAPKs). PI3-kinase has many downstream targets, including the kinase Akt, and activation of Akt promotes survival, proliferation, and growth.
IGF-1 has been extensively studied for its therapeutic potential in tissue repair and regeneration. IGF-1 is a small and highly diffusible protein that can act over long distances. However, systemic administration of IGF-1 has significant side effects as well as the potential to promote diabetic retinopathy and cancer. Therefore, local delivery of IGF-1 has been a longstanding challenge. Here, we describe the design of a new protein, formed by fusion of IGF-1 with the heparin-binding (HB) domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF binds selectively to glycosaminoglycans through its highly positively charged heparin-binding domain.

Thus, we hypothesized that engineering IGF-1 to bind to glycosaminoglycans could provide selective delivery of IGF-1 to cell surfaces or to specific tissues. We demonstrate that this heparin-binding IGF-1 (Xp-HB-IGF-1) can bind to cell surfaces as well as the proteoglycan-rich tissue of cartilage; furthermore, Xp-HB-IGF-1 prolongs the stimulation of chondrocyte biosynthesis, demonstrating its potential for tissue specific repair.

Purification of Xp-HB-IGF-1

Figure 1A—C shows the constructs for Xp-HB-IGF-1 and the control Xp-IGF-1 fusion proteins.

IGF-1 has 3 disulfide bonds and includes 70 amino acids. The IGF-1 fusion proteins both contain polyhistidine tags for protein purification and Xpress tags for protein detection. The expected molecular masses of Xp-HB-IGF-1 and Xp-IGF-1 are 14,018 and 11,548 Da, respectively. Xp-HB-IGF-1 has the HB domain on the N terminus of IGF-1. The HB domain has 21 amino acids and includes 12 positively charged amino acids. Final purification of the new fusion proteins after refolding was performed with RP-HPLC (Fig. 1D, E). Identification of the correctly folded protein was performed as described previously and confirmed with bioactivity assays. These 3 IGF-1s (Xp-HB-IGF-1, Xp-IGF-1, and unmodified IGF-1) yielded similar intensities.

Xp-HB-IGF-1 binds to heparin and cell surfaces

1. Xp-HB-IGF-1 binds selectively to heparin compared with Xp-IGF-1 (Fig. 2A).
2. Xp-HB-IGF-1 bound to 3T3 fibroblast cells when treated with 10 and 100 nM concentrations.
3. Xp-HB-IGF-1 binds with neonatal cardiac myocytes, with clear selective binding of Xp-HB-IGF-1 (Fig 2C)
4. These results are consistent with binding of this HB domain to heparan sulfate in the submicromolar range
5. Xp-HB-IGF-1 was readily detected on the surfaces of ES cells in embryoid bodies — which contain multiple cell types.
6. There is more Xpress epitope tag in Xp-HB-IGF-1 group than the Xp-IGF-1 group, suggesting that Xp-HB-IGF-1 binds with heparan sulfate on the cell surface.

Xp-HB-IGF-1 bioactivity

Bioassays for IGF-1 receptor phosphorylation and Akt activation were performed. Control IGF-1, Xp-HB-IGF-1, and Xp-IGF-1 all activated the IGF-1 receptor of neonatal cardiac myocytes dose-dependently and induced Akt phosphorylation identically (Fig. 3A), and they  activated Akt with a similar time course (Fig. 3B), indicating — addition of the heparin-binding domain does not interfere with the bioactivity of IGF-1.

  1. Xp-HB-IGF-1 transport in cartilage
  2. Cartilage is a proteoglycan-rich tissue, and chondrocytes respond to IGF-1 with increased extracellular matrix synthesis (19). Because prolonged local stimulation of IGF-1 signaling could thus be beneficial for cartilage repair, we studied the ability of Xp-HB-IGF-1 to bind to cartilage.
  3. Xp-HB-IGF-1 is selectively retained by cartilage, while Xp-IGF-1 is rapidly lost.
  4. Xp-HB-IGF-1 can bind to cartilage after chondroitin sulfate digestion

To explore the possibility of nonspecific binding of Xp-HB-IGF-1 to glycosaminoglycans other than heparan sulfate, we studied the binding of Xp-HB-IGF-1 after chondroitinase ABC digestion.
Xp-HB-IGF-1 retention is not mediated by the pool of chondroitin sulfated proteoglycans in the cartilage matrix.

  1. Xp-HB-IGF-1 increases chondrocyte biosynthesis
  2. Xp-HB-IGF-1, which is selectively retained in the cartilage, stimulates chondrocyte biosynthesis over a more sustained period.

DISCUSSION

In this study, we describe a novel IGF-1 protein, Xp-HB-IGF-1, which binds to proteoglycan-rich tissue and cell surfaces but has the same bioactivity as IGF-1. Our data indicate that Xp-HB-IGF-1 can activate the IGF-1 receptor and Akt and thus that the heparin-binding domain does not interfere with interactions of IGF-1 and its receptor. IGF-1 has four domains: B domain (aa 1–29), C domain (aa 30 – 41), A domain (aa 42–62) and D domain (aa 63–70), with the C domain playing the most important role in binding to the IGF-1 receptor. Replacement of the entire C domain causes a 30-fold decrease in affinity for the IGF-1 receptor. Thus, the addition of the heparin-binding domain to the N terminus of IGF-1 was not anticipated to interfere with interactions with the IGF-1 C domain.
Both extracellular matrix and cell surfaces are rich in proteoglycans and can serve as reservoirs for proteoglycan-binding growth factors. A classic example is the fibroblast growth factor-2 (FGF-2) system, where a low-affinity, high-capacity pool of proteoglycan receptors serves as a reservoir of FGF-2 for its high-affinity receptor. Our experiments suggest that Xp-HB-IGF-1 could function in some circumstances in a similar manner, since Xp-HB-IGF-1 is selectively retained on cell surfaces. Many growth factors are known to interact with heparan sulfate, including HB-EGF (10-12), FGF-2 (26), vascular endothelial growth factor-A (VEGF-A), transforming growth factor beta (TGF-β) (28), platelet-derived growth factors (PDGFs), and hepatocyte growth factor (HGF). However, other proteins such as nerve growth factor (NGF), which induces differentiation and reduces apoptosis of neurons, does not have the heparin-binding domain. Thus, the strategy of engineering growth factors for selective matrix or cell surface binding could be used for other growth factors.
IGF-1 can also bind with extracellular matrix via IGF binding proteins (IGFBPs); in the circulation, at least 99% of IGF-1 is bound to IGFBPs (IGFBP-1 to −6). Further experiments are necessary to determine whether addition of a heparin-binding domain to IGF-1 changes interactions with IGFBPs and whether this changes its biological activity.
IGF-1 can promote the synthesis of cartilage extracellular matrix and inhibit cartilage degradation (19); however, a practical mode of IGF-1 delivery to cartilage has yet to be developed (33). Heparan sulfate proteoglycans are prevalent in the pericellular matrix of cartilage, particularly as chains on perlecan and syndecan-2, and are known to bind other ligands such as FGF-2 (34). Our experiments suggest that Xp-HB-IGF-1 protein can bind with matrix and increase local, long-term bioavailability to chondrocytes and thus may improve cartilage repair.

Selected References

Hameed M, Orrell RW, Cobbold M, Goldspink G, Harridge SD. Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise. J. Physiol 2003;547:247–254. [PubMed: 12562960]
Shavlakadze T, Winn N, Rosenthal N, Grounds MD. Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle. Growth Horm. IGF Res 2005;15:4–18. [PubMed: 15701567]
Milner SJ, Francis GL, Wallace JC, Magee BA, Ballard FJ. Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties. Biochem. J 1995;308(Pt 3):865–871. [PubMed: 8948444]
Milner SJ, Carver JA, Ballard FJ, Francis GL. Probing the disulfide folding pathway of insulin-like growth factor-I. Biotechnol. Bioeng 1999;62:693–703. [PubMed: 9951525]
Bonassar LJ, Grodzinsky AJ, Srinivasan A, Davila SG, Trippel SB. Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage. Arch. Biochem. Biophys 2000;379:57–63. [PubMed: 10864441]
Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005;16:421–439. [PubMed: 15936977]
Musaro A, Dobrowolny G, Rosenthal N. The neuroprotective effects of a locally acting IGF-1 isoform. Exp. Gerontol 2007;42:76–80. [PubMed: 16782294]
Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 1986;883:173–177. [PubMed: 3091074]
Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991;64:841– 848. [PubMed: 1847668]
Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276:75–81. [PubMed: 9082989]

Figure 1.  Construction and purification of a new Xp-HB-IGF-1 fusion protein.

Figure 1.  Construction and purification of a new Xp-HB-IGF-1 fusion protein.

A) The heparin binding domain of HB-EGF was inserted N-terminal to IGF-1 to generate the fusion protein. The construct included the hexahistidine and Xpress tags from the pTrcHis vector for purification and detection. B) The resulting amino acid sequence of HB-IGF-1. C) Schematic for the structure of HB-IGF-1. Red circles: positively charged amino acids; blue circles: negatively charged amino acids; yellow circles: cysteines. The arrow shows the HB domain. In this figure the epitope tags are not shown. D, E) Representative reverse-phase high-performance liquid chromatography (RP-HPLC) elution profiles with single peaks containing correctly folded protein. Readings of optical density at 214 nm are in blue; readings at 280 nm are in red; elution is by acetonitrile (ACN) gradient. F) After RP-HPLC, Coomassie blue staining and Western blot analysis demonstrate isolation of single bands containing Xpress-tagged protein. The right panel shows that the Western blot analysis of IGF-1, and the two engineered IGF-1 proteins yield similar results using an anti-IGF-1 antibody.

Protein Therapeutics for Cardiac Regeneration after Myocardial Infarction

Vincent F.M. Segers and Richard T. Lee
Provasculon Inc, 14 Cambridge Center, and Harvard Stem Cell Institute and the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA
J Cardiovasc Transl Res. 2010 October ; 3(5): 469–477.   http://dx.doi./10.1007/s12265-010-9207-5.

Although most medicines have historically been small molecules, many newly approved drugs are derived from proteins. Protein therapies have been developed for treatment of diseases in almost every organ system, including the heart. Great excitement has now arisen in the field of regenerative medicine, particularly for cardiac regeneration after myocardial infarction. Every year, millions of people suffer from acute myocardial infarction, but the adult mammalian myocardium has limited regeneration potential. Regeneration of the heart after myocardium infarction is therefore an exciting target for protein therapeutics.  

In this review, we discuss different classes of proteins that have therapeutic potential to regenerate the heart after myocardial infarction. Protein candidates have been described that induce angiogenesis, including fibroblast growth factors and vascular endothelial growth factors, although thus far clinical development has been disappointing. Chemotactic factors that attract stem cells, e.g. hepatocyte growth factor and stromal cell derived factor-1, may also be useful. Finally, neuregulins and periostin are proteins that induce cell cycle reentry of cardiomyocytes, and growth factors like IGF-1 can induce growth and differentiation of stem cells. As our knowledge of the biology of regenerative processes and the role of specific proteins in these processes increases, the use of proteins as regenerative drugs could develop as a cardiac therapy.
Keywords: protein therapeutics; myocardial infarction; regeneration; heart failure

The current standard of care for MI is early reperfusion of the occluded vessel with angioplasty or thrombolysis to reverse ischemia and increase the number of surviving myocytes. Efforts to decrease delays between onset of symptoms and reperfusion have resulted in decreased morbidity and mortality, but the maximal benefit of early reperfusion has reached a point close to practical limits. Besides early reperfusion therapy, ACE inhibitors and beta-blockers are used to prevent remodeling after MI and progression to heart failure. Both ACE inhibitors and beta-blockers improve long term survival but no therapies besides cardiac transplantation are currently available that restore cardiac function.
In the last decade, a large number of pre-clinical and clinical studies have been published on the potential use of stem cells for cardiac regeneration after MI. Different stem cell types have been shown to improve cardiac function in animal studies and can induce a small but potentially significant increase in ejection fraction in clinical studies. Stem cell therapy is a promising treatment option for heart failure, but numerous technical challenges and gaps in our understanding of stem cell behavior may limit translation to the clinic.
With the advent of biotechnology, protein and peptide drugs are becoming increasingly important in modern medicine. Drugs based on naturally-occurring proteins have the advantage of efficacy based on a mechanism of action refined by millions of years of biological evolution. Though promising as therapeutics, proteins might behave differently when used at pharmacological instead of physiological concentrations with an increase in adverse effects on other organs. Proteins used as therapeutics have been modified in different ways to limit immunogenicity and rapid degradation in plasma and tissues.
We discuss four different classes of proteins that could potentially benefit patients with MI (Figure 1); all of these proteins have been shown to improve cardiac function in animal models of MI or heart failure. They include angiogenic growth factors, proteins that increase recruitment of progenitor cells to the heart, proteins that induce mitosis of existing myocytes, and proteins that increase differentiation and growth of stem cells and myocytes. As more is learned about cardiac regeneration and why mammals lack sufficient myocardial regeneration, more proteins are likely to be added to this list of candidates.

A decade of extensive research on cardiac stem cell biology revealed 1 protein (G-CSF) that can be used to mobilize hematopoietic stem cells and just 2 proteins with chemotactic properties on stem cells: SDF-1 on endothelial progenitor cells and HGF on cardiac stem cells. Another protein that has been identified as a stem cell attractant is monocyte chemotactic protein-3 which attracts mesenchymal stem cells [42]. It is unknown if local administration of MCP-3 improves cardiac function. Identification of new stem cell chemotactic proteins is important because it could lead to the development of new and feasible therapeutics for treatment of MI and heart failure. At the same time, the true regenerative potential of most stem cells remains highly controversial; indicating that even if a chemotactic factor attracting stem cells to the heart is identified, formation of functional myocardial is still a challenging task.

Proteins like periostin and neuregulin which stimulate mitosis of surviving myocytes can partially restore the damage inflicted by MI. However, some requirements have to be met before this will result in a viable therapy. An inherent selectivity for myocytes would also allow for systemic delivery as opposed to the use of more complicated local delivery methods. An important factor to consider is the duration of the signal necessary to induce mitosis in a significant number of myocytes. A protein that induces cell cycle reentry in a significant fraction of myocytes with a single pulse has more therapeutical potential than a protein that needs sustained or repeated delivery. Ideally, pro-mitotic proteins will be not only specific for myocytes in general but might also be specific for myocytes in the border zone of the MI. This has drawbacks, among which is that formation of new myocytes, either by stem cell differentiation or by myocyte mitosis, carries an increased risk of ventricular arrhythmias.

Figure 1. Regeneration of the heart by 4 different classes of proteins

Figure 1. Regeneration of the heart by 4 different classes of proteins

See text for details. A) FGF and VEGF increase angiogenesis. B) G-CSF mobilizes bone marrow hematopoietic stem cells and SDF-1 attracts endothelial progenitor cells. HGF attracts cardiac stem cells. C) Neuregulin and periostin can induce division of adult cardiomyocytes. D) IGF-1 induces maturation and differentiation of cardiac stem cells.

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Dealing with the Use of the High Sensitivity Troponin (hs cTn) Assays: Preparing the United States for High-Sensitivity Cardiac Troponin Assays

Author and Curator: Larry H Bernstein, MD, FCAP
Author and Curator: Aviva Lev-Ari, PhD, RD

In this article we shall address the two following papers:
  1. Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI – Corus CAD, hs cTn, CCTA
  2. Frederick K. Korley, MD, Allan S. Jaffe, MD in Journal of the American College of Cardiology  J Am Coll Cardiol. 2013; 61(17):1753-1758.

In a previous posting I commented on the problem of hs cTn use and the on site ED performance of cardiac treadmill (done in Europe)

  • prior to a decision of CT scan (not done in US).

Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI – Corus CAD, hs cTn, CCTA

We examine the emergence of Alternatives to Angiography and PCI as most common strategy for ER admission with listed cause of Acute Chest Pain. The Goal is to use methods that will improve the process to identify for an Interventional procedure only the patients that a PCI is a must to have.

Alternative #1: Corus®  CAD

Alternative #2: High-Sensitivity Cardiac Troponins in Acute Cardiac Care

Alternative #3: Coronary CT Angiography for Acute Chest Pain
After presenting the the Three alternatives, the Editorial by R.F. Redberg, Division of Cardiology, UCSF, will be analyzed.
  • Alternative #1:  First-Line Test to Help Clinicians Exclude Obstructive CAD as a Cause of the Patient’s Symptoms

Corus®  CAD, a blood-based  gene expression test, demonstrated high accuracy with both a high negative predictive value (96 percent) and high sensitivity (89 percent) for assessing  obstructive coronary artery disease  (CAD) in a population of patients referred for stress testing with myocardial perfusion imaging (MPI).

COMPASS enrolled stable patients with symptoms suggestive of CAD who had been referred for MPI at 19 U.S. sites.  A blood sample was obtained in all 431 patients prior to MPI and Corus CAD gene expression testing was performed with study investigators blinded to Corus CAD test results.Following MPI, patients underwent either invasive coronary angiography orcoronary CT angiography, gold-standard anatomical tests for the diagnosis of coronary artery disease.

A Blood Based Gene Expression Test for Obstructive Coronary Artery Disease Tested in Symptomatic Non-Diabetic Patients Referred for Myocardial Perfusion Imaging: The COMPASS Study

https://pharmaceuticalintelligence.com/2012/08/14/obstructive-coronary-artery-disease-diagnosed-by-rna-levels-of-23-genes-cardiodx-heart-disease-test-wins-medicare-coverage/

  • Alternative #2: High-Sensitivity Cardiac Troponins in Acute Cardiac Care

Recommendations for the use of cardiac troponin (cTn) measurement in acute cardiac care have recently been published.[1] Subsequently, a high-sensitivity (hs) cTn T assay was introduced into routine clinical practice.[2] This assay, as others, called highly sensitive, permits measurement of cTn concentrations in significant numbers of apparently illness-free individuals. These assays can measure cTn in the single digit range of nanograms per litre (=picograms per millilitre) and some research assays even allow detection of concentrations <1 ng/L.[2–4] Thus, they provide a more precise calculation of the 99th percentile of cTn concentration in reference subjects (the recommended upper reference limit [URL]). These assays measure the URL with a coefficient of variation (CV) <10%.[2–4]The high precision of hs-cTn assays increases their ability to determine small differences in cTn over time. Many assays currently in use have a CV >10% at the 99th percentile URL limiting that ability.[5–7] However, the less precise cTn assays do not cause clinically relevant false-positive diagnosis of acute myocardial infarction (AMI) and a CV <20% at the 99th percentile URL is still considered acceptable.[8]

We believe that hs-cTn assays, if used appropriately, will improve clinical care. We propose criteria for the clinical interpretation of test results based on the limited evidence available at this time.

References

1. Thygesen K, Mair J, Katus H, Plebani M, Venge P, Collinson P, Lindahl B, Giannitsis E, Hasin Y, Galvani M, Tubaro M, Alpert JS, Biasucci LM, Koenig W, Mueller C, Huber K, Hamm C, Jaffe AS; Study Group on Biomarkers in Cardiology  of the ESC Working Group on Acute Cardiac Care. Recommendations  for the use of cardiac troponin measurement in acute cardiac care. Eur Heart J 2010;31:2197–2204.

2. Saenger AK, Beyrau R, Braun S, Cooray R, Dolci A, Freidank H, Giannitsis E, Gustafson S, Handy B, Katus H, Melanson SE, Panteghini M, Venge P, Zorn M, Jarolim P, Bruton D, Jarausch J, Jaffe AS. Multicenter analytical evaluation of a high sensitivity troponin T assay. Clin Chim Acta 2011;412:748–754.

3. Zaninotto M, Mion MM, Novello E, Moretti M, Delprete E, Rocchi MB, Sisti D, Plebani M. Precision performance at low levels and 99th percentile concentration of the Access AccuTnI assay on two different platforms. Clin Chem Lab Med 2009; 47:367–371.

4. Todd J, Freese B, Lu A, Held D, Morey J, Livingston R, Goix P. Ultrasensitive flow based immunoassays using single-molecule counting. Clin Chem 2007; 53:1990–1995.

5. van de Kerkhof D, Peters B, Scharnhorst V. Performance of Advia Centaur second-generation troponin assay TnI-Ultra compared with the first-generation cTnI assay. Ann Clin Biochem 2008; 45:316–317.

6. Lam Q, Black M, Youdell O, Spilsbury H, Schneider HG. Performance evaluation and subsequent clinical experience with the Abbott automated Architect STAT Troponin-I assay. Clin Chem 2006; 52:298–300.

7. Tate JR, Ferguson W, Bais R, Kostner K, Marwick T, Carter A. The determination of the 99th percentile level for troponin assays in an Australian reference population. Ann Clin Biochem 2008; 45:275–288.

8. Jaffe AS, Apple FS, Morrow DA, Lindahl B, Katus HA. Being rational about (im)-precision: a statement from the Biochemistry Subcommittee of the Joint European Society of Cardiology/American College of Cardiology Foundation/American Heart Association/World Heart Federation Task Force for the definition of myocardial infarction. Clin Chem 2010; 56:921–943.

To the Editor:

Hoffmann et al. (July 26 issue)1 conclude that, among patients with low-to-intermediate-risk acute coronary syndromes, the incorporation of coronary computed tomographic angiography (CCTA) improves the standard evaluation strategy.2 However, it may be difficult to generalize their results, owing to different situations on the two sides of the Atlantic and the availability of high-sensitivity troponin T assays in Europe. In the United States, the Food and Drug Administration has still not approved a high-sensitivity troponin test, and patients in the Rule Out Myocardial Infarction/Ischemia Using Computer Assisted Tomography (ROMICAT-II) trial only underwent testing with the conventional troponin T test. As we found in the biomarker substudy in the ROMICAT-I trial, a single high-sensitivity troponin T test at the time of CCTA accurately ruled out acute myocardial infarction (negative predictive value, 100%) (Table 1TABLE 1Results of High-Sensitivity Troponin T Testing for the Diagnosis of Acute Coronary Syndromes in ROMICAT-I.).3 In addition, patients with acute myocardial infarction can be reliably identified, with up to 100% sensitivity, with the use of two high-sensitivity measurements of troponin T within 3 hours after admission.4,5

It seems plausible to assume that the incorporation of high-sensitivity troponin T assays in this trial would have outperformed CCTA. Therefore, it is important to assess the performance of such testing and compare it with routine CCTA testing in terms of length of stay in the hospital and secondary end points, especially cumulative costs and major adverse coronary events at 28 days.

Mahir Karakas, M.D.
Wolfgang Koenig, M.D.
University of Ulm Medical Center, Ulm, Germany
wolfgang.koenig@uniklinik-ulm.de

References

  1. Hoffmann U, Truong QA, Schoenfeld DA, et al. Coronary CT angiography versus standard evaluation in acute chest pain. N Engl J Med 2012;367:299-308

  2. Redberg RF. Coronary CT angiography for acute chest pain. N Engl J Med 2012;367:375-376

  3. Januzzi JL Jr, Bamberg F, Lee H, et al. High-sensitivity troponin T concentrations in acute chest pain patients evaluated with cardiac computed tomography. Circulation2010;121:1227-1234

  4. Keller T, Zeller T, Ojeda F, et al. Serial changes in highly sensitive troponin I assay and early diagnosis of myocardial infarction. JAMA 2011;306:2684-2693

  5. Thygesen K, Mair J, Giannitsis E, et al. How to use high-sensitivity cardiac troponins in acute cardiac care. Eur Heart J 2012;33:2252-2257

Author/Editor Response

In response to Karakas and Koenig: we agree that high-sensitivity troponin T assays may permit more efficient care of low-risk patients presenting to the emergency department with acute chest pain1 and may also have the potential to identify patients with unstable angina because cardiac troponin T levels are associated with the degree and severity of coronary artery disease.2 Hence, high-sensitivity troponin T assays performed early may constitute an efficient and safe gatekeeper for imaging. CCTA, however, may be useful for ruling out coronary artery disease in patients who have cardiac troponin T levels above the 99th percentile but below levels that are diagnostic for myocardial infarction. The hypothesis that high-sensitivity troponin T testing followed by CCTA, as compared with other strategies, may enable safe and more efficient treatment of patients in the emergency department who are at low-to-moderate risk warrants further assessment. The generalizability of our data to clinical settings outside the United States may also be limited because of differences in the risk profile of emergency-department populations and the use of nuclear stress imaging.3

Udo Hoffmann, M.D., M.P.H.
Massachusetts General Hospital, Boston, MA
uhoffmann@partners.org

W. Frank Peacock, M.D.
Baylor College of Medicine, Houston, TX

James E. Udelson, M.D.
Tufts Medical Center, Boston, MA

Since publication of their article, the authors report no further potential conflict of interest.

References

  1. Than M, Cullen L, Reid CM, et al. A 2-h diagnostic protocol to assess patients with chest pain symptoms in the Asia-Pacific region (ASPECT): a prospective observational validation study. Lancet 2011;377:1077-1084

  2. Januzzi JL Jr, Bamberg F, Lee H, et al. High-sensitivity troponin T concentrations in acute chest pain patients evaluated with cardiac computed tomography. Circulation2010;121:1227-1234

  3. Peacock WF. The value of nothing: the consequence of a negative troponin test. J Am Coll Cardiol 2011;58:1340-1342

  • Alternative #3: Coronary CT Angiography for Acute Chest Pain

The Study concluded:

There was increased diagnostic testing and higher radiation exposure in the CCTA group, with no overall reduction in the cost of care. 

Coronary CT Angiography versus Standard Evaluation in Acute Chest Pain

Udo Hoffmann, M.D., M.P.H., Quynh A. Truong, M.D., M.P.H., David A. Schoenfeld, Ph.D., Eric T. Chou, M.D., Pamela K. Woodard, M.D., John T. Nagurney, M.D., M.P.H., J. Hector Pope, M.D., Thomas H. Hauser, M.D., M.P.H., Charles S. White, M.D., Scott G. Weiner, M.D., M.P.H., Shant Kalanjian, M.D., Michael E. Mullins, M.D., Issam Mikati, M.D., W. Frank Peacock, M.D., Pearl Zakroysky, B.A., Douglas Hayden, Ph.D., Alexander Goehler, M.D., Ph.D., Hang Lee, Ph.D., G. Scott Gazelle, M.D., M.P.H., Ph.D., Stephen D. Wiviott, M.D., Jerome L. Fleg, M.D., and James E. Udelson, M.D. for the ROMICAT-II Investigators

N Engl J Med 2012; 367:299-308 July 26, 2012  http://dx.doi.org/10.1056/NEJMoa1201161

BACKGROUND

It is unclear whether an evaluation incorporating coronary computed tomographic angiography (CCTA) is more effective than standard evaluation in the emergency department in patients with symptoms suggestive of acute coronary syndromes.

METHODS

In this multicenter trial, we randomly assigned patients 40 to 74 years of age with symptoms suggestive of acute coronary syndromes but without ischemic electrocardiographic changes or an initial positive troponin test to early CCTA or to standard evaluation in the emergency department on weekdays during daylight hours between April 2010 and January 2012. The primary end point was length of stay in the hospital. Secondary end points included rates of discharge from the emergency department, major adverse cardiovascular events at 28 days, and cumulative costs. Safety end points were undetected acute coronary syndromes.

RESULTS

The rate of acute coronary syndromes among 1000 patients with a mean (±SD) age of 54±8 years (47% women) was 8%. After early CCTA, as compared with standard evaluation, the mean length of stay in the hospital was reduced by 7.6 hours (P<0.001) and more patients were discharged directly from the emergency department (47% vs. 12%, P<0.001). There were no undetected acute coronary syndromes and no significant differences in major adverse cardiovascular events at 28 days. After CCTA, there was more downstream testing and higher radiation exposure. The cumulative mean cost of care was similar in the CCTA group and the standard-evaluation group ($4,289 and $4,060, respectively; P=0.65).

CONCLUSIONS

In patients in the emergency department with symptoms suggestive of acute coronary syndromes, incorporating CCTA into a triage strategy improved the efficiency of clinical decision making, as compared with a standard evaluation in the emergency department, but it resulted in an increase in downstream testing and radiation exposure with no decrease in the overall costs of care. (Funded by the National Heart, Lung, and Blood Institute; ROMICAT-II ClinicalTrials.gov number, NCT01084239.)

http://www.nejm.org/doi/full/10.1056/NEJMoa1201161#t=abstract

REFERENCES

  1. Roe MT, Harrington RA, Prosper DM, et al. Clinical and therapeutic profile of patients presenting with acute coronary syndromes who do not have significant coronary artery disease. Circulation 2000;102:1101-1106

  2. Miller JM, Rochitte CE, Dewey M, et al. Diagnostic performance of coronary angiography by 64-row CT. N Engl J Med 2008;359:2324-2336

  3. Budoff MJ, Dowe D, Jollis JG, et al. Diagnostic performance of 64-multidetector row coronary computed tomographic angiography for evaluation of coronary artery stenosis in individuals without known coronary artery disease: results from the prospective multicenter ACCURACY (Assessment by Coronary Computed Tomographic Angiography of Individuals Undergoing Invasive Coronary Angiography) trial. J Am Coll Cardiol 2008;52:1724-1732

  4. Marano R, De Cobelli F, Floriani I, et al. Italian multicenter, prospective study to evaluate the negative predictive value of 16- and 64-slice MDCT imaging in patients scheduled for coronary angiography (NIMISCAD-Non Invasive Multicenter Italian Study for Coronary Artery Disease). Eur Radiol 2009;19:1114-1123
  5. Meijboom WB, Meijs MF, Schuijf JD, et al. Diagnostic accuracy of 64-slice computed tomography coronary angiography: a prospective, multicenter, multivendor study. J Am Coll Cardiol 2008;52:2135-2144
  6. Hoffmann U, Bamberg F, Chae CU, et al. Coronary computed tomography angiography for early triage of patients with acute chest pain: the ROMICAT (Rule Out Myocardial Infarction using Computer Assisted Tomography) trial. J Am Coll Cardiol 2009;53:1642-1650

  7. Hollander JE, Chang AM, Shofer FS, et al. One-year outcomes following coronary computerized tomographic angiography for evaluation of emergency department patients with potential acute coronary syndrome. Acad Emerg Med 2009;16:693-698

  8. Rubinshtein R, Halon DA, Gaspar T, et al. Usefulness of 64-slice cardiac computed tomographic angiography for diagnosing acute coronary syndromes and predicting clinical outcome in emergency department patients with chest pain of uncertain origin. Circulation2007;115:1762-1768

  9. Schlett CL, Banerji D, Siegel E, et al. Prognostic value of CT angiography for major adverse cardiac events in patients with acute chest pain from the emergency department: 2-year outcomes of the ROMICAT trial. JACC Cardiovasc Imaging 2011;4:481-491

  10. Goldstein JA, Chinnaiyan KM, Abidov A, et al. The CT-STAT (Coronary Computed Tomographic Angiography for Systematic Triage of Acute Chest Pain Patients to Treatment) trial. J Am Coll Cardiol 2011;58:1414-1422

  11. Litt HI, Gatsonis C, Snyder B, et al. CT angiography for safe discharge of patients with possible acute coronary syndromes. N Engl J Med 2012;366:1393-1403

  12. Shreibati JB, Baker LC, Hlatky MA. Association of coronary CT angiography or stress testing with subsequent utilization and spending among Medicare beneficiaries. JAMA2011;306:2128-2136

  13. Hoffmann U, Truong QA, Fleg JL, et al. Design of the Rule Out Myocardial Ischemia/Infarction Using Computer Assisted Tomography: a multicenter randomized comparative effectiveness trial of cardiac computed tomography versus alternative triage strategies in patients with acute chest pain in the emergency department. Am Heart J2012;163:330-338

  14. Abbara S, Arbab-Zadeh A, Callister TQ, et al. SCCT guidelines for performance of coronary computed tomographic angiography: a report of the Society of Cardiovascular Computed Tomography Guidelines Committee. J Cardiovasc Comput Tomogr 2009;3:190-204

  15. Gerber TC, Carr JJ, Arai AE, et al. Ionizing radiation in cardiac imaging: a science advisory from the American Heart Association Committee on Cardiac Imaging of the Council on Clinical Cardiology and Committee on Cardiovascular Imaging and Intervention of the Council on Cardiovascular Radiology and Intervention. Circulation 2009;119:1056-1065

  16. von Ballmoos MW, Haring B, Juillerat P, Alkadhi H. Meta-analysis: diagnostic performance of low-radiation-dose coronary computed tomography angiography. Ann Intern Med2011;154:413-420[Erratum, Ann Intern Med 2011;154:848.]

  17. Achenbach S, Marwan M, Ropers D, et al. Coronary computed tomography angiography with a consistent dose below 1 mSv using prospectively electrocardiogram-triggered high-pitch spiral acquisition. Eur Heart J 2010;31:340-346

  18. Than M, Cullen L, Reid CM, et al. A 2-h diagnostic protocol to assess patients with chest pain symptoms in the Asia-Pacific region (ASPECT): a prospective observational validation study. Lancet 2011;377:1077-1084

In the EDITORIAL by Redberg RF. Dr. Redberg, Cardiology Division, UCSF made the following points in:

Coronary CT angiography for acute chest pain. N Engl J Med 2012;367:375-376

  • Six million people present to ER annually with Acute Chest Pain, most have other diseases that Heart.
  • Current diagnostic methods lead to admission to the hospital, unnecessary stays and over-treatment – improvement of outcomes is needed.
  • Rule Out Myocardial Infarction Using Computer Assisted Tomography II (ROMICAT-II) 100 patients were randomly assigned to CCTA group or Standard Diagnosis Procedures Group in the ER which involved Stress Test in 74%.

CRITIQUE and Study FLAWS in MGH Study:

  • ROMICAT-II enrolled patients only during “weekday daytime hours, no weekend or nights when the costs are higher.
  • Assumption that a diagnostic test must be done before discharge for low-to-intermediate-risk patients is unproven and probably unwarranted.. No evidence that the tests performed let to improved outcomes.
  • Events rate for patient underwent CCTA, Stress test or no testing at al were less that 1% to have an MI, no one died. Thus, it is impossible to assign a benefit to the CCTA Group. So very low rates were observed in other studies
  • CCTA patients were exposed to substantial dose of Radiation, , contrast die,
  • Patients underwent ECG and Negative Troponin, no evidence that additional testing further reduced the risk.
  • Average age of patients: 54, 47% women.Demographic Characteristics with low incidence of CAD, NEJM, 1979; 300:1350-8
  • Risk of Cancer from radiation in younger population is higher, same in women.
  • Hoffmann’s Study: Radiation burden was clinically significant: Standard Evaluation Group: (4.7+-8.4 mSv), CCTA: (13.9+-10.4 mSv), exposure of 10 mSv have been projected to lead to 1 death from Cancer per 2000 persons, Arch Intern Med 2009; 169:2071-7
  • Middle Age women, increased risk of Breast Cancer from radiation, Arch Intern Med 2012 June 11 (ePub ahead of Print)
  • ROMICAT-II study: discharge diagnosis Acute Coronary Syndrome – less than 10%
  • CCTA Group: more tests, more radiation, more interventions tht the standard-evaluation group.
  • Choose Wisely Campaign – order test only when the benefit will exceed the risks

Dr. Redberd advocates ECG and Troponin, if NORMAL, no further testing.

Epicrisis on Part 1

Redberg’s conclusions are correct for the initial screening. The issue has been whether to do further testing for low or intermediate risk patients.

The most intriguing finding that is not at all surprising is that the CCTA added very little in the suspect group with small or moderate risk. My original studies using a receiver operator characteristic curve were very good, although some patients with CRF or ESRD had extremely high values. The ultra sensitive troponin threw the Area Under the ROC out the window, under the assumption that a perfect assay would exclude AMI, or any injury to the heart. The improved assay does pick up minor elevations of troponin in the absence of MI as a result of plaque rupture. It is possible that 50% of these elevations need medical attention, but then the question is an out of hospital referral or admission and further workup. I have discussed this at some length on several occasions with Dr. Jaffe at Mayo Clinic.

Many of those with minor or intermediate elevation have significant renal insufficiency, but they might also be in CKD Class 3 and not 1 or 2. The coexistence of Type 2 diabetes would go into the standard assessment, but is not mentioned in the study with respect to immediate admission or outcome 28 days after discharge.

The hs troponin I has been in daily use on the Ortho J&J (formerly Kodak) for about 2 years, and the QC standards are very high. I expected the Roche hs-TnT assay to be in use in US as well, but there may have been delays.  Januzzi , Jaffe, and Fred Aplle would be involved in the evaluation in the US, but Paul Collinson in UK, Katus and Mair in Germany, and other Europena centers certainly have been using the Roche Assay.

The biggest problem in these studies is as my mentor called my attention to – the frontrunners aren’t going to support a nose-to-nose up front study. Given that a diagnosis requires more information at minimal cost, especially when diagnosis of the heart that are not MI have to be evaluated as well, it is incomprehensibe to me that such information as

  1. mean arterial blood pressure,
  2. natriuretic peptides,
  3. the calculated EGFR are not used in the evaluation.

It is quite impossible to clear the deck when you have patients who don’t have

  1. ST elevation,
  2. depression, or
  3. T-wave inversion who are seen for vague

(not to mention long QT abnormalities).

  • predordial tightness or shortness of breath
  • pain that resembles gall bladder.

Is this an indication of the obsolescence of the RCT.

A Retrospective Quality and Cost Driven Audit on Effect of hs cTn Assay with On-Site CT Followup. (No treadmill availability)

A retrospective multisite study showed that doing the hs cTn followed by CT on-site was a good choice for US.

I also considered  the selective release of

  • low- moderate-risk patients cardiology followup in a timely manner.

This report is an excellent analysis of my point by Korley and Jaffe in Medscape, and satisfies some several years discussion

I have had with Dr. Jaffe, at Mayo Clinic.  He pointed out the importance of

  • Type 1 and Type 2 AMI

at a discussion with Dr. Fred Apple at a meeting of the Amer Assn for Clinical Chemistry that he fully elaborates on here.
It is really a refinement of other proposals that are being discussed.  It is also timely because hs cTnI is already being used
widely in the US, while there might be a holdup on the hs cTnT.

Highlights

  1. Need for a Universally Accepted Nomenclature
  2. Defining Uniform Criteria for Reference Populations
  3. Discriminating Between Acute and Nonacute Causes of hs-cTn Elevations
  4. Distinguishing Between Type 1 and Type 2 AMI
  5. Analytical Imprecision in Cardiac Troponin Assays
  6. Ruling Out AMI
  7. Investigating the Causes of Positive Troponin Values in Non-AMI Patients
  8. Risk Stratifying Patients With Nonacute Coronary Syndrome Conditions
  9. Conclusions

Abstract

It is only a matter of time before the use of high-sensitivity cardiac
troponin assays (hs-cTn) becomes common throughout the United
States. In preparation  for this inevitability, this article raises a number
of important issues regarding  these assays that deserve consideration.

These include: the need for

  • the adoption  of a universal nomenclature; the importance
  • of defining uniform criteria for reference populations;
  • the challenge of discriminating between acute and nonacute
    causes of hs-cTn elevations, and
  • between type 1 and type 2 acute myocardial infarction (AMI);

factors influencing the analytical precision of hs-cTn;

  • ascertaining the optimal duration  of the rule-out period for AMI;
  • the need for further evaluation to determine the causes
    of a positive hs-cTn in non-AMI patients; and
  • the use of hs-cTn to risk-stratify patients with disease conditions
    other than AMI.

This review elaborates on these critical issues  as a means of
educating clinicians and researchers about them.

Introduction

Recently, clinicians have begun to use the recommended cut-off values
for current generation cardiac troponin (cTn) assays:

  • the 99th percentile upper reference limit (URL).

Previously, there was reluctance to use these cut-off values because

  • of  cTn elevations from non-acute ischemic heart disease conditions.

Thus, there was a tendency to use cut-off values for troponin that equated with the

  • prior gold standard diagnosis developed with less sensitive markers
    • creatinine kinase-MB isoenzyme (CK-MB) or
    • the lowest value at which assay achieved a 10%
      coefficient of variation (CV),

which would reduce false-positive elevations (without plaque rupture).

The use of the 99th percentile URL increases the ability of these assays to detect both

  •   acute myocardial infarction (AMI) and
  •   structural cardiac morbidities.[1]

This change in practice should not be confused with

  •   newer-generation high-sensitivity assays.

Improvements in the analytic performance of cTn assays have resulted in

  •   superior sensitivity and precision.

Improved sensitivity occurs because of

  •   more sensitive antigen binding and detection antibodies,
  •   increases in the concentration of the detection probes on the tag antibodies,
  •   increases in sample volume, and buffer optimization.[2]

Assays now are able to measure

  •   10-fold lower concentrations with high precision

(a CV <10% at the 99th percentile  of the URL).

The high-sensitivity cardiac troponin T (hs-cTnT) assay is already in clinical use
throughout most of the world. It is only a matter of time before high- sensitivity
assays are approved for use in the United States. In preparation for this, as well as

  •   using the 99th percentile URL with contemporary assays,

there are a number of important issues that deserve consideration. Key concepts are included in (Table 1).

Table 1: Key ConceptsThere is a need to develop a universal nomenclature for troponin assays.There is a need for uniform criteria for selecting reference populations.The optimal delta criteria for distinguishing between acute and chronic cardiac injury remain unclear and are likely to be assay-specific.Distinguishing between type 1 and type 2 AMI is challenging, and
more type 2 AMIs will be detected with hsTn assays.Factors affecting the analytical precision of troponin assays (including how we collect samples) will become more important with the use of hs-cTn assays.The optimal duration for ruling out AMI remains unclear;

  • novel approaches to this issue are being developed.

Elevated hs-cTn, regardless of the cause, has important

  • prognostic implications and deserves additional evaluation; 

Many cases of chronic elevations can be evaluated in an outpatient setting.

Hs-cTn can be used to

  • risk-stratify patients with non-ACS cardiovascular comorbidities.

Need for a Universally Accepted Nomenclature

The literature is replete with terms used to refer to cTn assays.
We advocate the use of the term “high-sensitivity cardiac troponin assays”  (hs-cTn) for

  • cTn assays that v   measure cardiac troponin values in
  • in  at least 50% of a reference population.[2,3]

This policy has now been embraced by the journal Clinical Chemistry. High-sensitivity
assays can be further categorized as well (Table 2) with respect to generations of cTn.

Table 2.  Classification of High-Sensitivity Cardiac Troponin Assays 

Category

Description

First Generation                                   Able to measure cTn in
50%–75% of                                       a reference population
Second Generation                              Able to measure cTn in
75%–95% of                                       a reference population
Third Generation                                 Able to measure cTn in
> 95%                                               a reference population
Adapted from Apple and Collinson (3)
  • Ideally, assays should have a CV of <10% at the 99th percentile value.

Assays that do not achieve this level are less sensitive which protects against
false-positive results, and they can be used.[4]

Defining Uniform Criteria for Reference Populations
There is a lack of consistency in the types and numbers of subjects that constitute a reference
population.[2] Often, participants are included after simple screening by check list but without a

  • physical examination,
  • electrocardiogram, or
  • laboratory testing.

At other times, a

  • normal creatinine and/or a normal natriuretic peptide value is required.
  • Imaging to detect structural heart disease is rarely used. 

Because it is known that

  • gender,
  • age,
  • race,
  • renal function,
  • heart failure, and
  • structural heart disease, including
  • increased left ventricular (LV) mass

are associated with increased cTn concentrations,[5,6,7] An assay’s 99th percentile value depends on the composition of the reference group. Thus, the more criteria used, the lower the reference values (Figure 1).[5]

http://img.medscape.com/article/803/159/803159-fig1.jpg

Have no history of

  • vascular disease or diabetes, and
  • not taking cardioactive drugs,
    • based on questionnaire.
Normal defined as those individuals who had
  • no history of vascular or cardiovascular disease,
  • diabetes mellitus,
  • hypertension, or
  • heavy alcohol intake and who were
  • receiving no cardiac medication AND
  • had blood pressure ≤140/90 mmHg;
  • fasting glucose  <110 mg/dL;
  • eGFR >60mL/min;
  • LVEF > 50%; normal lung function; and no significant
  • valvular heart disease,
  • LVH,
  • diastolic HF, or
  • regional wall-motion abnormalities on ECHO.

The appropriate reference value to use clinically also is far from a settled issue.
It might be argued that

  • using a higher 99th percentile value for the elderly
  • allows comparison of the patient to his or her peers, but

in raising the cut-off value, if the increases are caused by comorbidities,

  • those who are particularly healthy will be disadvantaged.[8]

Gender and ethnicity are not comorbidities, and we would urge that those should be taken into account.
Regardless of the assay, there will need to be

  • 99th percentile values for men that are different for women.[2]

The reference population for assay validation studies should ideally be based on  –
demographic characteristics that mirror the U.S. population and include subjects whose

  • blood pressure,
  • serum glucose, and
  • creatinine and
  • natriuretic peptide values are
  • within the normal reference range and
  • who take no cardiac  medications.

These subjects should be

  • free from structural heart disease,
  • documented by echocardiography,
  • cardiac magnetic resonance imaging (MRI) or
  • computed tomography (CT) angiography.

Meeting these criteria will be a major challenge, especially for older individuals.
A conjoint pool of samples collected with manufacturers’ support so that all methods were derived from an

  • identical patient population for their reference ranges would be ideal.

[However, the method of collection and possible freeze-thaw effects is unavoidable].

One large national effort might be advantageous over multiple efforts.

 Discriminating Between Acute and Nonacute Causes of hs-cTn Elevations

With the ability to precisely measure small concentrations of cTn,

  • clinicians will be faced with the challenge of distinguishing patients
    • who have acute problems from those with chronic elevations from other causes.

Using the fourth-generation cTnT assay, approximately 0.7% of patients in
the general population have modest elevations >99th percentile URL.[11]

In the same population, this number was 2% with the hs-cTnT assay.[6]  Only

  • half of them had documentation (even with imaging) of cardiac abnormalities.

If the prevalence of a positive cTnT is 2% in the general population,

  • it will likely be 10% or 20% in the emergency department (ED)
  • and even higher in hospitalized patients, as
  • these patients often have cardiac comorbidities.

Measurement of changes in hs-cTn over time (δ hs-cTn)

  • improves the specificity of hs-cTn for the diagnosis of acute cardiac injury.[12,13]

However, it does so at the cost of sensitivity. With contemporary assays, differences

  • in analytical variation have been used to define an increasing pattern.

At elevated values, CV for most assays is in the range of 5% to 7%, so

  • a change of 20% ensures that a given change is not caused

by analytical variation alone.[10]

At values near the 99th percentile URL, higher change values are necessary.[13]  The situation with hs-cTn assays is much more complex, as follows:

1. Change criteria are unique for each assay.
2. It will be easy to misclassify patients with coronary artery disease who may present with a noncardiac cause of chest pain

  • but have elevated values.

They could be having unstable ischemia or elevations caused by structural cardiac abnormalities and noncardiac discomfort.

If hs-cTn is rising significantly, the issue is easy but

  • if the values are not rising, a diagnosis of AMI still might be made.
  • If so, some patients may be included as having AMI without a changing pattern.
  • This occurred in 14% patients studied by Hammarsten et al.[14]

If patients with elevated hs-cTn without a changing pattern are not called AMI,

  • should they be called patients with “unstable angina and cardiac injury” or patients with structural heart disease and noncardiac chest pain?

Perhaps both exist?

3. The release of biomarkers is flow-dependent.Thus, there may not always be rapid access to the circulation. An area of injury distal to a totally occluded vessel (when collateral channels close) may be different in terms of the dynAMIcs of

  • hs-cTn change than an intermittently occluded coronary artery.
4. Conjoint biological and analytical variation can be measured.

  • They are assay-dependent, and the reference change values range from 35% to 85%.[2]

The use of criteria less than that (which may be what is needed clinically) will thus
likely include individuals with changes caused by

  • conjoint biological and analytical variation alone.

This has been shown to be the case in

  • many patients with nonacute cardiovascular diagnoses.[14,15]
5. Most evaluations have attempted to define the optimal delta, often with receiver operator curve analysis. Such an approach is based on the concept that sensitivity and specificity deserve equivalent weight.[But higher deltas improve specificity more and lower ones improve sensitivity and it is not clear that all physicians want the same tradeoffs in this regard.]ED physicians often prefer high-sensitivity so that their miss rate is low (<1%),[16] whereas hospital clinicians want increased specificity. This tension will need to be addressed in defining the optimal delta.
6. The delta associated with AMI may be different from that associated with other cardiac injury.[14] In addition, women have less marked elevations of cTn in response to coronary artery disease[17] and in earlier studies were less apt to have elevated values.[18] Given their pathology is at times different,

  • it may be that different metrics may be necessary based on gender
7. Some groups have assumed that if a change is of a given magnitude over 6 hours, it can be divided by 6 and the 1-h values can be used.

  • This approach is not data driven, and biomarker release is more likely to be discontinuous rather than continuous.[19]

In addition, the values obtained with this approach are too small to be distinguished from a lack of change with most assays.

These issues pose a major challenge even for defining the ideal delta change value and provide the reasons why

  • the use of this approach will reduce sensitivity[20,21] (Figure 2).

http://img.medscape.com/article/803/159/803159-fig2.jpg

Defining the Optimal Delta: Tension Between Sensitivity and Specificity

There is a reciprocal relationship between sensitivity and specificity. With marked percentage changes,

  • specificity is improved at the expense of sensitivity, and
  • at lower values, the opposite occurs.

In addition, there is controversy in regard to the metrics that should be used with high-sensitivity assays.
The Australian-New Zealand group proposed

  • a 50% change for hs-cTnT for values below 53 ng/l and
  • a 20% change above that value.[22]
  • The 20% change is much less than conjoint biological and analytical variation.

A number of publications have suggested the superiority of

  • absolute δ cTn compared to relative δ cTn in discriminating between AMI and non-AMI causes of elevated cTn.[23,24,25]
  • The utility of the absolute or relative δ cTn appears to depend on the initial cTn concentration, and
  • the major benefit may be at higher values.[23]

A recent publication by Apple et al.[26] calculates deltas in several different ways with a contemporary assay and

  • provides a template for how to do such studies optimally.[26]

If all studies were carried out in a similar fashion, it would help immensely. In the long run, institutions will need to
define the approach they wish to take. We believe this discussion is a critical one and should include

  • laboratory,
  • ED, and
  • cardiology professionals.

Distinguishing Between Type 1 and Type 2 AMI

Although δ cTn is helpful in distinguishing between AMI and nonacute causes of Tn release,

  • it may or may not be useful in discerning type 1 from type 2 AMI.

As assay sensitivity increases, it appears that the frequency of type 2 AMI increases.
Making this distinction is not easy.

Type 1 AMI is caused by a primary coronary event, usually plaque rupture.

  • It is managed acutely with aggressive anticoagulation and
  • revascularization (percutaneous coronary intervention or coronary artery bypass).[10]

Type 2 AMI typically evolves secondary to ischemia from an oxygen demand/supply mismatch

  • severe tachycardia and
  • hypo- or hypertension and the like,
  • with or without a coronary abnormality.

These events usually are treated by addressing the underlying abnormalities.

They are particularly common in patients who are

  • critically ill and those who
  • are postoperative.[27]

However, autopsy studies from patients with postoperative AMI often manifest plaque rupture.[28]
Thus, the more important events, even if less common, may be type 1 AMIs. Type 2 events
seem more common in women,  who tend to have

  • more endothelial dysfunction,
  • more plaque erosion, and
  • less fixed coronary artery disease.[28-30]

Additional studies are needed to determine how best to make this clinical distinction.
For now, clinical judgment is recommended.

Analytical Imprecision in Cardiac Troponin Assays

All analytical problems will be more critical with hs-cTn assays. Cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are measured using enzyme linked immune- sorbent assays.

  •   quantification of hs-cTn can be influenced by interference by reagent antibodies to analyte (cTn), leading to false- positive or negative results.[31]
  •   Autoantibodies to cTnI or cTnT are found in 5% to 20% of individuals and can reduce detection of cTn.[32,33]
  •   Additionally, fetal cTn isoforms can be re-expressed in diseased skeletal muscle and detected by the cTnT assays, resulting in false-positive values.[34]

Several strategies, including the use of

  •   blocking reagents,
  •  assay redesign, and use of
  •  antibody fragments,

have been used to reduce interference.[35–36]

There are differences in measured cTn values based on specimen type (serum versus heparinized plasma versus EDTA plasma).
In addition, hemolysis may affect the accuracy of cTn measurement,[37] and with blood draws from peripheral IV lines, common in ICU.

Ruling Out AMI

Studies evaluating the diagnostic performance of hs-cTn assays for the early diagnosis of AMI usually define AMI on

  • the basis of a rising and/or falling pattern of current generation cTn values.[21,38]

However, defining AMI on the basis of the less sensitive current generation assay results in an underestimation of the true prevalence of AMI and

  • an overestimation of negative predictive value of the experimental assay.
  • shortens the time it takes to rule in all the AMIs and
  • to definitively exclude AMI as it
  • ignores the new AMIs more sensitively detected by the hs-cTn assay.

Thus, in the study by Hammarsten et al.,[14]

  • the time to exclude all AMIs was 8.5 hours when all of the AMIs detected
    with the high-sensitivity assay were included, whereas
  • others that do not include these additional events report this can be done
    in 3 to 4 hours.[21,29,38]

In our view, Hammarsten is correct.

This does not mean that hs-cTn cannot help in excluding AMI. Body et al.[39] reported that patients who present with undetectable values (less than the LOB of the hs-cTnT assay) were unlikely to have adverse events during follow-up. If that group of patients is added to those who present later than 6 hours, then perhaps a significant proportion of patients

 

  • with possible acute coronary syndrome (ACS) could
  • have that diagnosis excluded with the initial value.[40]
    • studies need to continue to evaluate cTn values for at least 6 h
      to define the frequency of additional AMIs detected in that manner.

Using follow-up evaluations of patients with small event rates

  • who are likely to have additional care during the follow-up period are likely to be underpowered.

It may be that better initial risk stratification may help with this, as recently reported.[16,41]
Low-risk patients who have good follow-up after an ED visit

  • may be a group that can be released as early as 2 h after presentation.[16]

Investigating the Causes of Positive Troponin Values in Non-AMI Patients

Elevated Tn values (including those obtained with high-sensitivity assays) are associated with

  • a 2-fold higher risk for longer-term all-cause mortality and
  • cardiovascular death than a negative troponin values.[6,42-44]

This association is dose-dependent.

  • If values are rising, they are indicative of acute cardiac injury.

Those patients should be admitted because the risk is often short-term. However,

  • if the values are stable, assuming the timing of any acute event would
    allow detection of a changing pattern,
  • the risk, although substantive, in our view, often plays out in the longer term.[44]
  • Many of these individuals, assuming they are doing well clinically, can be
    evaluated outside of the hospital, in our view.
  • However, because such elevations are an indicator of a subclinical
    cardiovascular injury,  such evaluations should be early and aggressive.

Data from several studies suggest that there may well be risk far below the 99th percentile URL value.
Thus, it may evolve that patients in the upper ranges of the normal range also require some degree of cardiovascular evaluation.

Risk Stratifying Patients With Nonacute Coronary Syndrome
Conditions

Patients who have a rising pattern of values have a higher risk of mortality than those with negative values regardless of the cause.
Investigations are ongoing to determine how well results from hs-cTn testing help to risk-stratify patients with

  • pulmonary embolism,[45]
  • congestive heart failure,[46]
  • sepsis,[47]
  • hypertensive emergency,[48] and
  • chronic obstructive pulmonary disease.[49]

Presently, the studies suggest that cTn values classify patients into clinically relevant  risk subgroups. Studies are needed

  • to evaluate the incremental prognostic benefit of hs-cTn.

Conclusions

Routine use of hs-cTn assays in the United States is inevitable. These assays hold
the promise of

  • improving the sensitivity of AMI diagnoses,
  • shortening the duration of AMI evaluation and
  • improving the risk stratification of other noncardiac diagnoses.

However, to be able to fully realize their potential, additional studies are needed to address the

  • knowledge gaps we have identified. In the interim, clinicians need to
    • learn how to use the 99th% URL and
    • the concept of changing values

John Adan, MD, FACC

In 2008 CMS commissioned Yale University to analyze 30 days mortality after myocardial infarction in their hospitals.

The study has been based on review of medical records. Consensus criteria for diagnosis of myocardial infarction include

  • clinical symptoms,
  • EKG,
  • troponins,
  • CK MB,
  • ECHO,
  • cath,
  • histopathology, etc.

How the reviewed hospitals performed diagnostic coding is unknown. In clinical practice we are bombarded by consults

  • for elevated troponins due to causes other than myocardial infarction, like
    • pneumonia,
    • accelerated hypertension,
    • arrhythmias,
    • renal failure, etc.

The metric started out over 19%. Now it is below 15%, on average.

CT Angiography (CCTA) Reduced Medical Resource Utilization compared to Standard Care reported in JACC
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/05/16/ct-angiography-ccta-reduced-medical-resource-utilization-compared-
to-standard-care-reported-in-jacc/?goback=%2Egde_4346921_member_241569351

typical changes in CK-MB and cardiac troponin ...

typical changes in CK-MB and cardiac troponin in Acute Myocardial Infarction (Photo credit: Wikipedia)

Phosphotungstic acid-haematoxylin staining dem...

Phosphotungstic acid-haematoxylin staining demonstrating contraction band necrosis in an individual that had a myocardial infarction (heart attack). (Photo credit: Wikipedia)

English: Troponin(SVG Version) 日本語: トロポニン(SVG修正版)

English: Troponin(SVG Version) 日本語: トロポニン(SVG修正版) (Photo credit: Wikipedia)

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