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Ischemic Stable CAD: Medical Therapy and PCI no difference in End Point: Meta-Analysis of Contemporary Randomized Clinical Trials
Reporter: Aviva Lev-Ari, PhD, RN
SOURCE
Stergiopoulos K, Boden WE, Hartigan P, et al. Percutaneous coronary intervention outcomes in patients with stable obstructive coronary artery disease and myocardial ischemia: A collaborative meta-analysis of contemporary randomized clinical trials. JAMA Intern Med 2013; DOI:10.1001/jamainternmed.2013.12855. Available at:http://www.jamainternalmedicine.com.
NEW YORK, NY — A new analysis is calling into question the de facto rationale for many of the revascularization procedures taking place today, at least in patients with stable coronary artery disease[1]. In a meta-analysis of more than 5000 patients, PCI was no better than medical therapy in patients with documented ischemia by stress testing or fractional flow reserve (FFR).
“Cardiology has a long history of finding a marker of a bad outcome and treating that marker of that bad outcome as if it were the cause of the bad outcome,” senior author on the study, Dr David Brown (State University of New York [SUNY]–Stony Brook School of Medicine), told heartwire. In the case of proceeding to PCI on the basis of documented ischemia, that stems from evidence that patients with ischemia have a worse prognosis than patients who don’t.”It has gotten to the point that a positive stress test [is the gateway] to doing an intervention, even if the ischemia is not in the same ischemic territory as the vessel being treated,” he said. “The medical/industrial complex in cardiology is now focused on finding and treating ischemia, and I think that’s not justified, and these data suggest that that’s not justified.”
Brown and colleagues, with first author Dr Kathleen Stergiopoulus (SUNY–Stony Brook School of Medicine), reviewed the literature for randomized clinical trials of PCI and medical therapy for stable CAD conducted over the past 40 years, ultimately including five trials of 5286 patients. These were a small German trial published in 2004, plus MASS II, COURAGE,BARI 2D, and FAME 2. In all, 4064 patients had myocardial ischemia documented by exercise, nuclear or echo stress imaging, or FFR.
Over a median follow-up of five years, mortality, nonfatal MI, unplanned revascularization, and angina were no different between patients treated medically vs those treated with PCI.
Odds Ratio, PCI vs Medical Therapy
Outcome
Odds ratio
95% CI
Death
0.90
0.71–1.16
Nonfatal MI
1.24
0.99–1.56
Unplanned revascularization
0.64
0.35–1.17
Angina
0.91
0.57–1.44
“These findings are unique in that this is the first meta-analysis to our knowledge limited to patients with documented, objective findings of myocardial ischemia, almost all of whom underwent treatment with intracoronary stents and disease-modifying secondary-prevention therapy,” Stergiopoulus et al write.
The findings, they continue, “strongly suggest that the relationship between ischemia and mortality is not altered or ameliorated by catheter-based revascularization of obstructive, flow-limiting coronary stenosis.”
To heartwire, Brown pointed out that their analysis could not separate out patients who had small amounts of ischemia from those with larger ischemic territories. “Maybe that’s where the differentiating factor will be,” he acknowledged, adding that the 8000-patient ISCHEMIAtrial, still ongoing, will hopefully yield some insights.
Current practice, however, is to check for ischemia and to proceed with catheterization and, usually, revascularization when ischemia is confirmed by stress testing or during FFR. “But if that doesn’t improve outcomes, why are we doing it?” Brown asked. “We think that needs to be rethought.”
Commenting on the study for heartwire, Dr Peter Berger(Geisinger Health System, Danville, PA) pointed out: “There is no question that PCI is more effective than medical therapy for relief of symptoms: the more severe the angina and the more active the patient, the greater the superiority of PCI.” And, as Berger noted, most of the studies included in this analysis documented ischemia but did not report on the frequency or severity of angina at baseline.
That said, “Patients with minimal angina—and certainly those with silent ischemia but no angina—are unlikely to have a significantly greater reduction of symptoms with PCI, and PCI is rarely beneficial in such patients.”
Moreover, Berger continued, it has been clearly established that PCI does not reduce the risk of death or MI in most such patients.
“I very much agree with the authors, however, that just because more severe ischemia has been shown to be associated with a worse long-term prognosis, reducing the ischemic burden ought not be assumed to reduce the likelihood of death or MI. In most such patients, it does not.”
Stergiopoulos and Brown had no disclosures. Disclosures for the coauthors are listed in the paper.
This contribution to the series on stem cells and regenerative medicine deals with transplantation of modified human adipose tissue to repair ischemic damaged skeletal muscle by apparently increase neovascularization, essentrial for laying down the circulation that feeds the tissue.
Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle
Evgeny K Shevchenko1*, Pavel I Makarevich12, Zoya I Tsokolaeva1,Maria A Boldyreva1, Veronika Yu Sysoeva2, Vsevolod A Tkachuk23 andYelena V Parfyonova12 1Laboratory of angiogenesis, Russian Cardiology Research and Production Complex; 2Lomonosov Moscow State University; 3Laboratory of molecular endocrinology, Russian Cardiology Research and Production Complex, Moscow, Russia. Journal of Translational Medicine 2013, 11:138. http://www.translational-medicine.com/content/11/1/138http://dx.doi.org/10.1186/1479-5876-11-138 This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/2.0
Abstract
Background
Modified cell-based angiogenic therapy has become a promising novel strategy for ischemic heart and limb diseases. Most studies focused on myoblast, endothelial cell progenitors or bone marrow mesenchymal stromal cells transplantation. Yet adipose-derived stromal cells (in contrast to bone marrow) are abundantly available and can be easily harvested during surgery or liposuction. Due to high paracrine activity and availability ADSCs appear to be a preferable cell type for cardiovascular therapy. Still neither genetic modification of human ADSC nor in vivo therapeutic potential of modified ADSC have been thoroughly studied. Presented work is sought to evaluate angiogenic efficacy of modified ADSCs transplantation to ischemic tissue.
Materials and methods
Human ADSCs were transduced using recombinant adeno-associated virus (rAAV) serotype 2 encoding human VEGF165. The influence of genetic modification on functional properties of ADSCs and their angiogenic potential in animal models were studied.
Results
We obtained AAV-modified ADSC with substantially increased secretion of VEGF (VEGF-ADSCs). Transduced ADSCs retained their adipogenic and osteogenic differentiation capacities and adhesion properties.
The level of angiopoetin-1 mRNA was significantly increased in VEGF-ADSC compared to unmodified cells yet
expression of FGF-2, HGF and urokinase did not change.
Using matrigel implant model in mice it was shown that
VEGF-ADSC substantially stimulated implant vascularization with paralleling increase of capillaries and arterioles.
In murine hind limb ischemia test we found
significant reperfusion and revascularization after intramuscular transplantation of VEGF-ADSC compared to controls with no evidence of angioma formation.
Conclusions
Transplantation of AAV-VEGF- gene modified hADSC resulted in stronger therapeutic effects in the ischemic skeletal muscle and may be a promising clinical treatment for therapeutic angiogenesis.
Despite advances in revascularization techniques, the treatment of ischemic heart and limb diseases remains a worldwide problem. Therapeutic angiogenesis represents alternative new strategy for ischemia resolution that utilizes regenerative capacity of human body and
Delivery of cDNA coding angiogenic factors via different expression mammalian vectors (plasmids, recombinant viruses) was found more feasible and allowed to achieve great improvement in some cases yet
efficacy was still not high enough especially in double blind placebo controlled trials [4].
Many authors discussed possible reasons of gene therapy low efficacy and most of them are univocal to emphasize transfection efficacy and transient transgene expression after plasmid delivery. This can be circumvented by administration of viral vectors but their use is limited due to possible danger of insertional mutagenesis and immune reactions [5,6].
Recently, autologous transplantation of bone marrow stromal cells or endothelial progenitor cells has been shown to enhance angiogenesis and peripheral blood flow [7–9]. However,
the regenerative capacity of these cells decreases with age and
in patients with co-morbidities such as diabetes mellitus which reduces efficacy of autologous cell administration, and
limited cell viability after transplantation into ischemic tissues also restricts their angiogenic potential [10–12].
It was shown in several experimental studies that this problem could be circumvented by gene modified cell therapy strategy utilizing stem or progenitor cells overexpressing angiogenic proteins [13,14]. To develop a feasible and potent gene modified cell therapy for ischemic diseases
the cells should be both effective and accessible in large numbers as well as
the chosen viral vector should be both safe and effective in terms of gene delivery.
The majority of experimental studies have evaluated gene modified bone marrow stromal cells or endothelial progenitor cells for ischemia treatment [15–17]. However, cells extracted from bone marrow or peripheral blood after mobilization are available in limited numbers and as for bone marrow cells painful aspiration procedure is required.
In contrast to bone marrow or myoblasts, stromal fraction of adipose tissue contains an abundant population of multipotent stem cells that can be easily harvested in high numbers by minimally invasive surgical techniques [18–21]. These adipose –derived stromal cells (ADSCs)
share common properties with bone marrow stromal cells and represent a very convenient object for therapeutic use.
However the best development of ADSC for angiogenic therapy still needs to be determined.
As for genetic modification of cells the choice of safe and effective gene transfer vector as well as the appropriate transgene determines the quality and safety of the cell product affecting the efficacy of modified cell based therapy. Recombinant adeno-associated viruses (rAAV)are one of the most promising and versatile tools in this field due to
low immunogenicity and
high transduction potency in vitro
in many types of both – dividing and non-dividing mammalian cells. Besides that until now no human disease caused by AAV has been identified [22].
In this study we genetically modified human ADSCs with a key regulator of angiogenesis – VEGF165 [23] via rAAV-transduction and then evaluated effects of rAAV-transduction and VEGF165 overexpression on human ADSC
growth,
differentiation capacity,
adhesion and
angiogenic factor expression as well as
revascularization and
functional improvement
after intramuscular injection in a mice hind limb model.
Methods
Cell culture
(refer to doi:10.1186/1479-5876-11-138)
DNA constructs production of rAAV particles and cell transduction
(refer to doi:10.1186/1479-5876-11-138)
Western blotting and ELISA
(refer to doi:10.1186/1479-5876-11-138)
ADSC proliferation activity assay
To assess population doubling time (PDT) of gene modified (transduced with rAAV at passage 1) or untreated ADSC (passage 2) seeded on 6-well plates (2 × 104 cells/well). After a 9 day incubation average cell numbers for three wells were obtained using a hemocytometer chamber. PDT was calculated as follows:
PDT=(log2)*t/(log(Nt/N0))
where t is period of incubation (hours), Nt – endpoint amount of cells, N0 – initial number of cells.
ADSC cell cycle stage analysis by flow cytometry
(refer to doi:10.1186/1479-5876-11-138)
ModFit LT 3.2 software (Verity Software House, USA) was used for analysis of cell distribution over cell cycle stages according to intensity of propidium iodide fluorescence in a wavelength range of 600–625 nm (excitation wavelength – 488 nm). Results are presented as a percentage of cells in S + G2/M stages.
Apoptosis assay
Analysis of spontaneous apoptosis frequency in ADSC culture was performed using Annexin-V FITC Apoptosis Kit (Invitrogen, USA) according to manufacturer’s protocol.
Adipogenic, osteogenic and endothelial differentiation of ADSC
To confirm adipogenesis intracellular lipid droplets were detected using Oil red O staining reagent (Millipore, USA) 2 weeks after induction. To confirm osteogenesis Alizarine Red C staining was used to detect extracellular matrix mineralization 2 and 3 weeks post induction. Endothelial cells were stained for CD31 and VEGFR2 surface antigens and cell counts were obtained using flow cytometry.
Cell attachment assay
(refer to doi:10.1186/1479-5876-11-138)
Flow cytometry
Antigen expression analysis was performed on cell sorter MoFlo (DakoCytomation, Denmark) or flow cytometry scanner BD FACS CantoTM II (BD Pharmingen, USA). 10 000 events were acquired and analyzed for antigen expression.
Quantitative polymerase chain reaction
Quantitative polymerase chain reaction (qPCR) was performed using primers specific for human VEGF165, ANGPT1, HGF, FGF2 and PLAU mRNAs.
Animals
8–10 week-old male BALB/c NUDE mice
Matrigel plug assay
(Refer to doi:10.1186/1479-5876-11-138)
Hind limb ischemia model
Ten week-old male BALB/с NUDE mice were anaesthetized by intraperitoneal injection of 0.3 ml of 2.5% avertin. Femoral artery was separated in its distal part and ligated proximal to its popliteal bifurcation (keeping v. femoralis and n. ischiadicus intact). ADSC, GFP-ADSC or VEGF-ADSC (5×105 cells per animal) were resuspended in 150 μl of PBS, and injected in 3 equally divided doses tom. tibialis anterior, m. gastrocnemius and m. biceps femoris to generate three experimental animal groups: “GFP-ADSC”, “VEGF-ADSC”, “ADSC” (14 animals per group). PBS (150 μl) was injected in negative control “PBS” group. Blood flow was subsequently measured by laser Doppler imaging.
Laser doppler imaging
(Refer to doi:10.1186/1479-5876-11-138)
Muscle explants
M. tibialis anterior explant culture was prepared on matrigel according to Jang et al. [26] protocol and cultured in M199 medium (Gibco, USA), containing 2% FBS. At day 3 and 7 medium was collected for determination of human VEGF165 concentration by ELISA.
Specimen preparation and histological analysis
At designated period (day 20 for muscles, day 14 for matrigel plugs) animals were sacrificed by lethal isoflurane dose followed by cervical dislocation. Afterwards m. tibialis anterior or matrigel implants respectively were harvested,
For muscle necrosis analysis we used routine hematoxylin-eosin staining of formalin-fixed muscle sections. Necrotic tissue was defined by loss of fiber morphology, cytoplasm disruption, inflammatory cells infiltration and fibrosis.
Statistical analysis
Results were analyzed in Statsoft Statistica 6.0 (Statsoft, USA).
Results
Effective transduction of human ADSC by adeno-associated virus serotype 2
Low-passage human ADSC obtained from different donors were transduced using rAAV encoding GFP to assess gene delivery efficacy. Transduced to total cells ratio was counted by flow cytometry. GFP-positive ADSC (GFP-ADSC) were detected as early as day 2 after viral infection. Maximum number of positive cells (65.6±3%) and highest GFP-fluorescence intensity was reached by day 4–5 (Figure 1). GFP signal was detectable for at least 30 days. At day 15 and 30 flow cytometry showed that 45±2% and 25±1.5% of ADSC were GFP-positive respectively.
Figure 1.Human ADSC transduction by recombinant adeno-associated virus
A. GFP-positive cell count by FACS in GFP-ADSC culture at day 4 after transduction by rAAV. B.Representative image of GFP-positive human ADSC (green) transduced by rAAV, 100 × magnification.
Increase of VEGF expression and secretion after rAAV transduction of human ADSC
To obtain gene modified ADSC we constructed rAAV vector encoding human VEGF165. In ADSC transduced by rAAV-VEGF (VEGF-ADSC) VEGF165 mRNA level increased 80±15-fold compared to basal expression in unmodified ADSC or GFP-ADSC (Figure 2A). Protein production was analyzed by Western blotting and ELISA. Data presented at Figure 2B, C shows that in VEGF-ADSC secretion of VEGF increased 45-50-fold (4.5±1.8 ng/ml/105 cells) compared to unmodified cells (0.1±0.02 ng/ml/105 cells) or GFP-ADSC (0.09±0.02 ng/ml/105cells). VEGF concentration in conditioned medium decreased over time during VEGF-ADSC cultivation but remained 30-fold higher (2.9±1.1 ng/ml/105 cells) than in controls (0.09 ± 0.02 ng/ml/105 cells) at day 30 post transduction. Material from a total of 10 donors was used to obtain mean values of VEGF expression increase.
Figure 2.Validation of VEGF165 expression in AAV-modified VEGF-ADSC.
A. VEGFA expression level in human ADSC 10 days after AAV transduction determined by quantitative PCR. B, C. Analysis of VEGF secretion by GFP-ADSC, VEGF-ADSC and unmodified cells using ELISA (B) and immunoblotting (C). In immunosorbent assay protein content was determined in conditioned media samples obtained at days 7 and 30 post genetic modification of ADSC.
rAAV-mediated modification of human ADSC suppresses their proliferation activity yet does not influence apoptosis
We found that proliferation rate of VEGF-ADSC and GFP-ADSC was reduced compared to unmodified cells (Figure 3A). ADSC population doubling time was 61.3±7 h, while for GFP-ADSC and VEGF-ADSC it was 116.9±11 and 145.4±12 h respectively (n=5, p<0.01 vs unmodified cells). At the same time spontaneous apoptosis rate in all three cell cultures was comparable and comprised about 2±0.5% of total cell population.
Figure 3.Proliferation of gene modified ADSC. Population doubling time in GFP-ADSC, VEGF-ADSC and ADSC cultures. Data of five serial runs. B. Cells distribution in S-G2 cell cycle stages according to cytometry analysis of GFP-ADSC, VEGF-ADSC and ADSC. Data of three serial runs.
Analysis of cell cycle stages distribution in ADSC, GFP-ADSC and VEGF-ADSC cultures (Figure 3B) showed that number of cells in S-G2 stages was more than 1.5-fold lower in modified cells: GFP-ADSC (16±4% cells) and VEGF-ADSC (13±6% cells) compared to unmodified ADSC (25±3% cells; n=3; p<0.05 vs unmodified cells).
ADSC adhesion does not change after genetic modification
Interactions with extracellular matrix proteins play important role in incorporation and integration to recipient’s tissue, cell viability and their functional properties upon transplantation. ADSC did adhere on main extracellular protein collagen type 1 as well as vitronectin and fibronectin while almost none of cells attached to laminin-coated plastic. We did not observe statistically significant differences in adhesion properties between ADSC, GFP-ADSC and VEGF-ADSC cultures (Figure 4).
Figure 4.Data from comparative study of ADSC, GFP-ADSC and VEGF-ADSC adhesion on culture plates coated by collagen 1, vitronectin, fibronectin or laminin (n=4).
Modified ADSC retain their adipogenic, osteogenic and endothelial differentiation potential in vitro
To analyze potential influence of viral transduction and transgene overexpression on differentiation capacity of gene modified cells we performed experiments on adipogenic and osteogenic differentiation of ADSC.
Microscopic analysis of gene modified and untreated ADSC stained with Oil Red O reagent after 14 days of incubation in adipogenic media showed >30% of differentiated (visualized by intracellular lipid droplets accumulation) cells (Figure 5). Oil Red O+ cell count did not reveal statistically significant differences in both GFP-ADSC (33.7±8.1%) and VEGF-ADSC (34.1±11.5%) as well as unmodified ADSC (34.3±11.7%). Similar results were obtained in osteogenic differentiation assay of ADSC. It was confirmed by Alizarin Red C staining that detects extracellular matrix mineralization. At 14 days of incubation in osteogenic media we detected dye-positive cells in ADSC, GFP-ADSC, VEGF-ADSC culture. At day 21 it was followed by dramatic increase of extracellular matrix calcification in both – modified and untreated cells without significant differences (Figure 5).
Figure 5.Adipogenic and osteogenic differentiation of gene modified ADSC
Representative images of ADSC and VEGF-ADSC cultures stained by Oil Red O (lipid droplets detection, kjadipogenic differentiation, 100 × magnification) and Alizarine Red C (matrix mineralization, osteogenic differentiation, 100 × magnification for “day 14” and 50 × magnification for “day 21”) reagents after incubation in specific differentiation medium, n=3.
Taking into account mitogenic activity of VEGF we analyzed possible effect of genetic modification and VEGF overexpression on endothelial cell fraction in VEGF-ADSC. Using flow cytometry we determined amount of cells that carry CD31 and VEGFR2 endothelial markers in ADSC, GFP-ADSC and VEGF-ADSC (rAAV-modified at passage 1) cultures at passage 2. Less than 1.5% of CD31, VEGFR2-positive cells were detected in all three populations. Subsequently modified and untreated ADSC at passage 2 that reached >90% confluency were subject to incubation in EGM-2 medium to stimulate endothelial differentiation. After 14 days of cultivation in EGM-2 repeated analysis of CD31 and VEGFR2 expression showed that percentage of endothelial marker-positive cells did not change and remained about 1% in all assayed cultures.
Level of angiopoietin-1 mRNA increases in VEGF-ADSC
Using qPCR we studied potential impact of genetic modification and augmented VEGF secretion on expression activity of hepatocyte growth factor (HGF), fibroblast growth factor-2 (FGF2), angiopoietin-1 (ANGPT-1) and urokinase (PLAU) genes in VEGF-ADSC. As shown in Figure 6 we did not find any changes in FGF2 and HGF expression in GFP-ADSC and VEGF-ADSC compared to ADSC. We found a 3-fold increase in urokinase expression in VEGF-ADSC yet it was not statistically significant. At the same time increase of ANGPT-1 expression in VEGF-ADSC was significant and 5.3±0.6-fold higher than in unmodified cells or GFP-ADSC (n=6, p<0.05).
Figure 6.Comparison of ANGPT1, FGF2, PLAU and HGF genes expression by quantitative PCR in GFP-ADSC, VEGF-ADSC and unmodified ADSC. Charts represent relative expression for assayed genes from a total of 6 runs.
Analysis of VEGF and PDGF receptors expression on ADSC surface
Analysis of VEGF receptors expression on human ADSC was carried out to assess possible autocrine action of VEGF on VEGF-ADSC functional properties. Flow cytometry of ADSC and VEGF-ADSC (at passage 1–2) from different donors stained for VEGF receptor 1 and 2 showed <1% of positive cells (Figure 7). Taking into account observation of Ball et al. which indicated platelet-derived growth factor receptors (PDGFRα and PDGFRβ) as facultative receptors for VEGF165 [27] we analyzed the presence of cells which expressed PDGFRβ in human ADSC culture. Using specific monoclonal antibodies and subsequent flow cytometry we found that >90% of human ADSC were positive for PDGFRβ (Figure 7).
Figure 7.Analysis of VEGF and PDGF receptors expression on ADSC surface. VEGFR1, VEGFR2 or PDGFRβ-positive cell count by flow cytometry in ADSC culture.
Increased vascularisation of matrigel implants after VEGF-ADSC transplantation
We used matrigel plug assay to determine angiogenic properties of gene modified ADSC in vivo. At day 14 matrigel implants were harvested and subject to histological analysis (Figure 8). In negative control group we found only small sporadic capillaries (<1 capillary per FOV) were detected while in “ADSC”, “GFP-ADSC” and “VEGF-ADSC” groups formation of vessel network was more evident. Vessel counts revealed a 2.7-fold increase of CD31-positive vessels in group “VEGF-ADSC” (88.1±10.4 vessels per FOV) compared to “GFP-ADSC” (31.3±6.2 vessels per FOV) and “ADSC” (34.5±11.6 per FOV). Number of smooth muscle actin (SMA)-positive vessels was also 2.5-fold higher in “VEGF-ADSC” (1.7±0.24 vessels per FOV) than in “GFP-ADSC” (0.7±0.3 vessels per FOV) and “ADSC” (0.7±0.2 vessels per FOV). Thus capillaries/SMA+vessels ratio did not vary among experimental groups.
Figure 8.Effect of VEGF-ADSC or ADSC on vascularization of matrigel implants in nude mice. A.Representative images of matrigel sections from “VEGF-ADSC” and “ADSC” groups stained by antibodies against murine CD31 and SMA, 100× magnification. B. Capillaries and arterioles count in matrigel implants.
Blood flow recovery after VEGF-ADSC transplantation into ischemic murine limb
Perfusion assessment in hind limb ischemia model showed maximum blood flow recovery in “VEGF-ADSC” group (Figure 9). By day 20 spontaneous reperfusion of ischemic limb in «PBS» group was feeble and did not exceed 30%. In contrast we observed evident augmentation of blood supply in three experimental groups that received cell injections. At the end of experiment perfusion in “ADSC” and “GFP-ADSC” groups reached 50% and 55% respectively. Blood flow recovery after VEGF-ADSC transplantation was much more effective. At day 12 perfusion in group “VEGF-ADSC” significantly exceeded values in “ADSC” and “GFP-ADSC” by 15-20% and towards the end of experiment (day 20) it reached 80-90%. Thus transplantation of ADSC overexpressing VEGF was more effective than of untreated or GFP-ADSC.
Figure 9.Reperfusion of murine ischemic limb after ADSC administration. A. Representative laser-doppler scans of subcutaneous blood flow in mice from “ADSC” and “VEGF-ADSC” groups obtained at days 4 and 20 after ischemia induction and cell transplantation. B. Dynamics of blood flow recovery in ischemic limbs within 20 days after intramuscular injection of ADSC, GFP-ADSC, VEGF-ADSC or PBS.
Transplantation of VEGF-ADSC reduces necrosis and stimulates stable vessel formation in ischemic muscle
Histological analysis of hematoxylin-eosin stained m. tibialis anterior specimens obtained at day 20 after and cell transplantation showed significant decrease in necrotic tissue span in «VEGF-ADSC» group (31.3±7%) compared to «ADSC» and «GFP-ADSC» groups (54.3±8.4% and 55.63±6.8%). Animals that received PBS injection as a negative control were characterized by the highest muscle necrosis span that reached 84±6.7% (Figure 10).
Figure 10.Morphometric analysis of tissue necrosis in ischemic muscle from study group animals. A. Images of hematoxylin-eosin stained m. tibialis anterior sections. Necrotic tissue is marked by black line. (N* – necrotic tissue, B* – border zone, H* – healthy or regenerating tissue). B. Representative images of muscle tissue from different zones of section. Labels: star – vasa in normal muscle tissue with; black dot – inflammatory demarcation zone between anucleic disrupted tissue and regenerating muscle fibers; triangle – regenerating round-shaped muscle fibers with multiple centrally located nuclei. C. Statistical data of necrotic tissue area in “PBS”, “ADSC”, “GFP-ADSC” and “VEGF-ADSC” groups. Measurements made in 4–5 animals per group.
To assess vascular density muscle tissue sections were stained by specific antibodies against mouse CD31 and SMA (Figure 11). Vessel count showed that in “ADSC” and “GFP-ADSC” groups capillary and arteriolar densities were similar reaching 129±11 and 125±14 capillaries/FOV, 1.35±0.12 and 1.37±0.09 arterioles/FOV respectively. In specimens from animals that received VEGF-ADSC capillary density was 189±19 per FOV (p<0.05) with arteriolar density of 3.1±0.2 per FOV (p<0.01). Furthermore, we found that arterioles/CD31+ vessels ratio was similar in all experimental groups and slightly higher in group “VEGF-ADSC” (1% vs 1.6%). In addition morphometric analysis of muscle tissue from group “VEGF-ADSC” did not reveal angioma or abnormal vessel formation.
Figure 11.Vascularization of murine ischemic muscles after ADSC administration. A. Representative images ofm. tibialis anterior sections from “VEGF-ADSC” and “ADSC” groups stained by antibodies against murine CD31 and SMA, 100× magnification. B. Capillaries and arterioles count in m. tibialis anterior sections. Counts made in 5–6 animals per group.
ADSC retain viability and transgene expression after transplantation into ischemic muscle
To evaluate viability of transplanted ADSC after injection into ischemic tissue m. tibialis anterior specimens from “GFP-ADSC” group were harvested at day 7 after induction of ischemia and cell transplantation. Frozen muscle sections were analyzed using fluorescence microscopy that allowed to detect GFP-positive cells distributed throughout muscle (Figure 12A).
Figure 12.Human ADSC viability and VEGF expression after transplantation to ischemic murine muscle.
A. Representative image of m. tibialis anterior section from “GFP-ADSC” group obtained at day 7 after ischemia induction and GFP-ADSC injection, 50× magnification. GFP-positive cells are distributed in tissue around injection site.B. Analysis of VEGF165 content by ELISA in explants culture medium from “ADSC”, “GFP-ADSC”, “VEGF-ADSC” groups obtained at days 3 and 20 after cell trasplantation.
Data from experimental studies indicates that prolonged expression of therapeutic transgene is essential for effective stimulation of angiogenesis and ischemic tissue recovery. Muscle explant model was carried out to confirm the presence of viable and functionally active human ADSC overexpressing VEGF in ischemic muscle at hind limb ischemia experiment endpoint. M. tibialis anteriorwere harvested from “ADSC”, “GFP-ADSC” and “VEGF-ADSC” group animals at day 3 and 20 after cell transplantation and cultured as explant in matrigel. In culture medium samples collected after 3 days of “VEGF-ADSC” explant incubation (obtained at day 3 after cell transplantation) human VEGF165 concentration determined by ELISA reached 2.86±0.21 ng/ml (Figure 12B). Protein concentration was expectedly lower (0.145±0.015 ng/ml) in conditioned medium from muscle explants harvested at day 20. In addition comparison of VEGF concentration in culture medium samples collected at day 3 and 7 post incubation of explant culture revealed accumulation of VEGF. It indirectly confirms presence of functionally active human VEGF-ADSC in ischemic muscle up to 20 days post transplantation. In contrast to “VEGF-ASDC” human VEGF165 concentration in explant cultures from “GFP-ADSC” and “ADSC” groups was below limit of detection.
Discussion
Gene modified cell-based therapy for ischemic disorders: myocardium infarction and limb ischemia is a rapidly evolving trend in experimental and regenerative medicine. Promoting angiogenesis in ischemic tissues via paracrine action of transplanted modified cells is an emerging alternative modality for patients who are unsuitable for surgical and interventional revascularization. Still choice of
appropriate cell type,
angiogenic factor and
gene delivery tool
are crucial issues for efficacy and safety of the method.
Regarding type of cells there are certain issues concerning their derivation and preparation prior to grafting. Thus, embryonic stem cells application is doubtful due to
ethical reasons,
potential risks of teratogenesis and
immune response to their differentiated progenies [28].
Use of endothelial progenitor cells from peripheral blood and bone marrow are limited by
expensive procedures of isolation and difficulties in obtaining sufficient amount of cells.
Regarding the latter point it is known that prolonged incubation of cells in vitro prior to transplantation is associated with
potential risks of malignancy,
proliferation decrease and
commitment to terminal differentiation.
Use of skeletal myoblasts or bone marrow derived mesenchymal stromal cells (BMMSC)is associated with painful isolation procedure of muscle biopsy and suprailiac puncture respectively.
ADSCused in our study share a lot of similar properties and characteristics with BMMSC, while they are easier to obtain in sufficient quantity using minimally invasive liposuction procedure. Various data suggests that up to 1.5 × 106 adipose stromal cells can be isolated from 1 ml of adipose tissue [29,30]. This allows to reduce the time of cell propagation in vitro prior to transplantation. As for therapeutic angiogenesis,
human ADSC produce a wide spectrum of biologically active molecules – angiogenic growth factors, cytokines, proteases etc. [31,32].
Multiple experimental studies accumulate data on relatively high therapeutic potential of ADSC for tissue regeneration and stimulation of angiogenesis [21,33,34]. However well-known reduction of cell regenerative potential with age and among patients with severe co-morbidities is also relevant for ADSC. Donor age-associated decrease of proliferation activity and differentiation capabilities was shown for human ADSC [35,36]. Angiogenic potential of ADSC also decreases with ageing and is characterized by reduced secretion of
Thus, attempts to improve regenerative potential of ADSC are reasonable.
We have shown high efficacy of rAAV-mediated genetic modification of human ADSC. Using rAAV encoding VEGF165 we obtained human ADSC with increased level of VEGF165 secretion which retained for at least 30 days. VEGF-A and particularly its most abundant 165-amino acid isoform triggers multiple reactions promoting new vessel formation and growth [23] that supported our choice of therapeutic gene in presented study. Observed gradual decrease of transgene expression can be attributed to proliferation activity of ADSC together with known episomal subsistence of rAAV [38]. Moreover cellular mechanism of addressed
methylation can be activated after transduction leading to
suppression of cytomegalovirus promoter which triggers
Potential influence of genetic modification and transgene expression on cell behavior and functional activity is frequently kept out of consideration while this issue is of great importance, especially for potential clinical application. We examined possible effects of rAAV-transduction and VEGF overexpression on functionalproperties of ADSC which included
proliferation,
spontaneous apoptosis,
adhesion and
differentiation capability.
We observed a decline in ADSC proliferation after modification by rAAVthat was evident by
increase of population doubling time as well as
decrease in number of cells in S–G2 stages of cell cycle.
At the same time spontaneous apoptosis rate did not exceed 2% in modified and unmodified cells. These results contribute to previously published data that showed transient cell cycle arrest after AAV transduction of embryonic fibroblasts and BMMSC [40]. This effect was observed whenever
wild-type,
recombinant or
genome-empty AAV particles were used.
It was suggested that changes in expression profile and decreased proliferation were related to initial stage of virus entry and caused by capsid proteins interaction with cellular signaling pathways [40]. Growth inhibitory effect was transientand
proliferation restored to normal level over time of cell passaging [41].
It appears that proliferation decline of rAAV-modified ADSC occurs by a common mechanism.
when cultured in special induction medium [42,43].
Analyzing data from our differentiation experiments we concluded that rAAV-mediated genetic modification of human ADSC and VEGF overexpression did not alter their adipogenic and osteogenic differentiation properties.
There are several observations indicating ability of ADSC for endothelial differentiation [44,45] as well as evidence for presence of small amount of endothelial cells in ADSC population at early passages [18,19]. In our experiments we did not find an increase in amount of cells positive for endothelial markers CD31 and VEGFR2 in VEGF-ADSC compared to unmodified ADSC population. This suggests that VEGF overexpression
neither induces endothelial differentiation of modified ADSC
or stimulates proliferation of preexisting endothelial cells in ADSC culture.
Adhesion tests conducted in our study were based on a fact that
interaction with extracellular matrix proteins is a key factor
that contributes to cell viability and integration into host tissue after transplantation [46].
We found that both modified and untreated ADSC showed very common adhesion on collagen type 1, vitronectin and fibronectin. Thus we can suggest that
rAAV-mediated genetic modification did not alter expression of adhesion molecules on cell surface of ADSC.
Our results showing low ADSC adhesion on laminin are not surprising taking into account published observations which indicate diminished or lack of α6, α7 and ß1 integrins expression in ADSC-components of α6/ß1 and α7/ß1 receptors for laminin [47,48].
Since VEGF can regulate multiple signaling pathways [23] we next determined whether expression of HGF, FGF2, urokinase and angiopoietin-1 might be altered in VEGF-ADSC. HGF and FGF2 are mitogens and chemoattractants for both endothelial and mural cells and directly participate in angio- and arteriogenesis [4]. Angiopoietin-1 is characterized as a stabilizing factor that provides formation of functionally mature vessel network [49]. Urokinase plasminogen activator is a key regulator of extracellular proteolysis which is
responsible for cleavage activation of growth factors and migration of endothelial cells during vessel growth [50,51].
We found almost 3-fold yet not statistically significant increase of urokinase expression while expression of HGF and FGF2 did not change. Another interesting finding is a 5-fold increase of angiopoietin-1 expression in VEGF–ADSC compared to GFP–ADSC or unmodified cells. We assumed that up-regulation of angiopoietin-1 expression occurs due to autocrine action of VEGF165 produced by VEGF-ADSC. However according to our data supported by other studies [30,52] cultured human ADSC population contains <1% of cells that express receptors to VEGF165 – VEGFR1 and VEGFR2. At the same time we found that
>90% of ADSC carry receptor to platelet-derived growth factor – PDGFRβ.
There is a published observation that
PDGFRα and PDGFRβ can act as a facultative receptor for VEGF [27].
it is also known that PDGFR activation leads to increase of angiopoietin-1 expression [53].
Considering that more than 90% of human ADSC are PDGFRβ-positive
we can speculate that increased expression of angiopoetin-1 in VEGF-ADSC could be attributed to PDGFRβ-mediated autocrine action of VEGF.
In our study we evaluated therapeutic potential of gene modified human ADSC in terms of their ability to induce angiogenesis in ischemic muscle tissue. It was found that matrigel implants after transplantation of VEGF-ADSC had higher vascular density than after delivery of untreated cells or ADSC transduced by areporter gene. Along with
capillary formation we also found
proportional increase in amount of mature blood vessels characterized by smooth-muscle wall.
This can occur due to the fact that cells transplanted in matrigel produce other angiogenic factors besides VEGF that can promote vessel maturation and stabilization.
Key angiogenic property of cell therapies in experimental study is ability to induce reperfusion of ischemic tissue in appropriate animal models. We used hind limb ischemia modelto show that
VEGF-ADSC transplantation led to significantly higher perfusion restoration than
after untreated of GFP-transduced cell administration.
It was also found that intramuscular injection of VEGF-ADSChad a tissue-protective effect and led to vivid decrement of necrosis span. VEGF is known to be significant antiapoptotic factor that can enhance cell survival.We suggest that
increased VEGF content during the first days after onset of acute ischemia and cells administration leads to promotion of cell survival and thus to reduction of necrotic disruption in muscle tissue.
We should also point that during the experiment we did not observe any blood flow decrease after cell administration or rapid “plateau” formation like it was previously described for plasmid-mediated gene delivery due to short-term transgene expression [4]. This can be explained by
presence of viable and functionally active ADSC that produced VEGF throughout the experiment.
In our muscle explant experiments we showed that VEGF-ADSC retain functional activity even at long terms after injection (up to 27 days) and produce VEGF in detectable quantities. Thus we can confidently attribute
tissue protection and restoration of blood flow in mice that received VEGF-ADSC to increased long-term VEGF production by modified cells.
As for decrease of human VEGF content in murine tissue by day 20 we suggest that cells undergo apoptosis over time. Besides that methylation of CMV promoter which drives VEGF expression in our vector could take place. Taking into account that Nude mice were used we find it hard to assume possible rejection of transplanted cells as far as this animal strain lacks T-cells immunity which plays a crucial role in graft rejection. Still, it seems that produced amount of VEGF is sufficient to trigger angiogenesis and relief tissue ischemia via restoration of blood flow.
Histological analysis of ischemic muscle injected with modified VEGF-ADSC revealed that
capillary density was significantly higher than in specimens from animals that received untreated cells or GFP-ADSC.
We noticed that this increase was not only due to higher capillary count, but also to SMA-positive blood vessels of arteriolar type. Furthermore arteriole/capillary ratio was constant throughout experimental groups that indicated formation of a stable mature vascular network. Thus, despite high level of VEGF produced by modified ADSCwe did not observe any evidence for abnormal tumour-like vascular structures in muscle as it was previously shown e.g. in studies of adenovirus-mediated delivery of VEGF gene [54]. In contrast to matrigel implants experiment in case of skeletal muscle we do not state that increase of vascular density in experimental groups was only due to de novo formed vessels. Besides promoting endothelial cell proliferation VEGF also prevents endothelial apoptosis leading to survival of preexisting vessels. There was surely a vast amount of persisted capillaries in the muscles due to VEGF anti-apoptotic effect of VEGF.
It is often speculated that low efficacy reported in clinical trials using gene delivery of VEGF alone can be explained by its high mitogenic activity which is not supported by vessel stabilizing stimuli and consequently ends up with dissociation of formed capillaries [55]. This led to a concept of combined gene delivery
indicating that combinations of angiogenic and vascular stabilizing factors should be used to treat ischemic tissues [55–58].
Cell therapy for ischemic disorders has a valuable advantage since transplanted cells produce a whole “cocktail” of biologically active molecules which render combined effect in impaired tissue. We suggest that stable vessel formation observed in our study is
mediated by aforementioned ADSC ability to produce a wide spectrum of angiogenic factors
including ones responsible for vessel stabilization and maturation: angiopoietin-1, TGF-β, PDGF,
which can act synergistically with increased production of VEGF165 by modified cells.
Besides that, genetic modification can alter cell’s expression profile. Observed increase in expression ofangiopoietin-1 in VEGF-ADSC can further contribute to
formation of mature vascular network that also
supports therapeutic effect of transplanted cells.
Increased concentration of VEGFin ischemic tissue plays a substantial role in vessel stabilization and therapeutic effect if maintained over a significant period of time, which was achieved in our study and exceeded a substantial term of 3 weeks.
Conclusions
Thus we can conclude that human ADSC with their accessibility and angiogenic paracrine activity is an appropriate and preferable type of cells for therapeutic angiogenesis. Obtained results indicate that relatively safe rAAV holds great potential for gene transfer into human ADSC. Taken together, we suggest that
the use of AAV-modified ADSC overexpressing VEGF165 is a feasible and effective approach for stimulation of stable vascular network formation in ischemic muscle and
can be implied for therapeutic angiogenesis or tissue-engineered transplants. Further study and improvements in vector design, regulated transgene expression, cell preparation and propagation conditions are still to be completed to allow clinical application of modified cell-based therapeuticals.
This article is a followup of the wonderful study of the effect of oxidation of a methionine residue in calcium dependent-calmodulin kinase Ox-CaMKII on stabilizing the atrial cardiomyocyte, giving protection from atrial fibrillation. It is also not so distant from the work reviewed, mostly on the ventricular myocyte and the calcium signaling by initiation of the ryanodyne receptor (RyR2) in calcium sparks and the CaMKII d isoenzyme.
We refer to the following related articles published in pharmaceutical Intelligence:
The material presented is very focused, and cannot be found elsewhere in Pharmaceutical Intelligence with respedt to genetics and heart disease. However, there are other articles that may be of interest to the reader.
PART 3. Determinants of Cardiovascular Diseases: Genetics, Heredity and Genomics Discoveries
3.2 Leading DIAGNOSES of Cardiovascular Diseases covered in Circulation: Cardiovascular Genetics, 3/2010 – 3/2013
The Diagnoses covered include the following – relevant to this discussion
MicroRNA in Serum as Bimarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis, diastolic dysfunction, and acute heart failure
Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy
Heredity of Cardiovascular Disorders Inheritance
3.2.1: Heredity of Cardiovascular Disorders Inheritance
The implications of heredity extend beyond serving as a platform for genetic analysis, influencing diagnosis,
prognostication, and
treatment of both index cases and relatives, and
enabling rational targeting of genotyping resources.
This review covers acquisition of a family history, evaluation of heritability and inheritance patterns, and the impact of inheritance on subsequent components of the clinical pathway.
3.2.2.1 MicroRNA in Serum as Biomarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis, diastolic dysfunction, and acute heart failure
Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage
Y Kuwabara, Koh Ono, T Horie, H Nishi, K Nagao, et al.
SOURCE: Circulation: Cardiovascular Genetics. 2011; 4: 446-454 http://dx.doi.org/10.1161/CIRCGENETICS.110.958975
3.2.2.2 Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease
3.2.4.2 Large-Scale Candidate Gene Analysis in Whites and African Americans Identifies IL6R Polymorphism in Relation to Atrial Fibrillation
The National Heart, Lung, and Blood Institute’s Candidate Gene Association Resource (CARe) Project
RB Schnabel, KF Kerr, SA Lubitz, EL Alkylbekova, et al.
SOURCE: Circulation: Cardiovascular Genetics.2011; 4: 557-564 http://dx.doi.org/10.1161/CIRCGENETICS.110.959197
Weighted Gene Coexpression Network Analysis of Human Left Atrial Tissue Identifies Gene Modules Associated With Atrial Fibrillation
N Tan, MK Chung, JD Smith, J Hsu, D Serre, DW Newton, L Castel, E Soltesz, G Pettersson, AM Gillinov, DR Van Wagoner and J Barnard
From the Cleveland Clinic Lerner College of Medicine (N.T.), Department of Cardiovascular Medicine (M.K.C., D.W.N.), and Department of Thoracic & Cardiovascular Surgery (E.S., G.P., A.M.G.); and Department of Cellular & Molecular Medicine (J.D.S., J.H.), Genomic Medicine Institute (D.S.), Department of Molecular Cardiology (L.C.), and Department of Quantitative Health Sciences (J.B.), Cleveland Clinic Lerner Research Institute, Cleveland, OH
Circ Cardiovasc Genet. 2013;6:362-371; http://dx.doi.org/10.1161/CIRCGENETICS.113.000133 http://circgenetics.ahajournals.org/content/6/4/362The online-only Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000133/-/DC1
Background—Genetic mechanisms of atrial fibrillation (AF) remain incompletely understood. Previous differential expression studies in AF were limited by small sample size and provided limited understanding of global gene networks, prompting the need for larger-scale, network-based analyses.
Methods and Results—Left atrial tissues from Cleveland Clinic patients who underwent cardiac surgery were assayed using Illumina Human HT-12 mRNA microarrays. The data set included 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without coronary artery disease (n=64), coronary artery disease without MV disease (n=57), and lone AF (n=35). Weighted gene coexpression network analysis was performed in the MV group to detect modules of correlated genes. Module preservation was assessed in the other 2 groups. Module eigengenes were regressed on AF severity or atrial rhythm at surgery. Modules whose eigengenes correlated with either AF phenotype were analyzed for gene content. A total of 14 modules were detected in the MV group; all were preserved in the other 2 groups. One module (124 genes) was associated with AF severity and atrial rhythm across all groups. Its top hub gene, RCAN1, is implicated in calcineurin-dependent signaling and cardiac hypertrophy. Another module (679 genes) was associated with atrial rhythm in the MV and coronary artery disease groups. It was enriched with cell signaling genes and contained cardiovascular developmental genes including TBX5.
Conclusions—Our network-based approach found 2 modules strongly associated with AF. Further analysis of these modules may yield insight into AF pathogenesis by providing novel targets for functional studies. (Circ Cardiovasc Genet. 2013;6:362-371.)
trial fibrillation (AF) is the most common sustained cardiac arrhythmia, with a prevalence of ≈1% to 2% in the general population.1,2 Although AF may be an isolated condition (lone AF [LAF]), it often occurs concomitantly with other cardiovascular diseases, such as coronary artery disease (CAD) and valvular heart disease.1 In addition, stroke risk is increased 5-fold among patients with AF, and ischemic strokes attributed to AF are more likely to be fatal.1 Current antiarrhythmic drug therapies are limited in terms of efficacy and safety.1,3,4 Thus, there is a need to develop better risk prediction tools as well as mechanistically targeted therapies for AF. Such developments can only come about through a clearer understanding of its pathogenesis.
Family history is an established risk factor for AF. A Danish Twin Registry study estimated AF heritability at 62%, indicating a significant genetic component.5 Substantial progress has been made to elucidate this genetic basis. For example, genome-wide association studies (GWASs) have identified several susceptibility loci and candidate genes linked with AF. Initial studies performed in European populations found 3 AF-associated genomic loci.6–9 Of these, the most significant single-nucleotide polymor-phisms (SNPs) mapped to an intergenic region of chromosome 4q25. The closest gene in this region, PITX2, is crucial in left-right asymmetrical development of the heart and thus seems promising as a major player in initiating AF.10,11 A large-scale GWAS meta-analysis discovered 6 additional susceptibility loci, implicating genes involved in cardiopulmonary development, ion transport, and cellular structural integrity.12
Differential expression studies have also provided insight into the pathogenesis of AF. A study by Barth et al13 found that about two-thirds of the genes expressed in the right atrial appendage were downregulated during permanent AF, and that many of these genes were involved in calcium-dependent signaling pathways. In addition, ventricular-predominant genes were upregulated in right atrial appendages of subjects with AF.13 Another study showed that inflammatory and transcription-related gene expression was increased in right atrial appendages of subjects with AF versus controls.14 These results highlight the adaptive responses to AF-induced stress and ischemia taking place within the atria.
Despite these advances, much remains to be discovered about the genetic mechanisms of AF. The AF-associated SNPs found thus far only explain a fraction of its heritability15; furthermore, the means by which the putative candidate genes cause AF have not been fully established.9,15,16 Additionally, previous differential expression studies in human tissue were limited to the right atrial appendage, had small sample sizes, and provided little understanding of global gene interactions.13,14 Weighted gene coexpression network analysis (WGCNA) is a technique to construct gene modules within a network based on correlations in gene expression (ie, coexpression).17,18 WGCNA has been used to study genetically complex diseases, such as metabolic syndrome,19 schizophrenia,20 and heart failure.21 Here, we obtained mRNA expression profiles from human left atrial appendage tissue and implemented WGCNA to identify gene modules associated with AF phenotypes.
Methods
Subject Recruitment
From 2001 to 2008, patients undergoing cardiac surgery at the Cleveland Clinic were prospectively screened and recruited. Informed consent for research use of discarded atrial tissues was obtained from each patient by a study coordinator during the presurgical visit. Demographic and clinical data were obtained from the Cardiovascular Surgery Information Registry and by chart review. Use of human atrial tissues was approved by the Institutional Review Board of the Cleveland Clinic.
Table S1: Clinical definitions of cardiovascular phenotype groups
Criterion Type
Mitral Valve (MV) Disease
Coronary Artery Disease (CAD)
Lone Atrial Fibrillation (LAF)
Inclusion Criteria
Surgical indication –
Surgical indication –
History of atrial fibrillation
mitral valve repair or replacement
coronary artery bypass graft
Surgical indication
– MAZE procedure
Preserved ejection fraction (≥50%)
Exclusion Criteria
Significant coronary artery disease:
Significant mitral valve disease:
Significant
coronary artery
– Significant (≥50%) stenosis
– Documented echocardiography
disease:
in at least
finding of
– Significant
one coronary artery
mitral regurgitation (≥3) or
(≥50%) stenosis in
via cardiac catheterization
mitral stenosis
at least one
– History of revascularization
– History of mitral valve
coronary artery via
(percutaneous coronary intervention or coronary artery bypass graft surgery)
repair or replacement
cardiac catheterization
– History of revascularization
(percutaneous coronary intervention or coronary artery bypass graft surgery)
Significant valvular heart disease:
-Documented echocardiography finding of valvular regurgitation (≥3) or stenosis
-History of valve repair or replacement
RNA Microarray Isolation and Profiling
Left atria appendage specimens were dissected during cardiac surgery and stored frozen at −80°C. Total RNA was extracted using the Trizol technique. RNA samples were processed by the Cleveland Clinic Genomics Core. For each sample, 250-ng RNA was reverse transcribed into cRNA and biotin-UTP labeled using the TotalPrep RNA Amplification Kit (Ambion, Austin, TX). cRNA was quantified using a Nanodrop spectrophotometer, and cRNA size distribution was assessed on a 1% agarose gel. cRNA was hybridized to Illumina Human HT-12 Expression BeadChip arrays (v.3). Arrays were scanned using a BeadArray reader.
Expression Data Preprocessing
Raw expression data were extracted using the beadarray package in R, and bead-level data were averaged after log base-2 transformation. Background correction was performed by fitting a normal-gamma deconvolution model using the NormalGamma R package.22 Quantile normalization and batch effect adjustment with the ComBat method were performed using R.23 Probes that were not detected (at a P<0.05 threshold) in all samples as well as probes with relatively lower variances (interquartile range ≤log2[1.2]) were excluded.
The WGCNA approach requires that genes be represented as singular nodes in such a network. However, a small proportion of the genes in our data have multiple probe mappings. To facilitate the representation of singular genes within the network, a probe must be selected to represent its associated gene. Hence, for genes that mapped to multiple probes, the probe with the highest mean expression level was selected for analysis (which often selects the splice isoform with the highest expression and signal-to-noise ratio), resulting in a total of 6168 genes.
Defining Training and Test Sets
Currently, no large external mRNA microarray data from human left atrial tissues are publicly available. To facilitate internal validation of results, we divided our data set into 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without CAD (MV group; n=64), CAD without MV disease (CAD group; n=57), and LAF (LAF group; n=35). LAF was defined as the presence of AF without concomitant structural heart disease, according to the guidelines set by the European Society of Cardiology.1 The MV group, which was the largest and had the most power for detecting significant modules, served as the training set for module derivation, whereas the other 2 groups were designated test sets for module reproducibility. To minimize the effect of population stratification, the data set was limited to white subjects. Differences in clinical characteristics among the groups were assessed using Kruskal–Wallis rank-sum tests for continuous variables and Pearson x2 test for categorical variables.
Weight Gene Coexpression Network Analysis
WGCNA is a systems-biology method to identify and characterize gene modules whose members share strong coexpression. We applied previously validated methodology in this analysis.17 Briefly, pair-wise gene (Pearson) correlations were calculated using the MV group data set. A weighted adjacency matrix was then constructed. I is a soft-thresholding parameter that provides emphasis on stronger correlations over weaker and less meaningful ones while preserving the continuous nature of gene–gene relationships. I=3 was selected in this analysis based on the criterion outlined by Zhang and Horvath17 (see the online-only Data Supplement).
Next, the topological overlap–based dissimilarity matrix was computed from the weighted adjacency matrix. The topological overlap, developed by Ravasz et al,24 reflects the relative interconnectedness (ie, shared neighbors) between 2 genes.17 Hence, construction of the network dendrogram based on this dissimilarity measure allows for the identification of gene modules whose members share strong intercon-nectivity patterns. The WGCNA cutreeDynamic R function was used to identify a suitable cut height for module identification via an adaptive cut height selection approach.18 Gene modules, defined as branches of the network dendrogram, were assigned colors for visualization.
Network Preservation Analysis
Module preservation between the MV and CAD groups as well as the MV and LAF groups was assessed using network preservation statistics as described in Langfelder et al.25 Module density–based statistics (to assess whether genes in each module remain highly connected in the test set) and connectivity-based statistics (to assess whether connectivity patterns between genes in the test set remain similar compared with the training set) were considered in this analysis.25 In each comparison, a Z statistic representing a weighted summary of module density and connectivity measures was computed for every module (Zsummary). The Zsummary score was used to evaluate module preservation, with values ≥8 indicating strong preservation, as proposed by Langfelder et al.25 The WGCNA R function network preservation was used to implement this analysis.25
Table S2: Network preservation analysis between the MV and CAD groups – size and Zsummary scores of gene modules detected.
Module
Module Size
ZSummary
Black
275
15.52
Blue
964
44.79
Brown
817
12.80
Cyan
119
13.42
Green
349
14.27
Green-Yellow
215
19.31
Magenta
239
15.38
Midnight-Blue
83
15.92
Pink
252
23.31
Purple
224
16.96
Red
278
17.30
Salmon
124
13.84
Tan
679
28.48
Turquoise
1512
44.03
Table S3: Network preservation analysis between the MV and LAF groups – size and Zsummary scores of gene modules detected
Module
Module Size
ZSummary
Black
275
13.14
Blue
964
39.26
Brown
817
14.98
Cyan
119
11.46
Green
349
14.91
Green-Yellow
215
20.99
Magenta
239
18.58
Midnight-Blue
83
13.87
Pink
252
19.10
Purple
224
8.80
Red
278
16.62
Salmon
124
11.57
Tan
679
28.61
Turquoise
1512
42.07
Clinical Significance of Preserved Modules
Principal component analysis of the expression data for each gene module was performed. The first principal component of each module, designated the eigengene, was identified for the 3 cardiovascular disease groups; this served as a summary expression measure that explained the largest proportion of the variance of the module.26 Multivariate linear regression was performed with the module ei-gengenes as the outcome variables and AF severity (no AF, paroxysmal AF, persistent AF, permanent AF) as the predictor of interest (adjusting for age and sex). A similar regression analysis was performed with atrial rhythm at surgery (no AF history, AF history in sinus rhythm, AF history in AF rhythm) as the predictor of interest. The false discovery rate method was used to adjust for multiple comparisons. Modules whose eigengenes associated with AF severity and atrial rhythm were identified for further analysis.
In addition, hierarchical clustering of module eigengenes and selected clinical traits (age, sex, hypertension, cholesterol, left atrial size, AF state, and atrial rhythm) was used to identify additional module–trait associations. Clusters of eigengenes/traits were detected based on a dissimilarity measure D, as given by
D=1−cor(Vi,Vj),i≠j (3)
where V=the eigengene or clinical trait.
Enrichment Analysis
Gene modules significantly associated with AF severity and atrial rhythm were submitted to Ingenuity Pathway Analysis (IPA) to determine enrichment for functional/disease categories. IPA is an application of gene set over-representation analysis; for each dis-ease/functional category annotation, a P value is calculated (using Fisher exact test) by comparing the number of genes from the module of interest that participate in the said category against the total number of participating genes in the background set.27 All 6168 genes in the current data set served as the background set for the enrichment analysis.
Hub Gene Analysis
Hub genes are defined as genes that have high intramodular connectivity17,20
Alternatively, they may also be defined as genes with high module membership21,25
Both definitions were used to identify the hub genes of modules associated with AF phenotype.
To confirm that the hub genes identified were themselves associated with AF phenotype, the expression data of the top 10 hub genes (by intramodular connectivity) were regressed on atrial rhythm (adjusting for age and sex). In addition, eigengenes of AF-associated modules were regressed on their respective (top 10) hub gene expression profiles, and the model R2 indices were computed.
Membership of AF-Associated Candidate Genes From Previous Studies
Previous GWAS studies identified multiple AF-associated SNPs.8,9,12,15,28 We selected candidate genes closest to or containing these SNPs and identified their module locations as well as their closest within-module partners (absolute Pearson correlations).
Sensitivity Analysis of Soft-Thresholding Parameter
To verify that the key results obtained from the above analysis were robust with respect to the chosen soft-thresholding parameter (I=3), we repeated the module identification process using I=5. The eigen-genes of the detected modules were computed and regressed on atrial rhythm (adjusting for age and sex). Modules significantly associated with atrial rhythm in ≥2 groups of data set were compared with the AF phenotype–associated modules from the original analysis.
Results
Subject Characteristics
Table 1 describes the clinical characteristics of the cardiac surgery patients who were recruited for the study. Subjects in the LAF group were generally younger and less likely to be a current smoker (P=2.0×10−4 and 0.032, respectively). Subjects in the MV group had lower body mass indices (P=2.7×10−6), and a larger proportion had paroxysmal AF compared with the other 2 groups (P=0.033).
Table 1. Clinical Characteristics of Study Subjects
A total of 14 modules were detected using the MV group data set (Figure 1), with module sizes ranging from 83 genes to 1512 genes; 38 genes did not share similar coexpression with the other genes in the network and were therefore not included in any of the identified modules
Figure 1. Network dendrogram (top) and colors of identified modules (bottom). The dendrogram was constructed using the topological overlap matrix as the similarity measure. Modules corresponded to branches of the dendrogram and were assigned colors for visualization.
Network Preservation Analysis Revealed Strong Preservation of All Modules Between the Training and Test Sets
All 14 modules showed strong preservation across the CAD and LAF groups in both comparisons, with Z [summary] scores of >10 in most modules (Figure 2). No major deviations in the Z [summary] score distributions for the 2 comparisons were noted, indicating that modules were preserved to a similar extent across the 2 groups
Figure 2. Preservation of modules between mitral valve (MV) and coronary artery disease (CAD) groups (left), and MV and lone atrial fibrillation (LAF) groups (right). A Zsummary statistic was computed for each module as an overall measure of its preservation relating to density and connectivity. All modules showed strong preservation in both comparisons with Zsummary scores >8 (red dotted line).
Regression Analysis of Module Eigengene Profiles Identified 2 Modules Associated With AF Severity and Atrial Rhythm
Table IV in the online-only Data Supplement summarizes the proportion of variance explained by the first 3 principal components for each module. On average, the first principal component (ie, the eigengene) explained ≈18% of the total variance of its associated module. For each group, the module eigengenes were extracted and regressed on AF severity (with age and sex as covariates). The salmon module (124 genes) eigengene was strongly associated with AF severity in the MV and CAD groups (P=1.7×10−6 and 5.2×10−4, respectively); this association was less significant in the LAF group (P=9.0×10−2). Eigengene levels increased with worsening AF severity across all 3 groups, with the greatest stepwise change taking place between the paroxysmal AF and persistent AF categories (Figure 3A). When the module eigen-genes were regressed on atrial rhythm, the salmon module eigengene showed significant association in all groups (MV: P=1.1×10−14; CAD: P=1.36×10−6; LAF: P=2.1×10−4). Eigen-gene levels were higher in the AF history in AF rhythm category (Figure 3B).
Table S4:Proportion of variance explained by the principal components for each module.
Dataset
Group
Principal
Component
Black
Blue
Brown
Cyan
Green
Green-
Yellow
Magenta
Mitral
1
20.5%
22.2%
20.1%
21.8%
21.4%
22.8%
19.6%
2
4.1%
3.6%
4.8%
5.7%
4.5%
5.9%
3.9%
3
3.4%
3.1%
3.8%
4.4%
3.9%
3.7%
3.7%
CAD
1
12.5%
18.6%
7.1%
16.8%
12.2%
20.3%
12.8%
2
6.0%
5.5%
5.0%
7.0%
5.5%
6.1%
6.4%
3
4.9%
4.1%
4.4%
6.5%
4.8%
4.4%
4.8%
LAF
1
14.0%
16.6%
11.7%
14.3%
14.7%
20.8%
20.2%
2
8.9%
8.5%
7.6%
9.3%
7.3%
11.1%
6.9%
3
6.5%
6.3%
5.5%
8.2%
6.1%
5.3%
6.2%
Dataset
Group
Principal
Component
Midnight- Blue
Pink
Purple
Red
Salmon
Tan
Turquoise
Mitral
1
28.5%
22.6%
18.7%
20.5%
22.3%
19.0%
25.8%
2
4.6%
6.0%
4.7%
4.1%
6.9%
4.0%
3.5%
3
4.2%
4.2%
4.2%
3.5%
4.0%
3.6%
3.3%
CAD
1
23.4%
17.1%
15.5%
15.0%
18.0%
14.6%
18.2%
2
7.4%
8.6%
6.0%
6.4%
7.2%
5.8%
6.6%
3
5.1%
5.4%
5.3%
5.4%
6.2%
5.1%
4.5%
LAF
1
23.5%
18.4%
12.0%
15.9%
16.9%
13.7%
16.5%
2
7.9%
8.5%
9.8%
9.4%
9.5%
9.1%
9.6%
3
6.7%
7.0%
6.6%
6.0%
6.9%
6.8%
6.3%
Figure 3. Boxplots of salmon module eigengene expression levels with respect to atrial fibrillation (AF) severity (A) and atrial rhythm (B).
A, Eigengene expression correlated positively with AF severity, with the largest stepwise increase between the paroxysmal AF and permanent AF categories. B, Eigengene expression was highest in the AF history in AF rhythm category in all 3 groups. CAD indicates coronary artery disease; LAF, lone AF; and MV, mitral valve.
The regression analysis also revealed statistically significant associations between the tan module (679 genes) eigengene and atrial rhythm in the MV and CAD groups (P=5.8×10−4 and 3.4×10−2, respectively). Eigengene levels were lower in the AF history in AF rhythm category compared with the AF history in sinus rhythm category (Figure 4); this trend was also observed in the LAF group, albeit with weaker statistical evidence (P=0.15).
Figure 4. Boxplots of tan module eigengene expression levels with respect to atrial rhythm. Eigengene expression levels were lower in the atrial fibrillation (AF) history in AF rhythm category compared with the AF history in sinus rhythm category. CAD indicates coronary artery disease; LAF, lone AF; and MV, mitral valve
Hierarchical Clustering of Eigengene Profiles With Clinical Traits
Hierarchical clustering was performed to identify relationships between gene modules and selected clinical traits. The salmon module clustered with AF severity and atrial rhythm; in addition, left atrial size was found in the same cluster, suggesting a possible relationship between salmon module gene expression and atrial remodeling (Figure 5A). Although the tan module was in a separate cluster from the salmon module, it was negatively correlated with both atrial rhythm and AF severity (Figure 5B).
Figure 5. Dendrogram (A) and correlation heatmap (B) of module eigengenes and clinical traits.
A, The salmon module eigengene but not the tan module eigengene clustered with atrial fibrillation (AF) severity, atrial rhythm, and left atrial size. B, AF severity and atrial rhythm at surgery correlated positively with the salmon module eigengene and negatively with the tan module eigengene. Arhythm indicates atrial rhythm at surgery; Chol, cholesterol; HTN, hypertension; and LASize, left atrial size.
IPA Enrichment Analysis of Salmon and Tan Modules
The salmon module was enriched in genes involved in cardiovascular function and development (smallest P=4.4×10−4) and organ morphology (smallest P=4.4×10−4). In addition, the top disease categories identified included endocrine system disorders (smallest P=4.4×10−4) and cardiovascular disease (smallest P=2.59×10−3).
The tan module was enriched in genes involved in cell-to-cell signaling and interaction (smallest P=8.9×10−4) and cell death and survival (smallest P=1.5×10−3). Enriched disease categories included cancer (smallest P=2.2×10−4) and cardiovascular disease (smallest P=4.5×10−4).
We identified hub genes in the 2 modules based on intramod-ular connectivity and module membership. For the salmon module, the gene RCAN1 exhibited the highest intramodular connectivity and module membership. The top 10 hub genes (by intramodular connectivity) were significantly associated with atrial rhythm, with false discovery rate–adjusted P values ranging from 1.5×10−5 to 4.2×10−12. These hub genes accounted for 95% of the variation in the salmon module eigengene.
In the tan module, the top hub gene was CPEB3. The top 10 hub genes (by intramodular connectivity) correlated with atrial rhythm as well, although the statistical associations in the lower-ranked hub genes were relatively weaker (false discovery rate–adjusted P values ranging from 1.1×10−1 to 3.4×10−4). These hub genes explained 94% of the total variation in the tan module eigengene.
The names and connectivity measures of the hub genes found in both modules are presented in Table 2.
Table 2. Top 10 Hub Genes in the Salmon (Left) and Tan (Right) Modules as Defined by Intramodular Connectivity and Module Membership
Salmon Module
Tan Module
Gene
IMC
Gene
MM
Gene
IMC
Gene
MM
RCAN1
8.2
RCAN1
0.81
CPEB3
43.3
CPEB3
0.85
DNAJA4
7.7
DNAJA4
0.81
CPLX3
42.4
CPLX3
0.84
PDE8B
7.7
PDE8B
0.80
NEDD4L
40.8
NEDD4L
0.83
PRKAR1A
6.9
PRKAR1A
0.77
SGSM1
40.7
SGSM1
0.82
PTPN4
6.7
PTPN4
0.75
UCKL1
39.0
UCKL1
0.81
SORBS2
6.0
FHL2
0.69
SOSTDC1
37.2
SOSTDC1
0.79
ADCY6
5.7
ADCY6
0.69
PRDX1
35.5
RCOR2
0.78
FHL2
5.7
SORBS2
0.68
RCOR2
35.4
EEF2K
0.77
BVES
5.4
DHRS9
0.67
NPPB
35.3
PRDX1
0.76
TMEM173
5.3
LAPTM4B
0.65
LRRN3
34.6
MMP11
0.76
A visualization of the salmon module is shown using the Cytoscape tool (Figure 6). A full list of the genes in the salmon and tan modules is provided in the online-only Data Supplement.
Figure 6. Cytoscape visualization of genes in the salmon module.
Nodes representing genes with high intramodu-lar connectivities, such as RCAN1 and DNAJA4, appear larger in the network. Strong connections are visualized with darker lines, whereas weak connections appear more translucent
Membership of AF-Associated Candidate Genes From Previous Studies
The tan module contained MYOZ1, which was identified as a candidate gene from the recent AF meta-analysis. PITX2 was located in the green module (n=349), and ZFHX3 was located in the turquoise module (n=1512). The locations of other candidate genes (and their closest partners) are reported in the online-only Data Supplement.
Sensitivity Analysis of Key Results
We repeated the WGCNA module identification approach using a different soft-thresholding parameter (β=5). One module (n=121) was found to be strongly associated with atrial rhythm at surgery across all 3 groups of data set, whereas another module (n=244) was associated with atrial rhythm at surgery in the MV and CAD groups. The first module overlapped significantly with the salmon module in terms of gene membership, whereas most of the second modules’ genes were contained within the tan module. The top hub genes found in the salmon and tan modules remained present and highly connected in the 2 new modules identified with the different soft-thresholding parameter.
Discussion
To our knowledge, our study is the first implementation of an unbiased, network-based analysis in a large sample of human left atrial appendage gene expression profiles. We found 2 modules associated with AF severity and atrial rhythm in 2 to 3 of our cardiovascular comorbidity groups. Functional analyses revealed significant enrichment of cardiovascular-related categories for both modules. In addition, several of the hub genes identified are implicated in cardiovascular disease and may play a role in AF initiation and progression.
In our study, WGCNA was used to construct modules based on gene coexpression, thereby reducing the net-work’s dimensionality to a smaller set of elements.17,21 Relating modulewise changes to phenotypic traits allowed statistically significant associations to be detected at a lower false discovery rate compared with traditional differential expression studies. Furthermore, shared functions and pathways among genes in the modules could be inferred via enrichment analyses.
We divided our data set into 3 groups to verify the reproducibility of the modules identified by WGCNA; 14 modules were identified in the MV group in our gene network. All were strongly preserved in the CAD and LAF groups, suggesting that gene coexpression patterns are robust and reproducible despite differences in cardiovascular comorbidities.
The use of module eigengene profiles as representative summary measures has been validated in a number of studies.20,26 Additionally, we found that the eigengenes accounted for a significant proportion (average 18%) of gene expression variability in their respective modules. Regression analysis of the module eigengenes found 2 modules associated with AF severity and atrial rhythm in ≥2 groups of data set. The association between the salmon module eigengene and AF severity was statistically weaker in the LAF group (adjusted P=9.0×10−2). This was probably because of its significantly smaller sample size compared with the MV and CAD groups. Despite this weaker association, the relationship between the salmon module eigengene and AF severity remained consistent among the 3 groups (Figure 3A). Similarly, the lack of statistical significance for the association between the tan module eigengene and atrial rhythm at surgery in the LAF group was likely driven by the smaller sample size and (by definition) lack of samples in the no AF category.
A major part of our analysis focused on the identification of module hub genes. Hubs are connected with a large number of nodes; disruption of hubs therefore leads to widespread changes within the network. This concept has powerful applications in the study of biology, genetics, and disease.29,30 Although mutations of peripheral genes can certainly lead to disease, gene network changes are more likely to be motivated by changes in hub genes, making them more biologically interesting targets for further study.17,29,31 Indeed,
the hub genes of the salmon and tan modules accounted for the vast majority of the variation in their respective module eigengenes, signaling their importance in driving gene module behavior.
The hub genes identified in the salmon and tan modules were significantly associated with AF phenotype overall. It was noted that this association was statistically weaker for the lower-ranked hub genes in the tan module. This highlights an important aspect and strength of WGCNA—to be able to capture module-wide changes with respect to disease despite potentially weaker associations among individual genes.
The implementation of WGCNA necessitated the selection of a soft-thresholding parameter 13. Unlike hard-thresholding (where gene correlations below a certain value are shrunk to zero), the soft-thresholding approach gives greater weight to stronger correlations while maintaining the continuous nature of gene–gene relationships. We selected a 13 value of 3 based on the criteria outlined by Zhang and Horvath.17 His team and other investigators have demonstrated that module identification is robust with respect to the 13 parameter.17,19–21 In our data, we were also able to reproduce the key findings reported with a different, larger 13 value, thereby verifying the stability of our results relating to 13.
The salmon module (124 genes) was associated with both AF phenotypes; furthermore, IPA analysis of its gene contents suggested enrichment in cardiovascular development as well as disease. Its eigengene increased with worsening AF severity, with the largest stepwise change occurring between the paroxysmal AF and persistent AF categories (Figure 3). Hence,
the gene expression changes within the salmon module may reflect the later stages of AF pathophysiology.
The top hub gene of the salmon module was RCAN1 (regulator of calcineurin 1). Calcineurin is a cytoplasmic Ca2+/ calmodulin-dependent protein phosphatase that stimulates cardiac hypertrophy via its interactions with NFAT and L-type Ca2+ channels.32,33RCAN1 is known to inhibit calcineurin and its associated pathways.32,34 However, some data suggest that RCAN1 may instead function as a calcineurin activator when highly expressed and consequently potentiate hypertrophic signaling.35 Thus,
perturbations in RCAN1 levels (attributable to genetic variants or mutations) may cause an aberrant switching in function, which in turn triggers atrial remodeling and arrhythmogenesis.
Other hub genes found in the salmon module are also involved in cardiovascular development and function and may be potential targets for further study.
DNAJA4 (DnaJ homolog, subfamily A, member 4) regulates the trafficking and maturation of KCNH2 potassium channels, which have a prominent role in cardiac repolarization and are implicated in the long-QT syndromes.36
FHL2 (four-and-a-half LIM domain protein 2) interacts with numerous cellular components, including
actin cytoskeleton,
transcription machinery, and
ion channels.37
FHL2 was shown to enhance the hypertrophic effects of isoproterenol, indicating that
FHL2 may modulate the effect of environmental stress on cardiomyocyte growth.38
FHL2 also interacts with several potassium channels in the heart, such as KCNQ1, KCNE1, and KCNA5.37,39
Additionally, blood vessel epicardial substance (BVES) and other members of its family were shown to be highly expressed in cardiac pacemaker cells. BVES knockout mice exhibited sinus nodal dysfunction, suggesting that BVES regulates the development of the cardiac pacemaking and conduction system40 and may therefore be involved in the early phase of AF development.
The tan module (679 genes) eigengene was negatively correlated with atrial rhythm in the MV and CAD groups (Figure 4); this may indicate a general decrease in gene expression of its members in fibrillating atrial tissue. IPA analysis revealed enrichment in genes involved in cell signaling as well as apoptosis. The top-ranked hub gene, cytoplasmic polyade-nylation element binding protein 3 (CPEB3), regulates mRNA translation and has been associated with synaptic plasticity and memory formation.41 The role of CPEB3 in the heart is currently unknown, so further exploration via animal model studies may be warranted.
Natriuretic peptide-precursor B (NPPB), another highly interconnected hub gene, produces a precursor peptide of brain natriuretic peptide, which
regulates blood pressure through natriuresis and vasodilation.42
(NPPB) gene variants have been linked with diabetes mellitus, although associations with cardiac phenotypes are less clear.42 TBX5 and GATA4, which play important roles in the embryonic heart development,43 were members of the tan module. Although not hub genes, they may also contribute toward developmental susceptibility of AF. In addition, TBX5 was previously reported to be near an SNP associated with PR interval and AF in separate large-scale GWAS studies.12,28 MYOZ1, another candidate gene identified in the recent AF GWAS meta-analysis, was found to be a member as well; it associates with proteins found in the Z-disc of skeletal and cardiac muscle and may suppress calcineurin-dependent hypertrophic signaling.12
Some, but not all, of the candidate genes found in previous GWAS studies were located in the AF-associated modules. One possible explanation for this could be the difference in sample sizes. The meta-analysis involved thousands of individuals, whereas the current study had <100 in each group of data set, which limited the power to detect significant differences between levels of AF phenotype even with the module-wise approach. Additionally, transcription factors like PITX2 are most highly expressed during the fetal phase of development. Perturbations in these genes (attributable to genetic variants or mutations) may therefore initiate the development of AF at this stage and play no significant role in adults (when we obtained their tissue samples).
Limitations in Study
We noted several limitations in this study. First, no human left atrial mRNA data set of adequate size currently exists publicly. Hence, we were unable to validate our results with an external, independent data set. However, the network preservation assessment performed within our data set showed strong preservation in all modules, indicating that our findings are robust and reproducible.
Although the module eigengenes captured a significant proportion of module variance, a large fraction of variability did remain unaccounted for, which may limit their use as representative summary measures.
We extracted RNA from human left atrial appendage tissue, which consists primarily of cardiomyocytes and fibroblasts. Atrial fibrosis is known to occur with AF-associated remodeling.44 As such, the cardiomyocyte to fibroblast ratio is likely to change with different levels of AF severity, which in turn influences the amount of RNA extracted from each cell type. Hence, true differences in gene expression (and coexpression) within cardiomyocytes may be confounded by changes in cellular composition attributable to atrial remodeling. Also, there may be significant regional heterogeneity in the left atrium with respect to structure, cellular composition, and gene expression,45 which may limit the generaliz-ability of our results to other parts of the left atrium.
All subjects in the study were whites to minimize the effects of population stratification. However, it is recognized that the genetic basis of AF may differ among ethnic groups.9 Thus, our results may not be generalizable to other ethnicities.
Finally, it is possible for genes to be involved in multiple processes and functions that require different sets of genes. However, WGCNA does not allow for overlapping modules to be formed. Thus,
this limits the method’s ability to characterize such gene interactions.
Conclusions
In summary, we constructed a weighted gene coexpression network based on RNA expression data from the largest collection of human left atrial appendage tissue specimens to date. We identified 2 gene modules significantly associated with AF severity or atrial rhythm at surgery. Hub genes within these modules may be involved in the initiation or progression of AF and may therefore be candidates for functional studies.
Refererences
1. European Heart Rhythm Association, European Association for Cardio-Thoracic Surgery, Camm AJ, Kirchhof P, Lip GY, Schotten U, et al. Guidelines for the management of atrial fibrillation: the task force for the management of atrial fibrillation of the European Society of Cardiology (ESC). Eur Heart J. 2010;31:2369–2429.
2. Lemmens R, Hermans S, Nuyens D, Thijs V. Genetics of atrial fibrillation and possible implications for ischemic stroke. Stroke Res Treat. 2011;2011:208694.
3. Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA III, et al; ACCF/AHA/HRS. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2011;57:223–242.
4. Dobrev D, Carlsson L, Nattel S. Novel molecular targets for atrial fibrillation therapy. Nat Rev Drug Discov. 2012;11:275–291.
5. Christophersen IE, Ravn LS, Budtz-Joergensen E, Skytthe A, Haunsoe S, Svendsen JH, et al. Familial aggregation of atrial fibrillation: a study in Danish twins. Circ Arrhythm Electrophysiol. 2009;2:378–383.
6. Gudbjartsson DF, Arnar DO, Helgadottir A, Gretarsdottir S, Holm H, Sig-urdsson A, et al. Variants conferring risk of atrial fibrillation on chromosome 4q25. Nature. 2007;448:353–357.
7. Ellinor PT, Lunetta KL, Glazer NL, Pfeufer A, Alonso A, Chung MK, et al. Common variants in KCNN3 are associated with lone atrial fibrillation. Nat Genet. 2010;42:240–244.
8. Benjamin EJ, Rice KM, Arking DE, Pfeufer A, van Noord C, Smith AV, et al. Variants in ZFHX3 are associated with atrial fibrillation in individuals of European ancestry. Nat Genet. 2009;41:879–881.
9. Sinner MF, Ellinor PT, Meitinger T, Benjamin EJ, Kääb S. Genome-wide association studies of atrial fibrillation: past, present, and future. Cardio-vasc Res. 2011;89:701–709.
10. Clauss S, Kääb S. Is Pitx2 growing up? Circ Cardiovasc Genet. 2011;4:105–107.
11. Kirchhof P, Kahr PC, Kaese S, Piccini I, Vokshi I, Scheld HH, et al. PITX2c is expressed in the adult left atrium, and reducing Pitx2c expression promotes atrial fibrillation inducibility and complex changes in gene expression. Circ Cardiovasc Genet. 2011;4:123–133.
12. Ellinor PT, Lunetta KL, Albert CM, Glazer NL, Ritchie MD, Smith AV, et al. Meta-analysis identifies six new susceptibility loci for atrial fibrillation. Nat Genet. 2012;44:670–675.
13. Barth AS, Merk S, Arnoldi E, Zwermann L, Kloos P, Gebauer M, et al. Reprogramming of the human atrial transcriptome in permanent atrial fibrillation: expression of a ventricular-like genomic signature. Circ Res. 2005;96:1022–1029.
Atrial fibrillation is the most common sustained cardiac arrhythmias in the United States. The genetic and molecular mechanisms governing its initiation and progression are complex, and our understanding of these mechanisms remains incomplete despite recent advances via genome-wide association studies, animal model experiments, and differential expression studies. In this study, we used weighted gene coexpression network analysis to identify gene modules significantly associated with atrial fibrillation in a large sample of human left atrial appendage tissues. We further identified highly interconnected genes (ie, hub genes) within these gene modules that may be novel candidates for functional studies. The discovery of the atrial fibrillation-associated gene modules and their corresponding hub genes provide novel insight into the gene network changes that occur with atrial fibrillation, and closer study of these findings can lead to more effective targeted therapies for disease management.
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
This is the Third article of a multipart series covering Ca(2+) signaling and the cytoskeleton, and two on Ca2+ in cardiac contractility governed by the activations involving a ryanodine (RyR2) receptor and a specific calmodulin protein CaKIIδ with B and C splice variants. In all of these discussions, Ca(2+) has a crucial role in many cellular events, not all of which are detailed, and its importance to cardiac function and function disorders is critical. We shall next undertake the difficult examination of Ca(2+) movements in the kidney, which has a special relationship to vitamin D and bone mineral metabolism that is not of interest here. Nor will we go into any depth on the importance of the kidney to maintenance of plasma H+ and K+ balance and metabolic acidosis. Whereas the lung has a large role in pH maintenance by the respiratory rate (under sympathetic control), it maintains the balance through the expiration of CO2, with H+ tied up in water via the carbonic anhydrase reaction.
(Part II) Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
(Part II) Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
(Part IV) The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part V: Heart Failure and Arrhythmia: Potential for Targeted Intervention — The Effects of Ca 2+ -calmodulin (Ca-CaM) phosphorylation/dephosphorylation/hyperphosphorylation
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses in the Human Heart
Basal-lateral-plasma-membrane vesicles and brush-border-membrane vesicles were isolated from rat kidney cortex by differential centrifugation followed by free-flow electrophoresis. Ca2+ uptake into these vesicles was investigated by a rapid filtration method. Both membranes show a considerable binding of Ca2+ to the vesicle interior, making the analysis of passive fluxes in uptake experiments difficult. Only the basal-lateral-plasma-membrane vesicles exhibit an ATP-dependent pump activity which can be distinguished from the activity in mitochondrial and endoplasmic reticulum by virtue of the different distribution during free-flow electrophoresis and its lack of sensitivity to oligomycin. The basal-lateral plasma membranes contain in addition a Na+/Ca2+-exchange system which mediates a probably rheogenic counter-transport of Ca2+ and Na+ across the basal cell border. The latter system is probably involved in the secondary active Na+-dependent and ouabain-inhibitable Ca2+ reabsorption in the proximal tubule, the ATP-driven system is probably more important for the maintenance of a low concentration of intracellular Ca2+.
In recent micropuncture studies using simultaneously tubular and capillary perfusion it could be demonstrated that in the rat kidney proximal tubule Ca2+ reabsorption is dependent on the presence of Na+- ions and sensitive to ouabain (Ullrich et al., 1976). On the other hand cell-fractionation studies on the distribution of plasma-membrane-bound enzymes in rat proximal tubular epithelial cells revealed a contraluminal localization of a Ca2+-stimulated ATPase (Kinne-Saffran & Kinne, 1974). These results suggested that both Na+-driven and ATP-driven Ca2+ transport systems might be involved in proximal tubular transepithelial Ca2+ transport. Considering the low concentration of intracellular Ca2+ one could expect that these active steps in Ca2+ reabsorption are located at the basal cell pole.
To our knowledge there have been two attempts to study the role of ATP in the Ca2+ transport of renal membranes. In one study increase in Ca2+ uptake by rabbit kidney membranes was observed, but this increase was attributed to a phosphorylation of the membranes and a concomitant binding of Ca2+ to the negative charges newly generated at the membrane surface. Moore et al. (1974) observed an ATP-dependent Ca2+ uptake distinct from that of the mitochondria in a crude fraction of renal plasma membranes as well as in rat renal microsomes. The two uptake systems differed in their capacity, their sensitivity to Na+ and their apparent Km values for Mg2+-ATP.
Experiments are described on the Ca2+ transport into brush-border-membrane vesicles and basal-lateral plasma-membrane vesicles isolated from rat renal cortex. The results show that a primary active ATP-driven Ca2+ pump and an Na+/Ca2+-exchange system are present in the basal-lateral plasma membranes, but not in the brush-border membrane.
These findings indicate that trans-epithelial Ca2+ transport in rat proximal tubule can be
primarily active via the ATP-driven system as well as
secondarily active if the Na+/Ca2+ exchange system is involved.
It appears that the Na+/Ca2+ exchange system
is responsible for the bulk flow of Ca2+ across the epithelium, whereas
the ATP-driven system might be involved in the fine regulation of the concentration of intracellular Ca2+.
(Gmaj P, Murer H, and Kinne R. 1979)
The Renal Na+/Ca2+ Exchange System of the Nephron
The movement of Ca2+ across the basolateral plasma membrane was studied from rabbit proximal and distal convoluted tubules and ATP-dependent Ca2+ uptake was found in both. But the activity was higher distal. The distal tubular membranes had a very active Na+/Ca2+ exchange system, which was absent in the proximal segment. The ATP-dependent Ca2+ uptake in the distal tubular membrane preparations was gradually inhibited by Na+ outside the vesicles, and was a function of the imposed Na+ gradient. The results indicate that an active Na+/Ca2+ exchange system is absent in the proximal tubule. Ramachandram & Brunette, 1989). Parathyroid hormone (PTH) and calcitonin increase Ca2+ uptake by purified distal tubular luminal membranes (DTLM), and both hormone stimulate adenylate cyclase and phospholipase C. Therefore, distal tubules were incubated with dibutyryl cAMP (dbcAMP) and the result was that dbcAMP increased the Ca2+ transport by luminal membranes, but phorbol 12-myristate 13 acetate (PMA) had no such effect. But when PMA was added to low concentrations of dbcAMP the uptake significantly increased. Protein kinase C inhibitors prevented the effect. This indicated that in the distal tubule Ca2+ transport required both the combined effect of PK A and C involves both components of the transport kinetics. (Hila, Claveau, Laclerc, Brunette, 1997)
In the rabbit, calcitonin enhances Ca2+ reabsorption in the distal tubule. Tubules were incubated with or in the absence of calcitonin, and the luminal or basolateral membranes were purified and Ca2+ transport was measured through the vesicles. The results were compared with those obtained from proximal tubule membranes, and the results were no effect of calcitonin on Ca2+ uptake in the proximal tubules. In the distal tubules there was the expected uptake, but the presence of Na+ in the suspension decreased the Ca2+ uptake. The uptake was partially restored by preincubation with calcitonin. Recall the experiment demonstrating a requirement for PK A and C in Ca2+ uptake indicating a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. Calcitonin stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Incubation of distal tubule suspensions with dbcAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. The researchers concluded that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms:
the opening of low affinity Ca2+ channels in the luminal membrane and
the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.
(Zuo Q, Claveau D, Hilal G, Leclerc M, Brunette MG. 1997)
Calcium (Ca2+) filtered in the glomerulus is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by hormones requires
binding of these hormones to the basolateral membrane,
interaction with G proteins,
liberation of messengers,
activation of kinases
opening of the channels at the opposite pole of the cells.
It follows that if the Ca2+ entry through the luminal membranes of proximal and distal tubules is a membrane-limited process, then G proteins have a regulatory role. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPγs or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPγs significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPγs increased 0.5 mM Ca2+ uptake in the absence (p < 0.02) and in the presence (p < 0.02) of Na+. The action of GTPγs, when present, was dose dependent. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPγs increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.
(Brunette MG, Hilal G, Mailloux J, Leclerc M. 2000)
We previously reported a dual kinetics of Ca2+ transport by the distal tubule luminal membrane of the kidney, suggesting the presence of several types of channels. We, therefore, examined the effects of specific inhibitors (i.e., diltiazem, an L-type channel; ω-conotoxin MVIIC, a P/Q-type channel; and mibefradil, a T-type channel antagonist) on Ca2+ uptake by rabbit nephron luminal membranes. None of these inhibitors influenced Ca2+ uptake by the proximal tubule membranes. In contrast, in the absence of sodium (Na+), the three channel antagonists decreased Ca2+ transport by the distal membranes, and their action depended on the substrate concentrations: (P < 0.05) without influencing 0.5 mM Ca2+transport, whereas ω-conotoxin MVIIC decreased 0.5 mM Ca2+ (P < 0.02) and 1 µM mibefradil decreased it (P < 0.05); the latter two inhibitors [P/Q type, T-type] left 0.1 mM Ca2+transport unchanged. Diltiazem [L-type] decreased the Vmax of the high-channels, whereas ω-conotoxin MVIIC and mibefradil influenced exclusively the Vmax of the low-affinity channels. These results not only confirm that the distal luminal membrane is the site of Ca2+ channels, but they suggest that these channels belong to the L, P/Q, and T types. (M G Brunette, M Leclerc, D Couchourel, J Mailloux, Y Bourgeois. 2000)
Calcium (Ca2+) transport by the distal tubule (DT) luminal membrane
Calcium (Ca2+) transport by the distal tubule (DT) luminal membrane is regulated by
the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers,
protein kinases, and
ATP as the phosphate donor.
Could ATP itself, when directly applied to the cytosolic surface of the membrane influence the Ca2+ channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca2+ uptake by these vesicles loaded with ATP or the carrier. The presence of 100 μM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca2+ uptake in the absence of Na+ (P < 0.01) and in the presence of 100 mM Na+ (P < 0.01). This effect was dose dependent with an EC50 value of approximately 40 μM. ATP action involved the high-affinity component of Ca2+ transport, decreasing the Km from 0.08 ± 0.01 to 0.04 ± 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPγs abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, they investigated the effect of intravesicular cytochalasin on Ca2+ transport. Cytochalasin B decreased 0.5 mM Ca2+ uptake (P< 0.01). However, when both ATP and cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca2+ uptake by the PT luminal membrane. They conclude from this that the high-affinity Ca2+ channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca2+ channels within this membrane. (MG. Brunette*, J Mailloux, G Hilal. 1999)
Proximal tubular sodium-calcium exchanger
The functional expression of the renal sodium-calcium exchanger has been amply documented in studies on renal cortical basolateral membranes. In perfused renal tubules, other investigators have shown sodium-calcium exchange activity in the
proximal convolution
in the distalconvolution,
the connecting tubule, and
the collecting tubule of the rabbit.
In rat proximal tubules, we found that the sodium-calcium exchanger is an important determinant of cytosolic calcium homeostasis, since
inhibition of sodium-dependent calcium efflux mode caused a large accumulation of tubular calcium.
In membranes from rat proximal tubulessodium-calcium activity was high, and in intact proximal tubules,
the tubular sodium-calcium exchanger exhibited a high affinity for cytosolic calcium
and had a substantial transport capacity, which may be absolute requirements for the maintenance of stable cytosolic calcium in proximal tubules. (Dominguez JH, Juhaszova M, Feister HA. 1992.)
Proximal tubule Na(+)-Ca2+ exchanger protein is same as the cardiac protein
The activity of the Na(+)-Ca2+ exchanger, a membrane transporter that mediates Ca2+ efflux, has been described in amphibian and mammalian renal proximal tubules. However, demonstration of cell-specific
expression of the Na(+)-Ca2+ exchanger in proximal renal tubules has been restricted to functional assays.
In this work, Na(+)-Ca2+ exchanger gene expression in rat proximal tubules was characterized by three additional criteria:
functional assay of transport activity in membrane vesicles derived from proximal tubules, expression of
specific Na(+)-Ca2+ exchanger protein detected on Western blots, and
determination of specific mRNA encoding Na(+)-Ca2+ exchanger protein on Northern blots.
A new transport activity assay showed that proximal tubule membranes
contained the highest Na(+)-Ca2+ exchanger transport activity reported in renal tissues.
In dog renal proximal tubules and sarcolemma, a specific protein of approximately 70 kDa was detected, whereas in rat proximal tubules and sarcolemma, the specific protein approximated 65 kDa and was localized to the basolateral membrane. On Northern blots, a single 7-kb transcript isolated from rat
proximal tubules,
whole kidney, and
heart
hybridized with rat heart cDNA.
These data indicate that Na(+)-Ca2+ exchanger protein expressed in rat proximal tubule is similar, if not identical, to the cardiac protein. We suggest that the tubular Na(+)-Ca2+ exchanger characterized herein represents the Na(+)-Ca2+ exchanger described in functional assays of renal proximal tubules. (Dominguez JH, Juhaszova M, Kleiboeker SB, Hale CC, Feister HA. 1992.)
Calcium reabsorption regulated by the distal tubules
Extracellular calcium homeostasis involves coordinated calcium absorption by
the intestine,
calcium resorption from bone, and
calcium reabsorption by the kidney.
This review addresses the mechanism and regulation of renal calcium transport. Calcium reabsorption occurs throughout the nephron. However, distal tubules are the nephron site at which calcium reabsorption is regulated by
parathyroid hormone,
calcitonin, and
1 alpha,25-dihydroxyvitamin D3 and
where the magnitude of net reabsorption is largely determined. These and related observations underscore the view that distal tubules are highly specialized
to permit fine regulation of calcium excretion in response to
alterations in extracellular calcium levels.
Progress in understanding the mechanism and regulation of calcium transport has emerged from application of
single cell fluorescence,
patch clamp, and
molecular biological approaches.
These techniques permit the examination of
ion transport at the cellular level and
its regulation at subcellular and molecular levels.
This editorial review focuses on recent and emerging observations and attempts to integrate them into models of cellular calcium transport. (Friedman PA , Gesek FA. 1993)
Calcium-Sensing Receptor (CSR)
Renal tubular calcium reabsorption is a critical determinant of extracellular fluid (ECF) calcium concentration; for the need of constancy of ECF calcium concentration,
the renal tubular handling of calcium is tightly controlled
in order to match renal calcium excretion to the net amount of calcium entering the ECF.
Both parathyroid hormone (PTH) and vitamin D metabolites are involved in
the control of renal tubular calcium reabsorption and
ECF calcium concentration [1].
Besides this hormonal control, it has been recognized recently that
ECF calcium is able to regulate its own reabsorption by the mammalian tubule.
Indeed, a large body of evidence supports the view that ECF calcium exerts this action
by activating the calcium/polyvalent cation-sensing receptor (CaSR)
located in the plasma membrane of many tubular cell types.
First, increasing ECF calcium concentration
elicits a marked increase in urinary calcium (and magnesium) excretion [2,3] and
this occurs independently of any change in the calcium-regulating hormones [2,3].
Second, the inhibitory effect of ECF calcium on its own reabsorption is shared by other CaSR agonists, e.g. magnesium [4].
Third, the relationship between ECF calcium and urinary calcium excretion
is altered in patients bearing mutations of the CASR gene: renal tubular calcium reabsorption
is enhanced in patients with inactivating mutations [5,6]
and decreased in patients with activating mutations.
Therefore, there is abundant evidence that renal tubular CaSR plays a role
in the control of divalent cations reabsorption under
both normal and pathological conditions.
Localization of the extracellular CaSR
Transcripts of the CASR gene are expressed in many nephron segments of rat kidney, extending from glomeruli to the inner medullary collecting duct (IMCD) [7]. The CaSR protein is expressed in
the proximal tubule,
medullary and cortical thick ascending limb (TAL) segments,
macula densa cells,
distal convoluted tubule (DCT) and
type-A intercalated cells in the distal tubule and cortical collecting duct [8]
and in inner medullary collecting duct cells [9].
The polarity of expression varies from segment to segment, the protein being expressed in
the apical membrane of proximal tubule and
IMCD cells and
in the basolateral membrane of TAL and DCT cells [8,9].
Interestingly, the highest density of protein expression has been observed in the cortical TAL (cTAL),
known to reabsorb calcium and magnesium in a regulated manner.
CaSR under physiological conditions
Consistent with its polarized plasma membrane localization,
CaSR has been shown to be involved in the control of thick ascending limb (TAL) calcium and magnesium reabsorption.
In the mouse and rat TAL,
both calcium and magnesium are reabsorbed selectively in the cortical portion (cTAL) [10]
and this reabsorption is passive along an electrical gradient
through the paracellular pathway [10,11]. The electrical gradient is related to
transcellular NaCl reabsorption.
The first step is NaCl entry into the cell via
the electroneutral apical Na- K-2Cl co-transporter BSC1 (NKCC2).
Subsequently, most of the potassium recycles back to the lumen, through an apical potassium channel,
necessary to maintain NaCl absorption via BSC1 (NKCC2).
In the absence of recycling, NaCl absorption is inhibited because of
the low availability of potassium in luminal fluid.
In addition, potassium recycling hyperpolarizes the apical membrane.
Chloride exits the cell
across the basolateral membrane
mainly via the CLC-Kb channel,
which depolarizes the basolateral membrane.
The overall consequence is a lumen-positive transepithelial voltage that
drives calcium, magnesium and also sodium through the paracellular pathway.
The pathway permeability for calcium and magnesium requires the presence of a specific protein,
paracellin-1 (also known as claudin-16),
co-expressed with occludin
in the tight junctions of thick ascending limb (TAL) [12].
Inactivating mutations of the paracellin-1 gene cause a specific
decrease in cTAL calcium and magnesium reabsorption and
renal loss of both cations without renal sodium loss,
which is the landmark of an inherited disease referred to as hypercalciuric hypomagnesaemia with nephrocalcinosis [4].
Calcium and magnesium reabsorption in the cTAL is tightly regulated. Micropuncture studies have shown that peptide hormones, such as
PTH,
arginine vasopressin,
calcitonin and
glucagon,
stimulate NaCl as well as calcium and magnesium reabsorption in the loop of Henle and decrease their excretion in final urine. PTH, the most important peptide hormone for the stimulation of renal calcium transport, elicits an increase in calcium and magnesium reabsorption cTAL.
Wittner et al. [14] demonstrated that PTH stimulation of calcium and magnesium transport
involves an increase in paracellular pathway permeability.
The activation of CaSR also affects a number of intracellular events in TAL cells and
modulates transport processes along the cTAL epithelium.
Activating CaSR increases intracellular free calcium concentration in
cTAL,
DCT and
cortical as well as
outer medullary collecting duct.
This also decreases hormone-dependent cAMP accumulation in cTAL by
inhibition of type-6 adenylyl cyclase [20],
increases inositol phosphate formation [21] and
elicits an increase in phospholipase A2 activity and
in intracellular cellular production of 20-hydroxyeicosatetraenoic acid [22]. ….
In conclusion, a large body of evidence supports the view that CaSR is
a major regulator of calcium and magnesium reabsorption in the cTAL and,
of overall tubular divalent cation handling.
However, several issues remain unresolved. It is still unclear whether CaSR activation in the cTAL decreases NaCl reabsorption in this segment or not. The mechanism through which CaSR activation could alter the function of paracellin-1 and the paracellular pathway permeability also remains unsettled. Finally, the role of CaSR in the medullary part of TAL should be investigated: a CaSR-dependent inhibition of NaCl reabsorption could explain at least part of the polyuria that accompanies hypercalcaemic states. (P Houillier and M Paillar. 2003)
Alpha-Kloth and FGf23
Recent advances that have given rise to marked progress in clarifying actions of alpha(α)-Klotho (alpha-Kl) and FGf23 can be summarized as follows ;
(i) α-Kl binds to Na(+), K(+)-ATPase, and Na(+), K(+)-ATPase is recruited to the plasma membrane by a novel α-Kl dependent pathway in correlation with cleavage and secretion of α-Kl in response to extracellular Ca(2+) fluctuation.
(ii) The increased Na(+) gradient created by Na(+), K(+)-ATPase activity drives the transepithelial transport of Ca(2+) in the choroid plexus and the kidney, this is defective in α-kl(-/-) mice.
(iii) The regulated PTH secretion in the parathyroid glands is triggered via recruitment of Na(+), K(+)-ATPase to the cell surface in response to extracellular Ca(2+) concentrations.
(iv) α-Kl, in combination with FGF23, regulates the production of 1,25 (OH) (2)D in the kidney. In this pathway, α-Kl binds to FGF23, and α-Kl converts the canonical FGF receptor 1c to a specific receptor for FGF23, enabling the high affinity binding of FGF23 to the cell surface of the distal convoluted tubule where α-Kl is expressed.
(v) FGF23 signal down-regulates serum phosphate levels, due to decreased NaPi-IIa abundance in the apical membrane of the kidney proximal tubule cells.
(vi) α-Kl in urine increases TRPV5 channel abundance at the luminal cell surface by hydrolyzing the N-linked extracellular sugar residues of TRPV5, resulting in increased Ca(2+) influx from the lumen.
These findings revealed a comprehensive regulatory scheme of mineral homeostasis that is illustrated by the mutually regulated positive/negative feedback actions of α-Kl, FGF23, PTH and 1,25 (OH) (2)D. In this regard, α-Kl and FGF23 might play pivotal roles in mineral metabolism as regulators that integrate calcium and phosphate homeostasis, although this concept requires further verification in the light of related findings. Here, the unveiling of the molecular functions of α-Klotho and FGF23 has recently given new insight into the field of calcium and phosphate homeostasis. Unveiled molecular functions of α-Kl and FGF23 provided answers for several important questions regarding the mechanisms of calcium and phosphate homeostasis that remained to be solved, such as :
(i) what is the non-hormonal regulatory system that directly responds to the fluctuation of extracellular Ca(2+),
(ii) how is Na(+), K(+)-ATPase activity enhanced in response to low calcium stimuli in the parathyroid glands,
(iii) what is the exact role of FGF23 in calcium and phosphorus metabolism,
(iv) how is Ca(2+) influx through TRPV5 controlled in the DCT nephron, and finally
(v) how is calcium homeostasis regulated in cerebrospinal fluid. However, several critical questions still remain to be solved. So far reported,
α-Kl binds to Na(+),
K(+)-ATPase,
FGF receptors and FGF23, and
α-Kl hydrolyzes the sugar moieties of TRPV5.
The following questions are unresolved:
Does alpha-Kl recognize these proteins directly or indirectly?
Is there any common mechanism?
How can we reconcile such diverse functions of alpha-Kl?What is the Ca(2+) sensor machinery and how can we isolate it?
How do hypervitaminosis D and the subsequently altered mineral-ion balance lead to the multiple phenotypes?
What is the phosphate sensor machinery and how can we isolate it?
How does the Fgf23/α-Kl system regulate phosphorus homeostasis?
How are serum concentrations of Ca(2+) and phosphate mutually regulated?
(Nabeshima Y. 2008)
Cilium and Calcium Signal
We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca2+-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP3-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. (Praetorius HA, Spring KR. 2001)
Fgf23 regulation in chronic renal disease
The mechanism of FGF23 action in calcium/phosphorus metabolism of patients with chronic kidney disease (CKD) was studied using a mathematical model and clinical data in a public domain. We have previously built a physiological model that describes interactions of PTH, calcitriol, and FGF23 in mineral metabolism encompassing organs such as bone, intestine, kidney, and parathyroid glands. Since an elevated FGF23 level in serum is a characteristic symptom of CKD patients, we evaluate herein potential metabolic alterations in response to administration of a neutralizing antibody against FGF23. Using the parameters identified from available clinical data, we observed that a transient decrease in the FGF23 level elevated the serum concentrations of PTH, calcitriol, and phosphorus. The model also predicted that the administration reduced a urinary output of phosphorous. This model-based prediction indicated that the therapeutic reduction of FGF23 by the neutralizing antibody did not reduce phosphorus burden of CKD patients and decreased the urinary phosphorous excretion. Thus, the high FGF23 level in CKD patients was predicted to be a failure of FGF23-mediated phosphorous excretion. The results herein indicate that it is necessary to understand the mechanism in CKD in which the level of FGF23 is elevated without effectively regulating phosphorus.
A traditional, physiological model with PTH and calcitriol needs to be rebuilt in accordance with the emerging role of FGF23 and its interacting molecules. To understand probable interactions among FGF23, PTH and calcitriol, we previously developed a minimum physiological model of calcium/phosphorus metabolism and investigated potential influences of FGF23 on the observable state variables such as the serum concentrations of PTH, calcitriol, calcium (Ca), and phosphorous (P), as well as the urinary excretion of Ca and P.3 In this study, we extended the model and evaluated the mechanism of FGF23-mediated regulation in chronic kidney diseases (CKD).
The FGF23 gene was identified by its mutations associated with autosomal dominant hypophosphatemic rickets (ADHR), which is an inherited phosphate wasting disorder.4 Thereafter, a variety of disorders resulting from gain or loss of FGF23 bioactivity have been reported.5 These disorders, which are caused by mutations in the genes that directly or indirectly interact with FGF23, include hyperphosphatemic familial tumoral calcinosis (HFTC), hereditary hypo-phosphatemic rickets with hypercalciuria (HHRH), autosomal recessive hypophosphatemic rickets (ARHR), and X-linked dominant hypophosphatemic rickets (XLH, HYP). CKD patients who need dialysis have very high levels of FGF23 in serum that are linked with increased rates of death.6
We examined the effect of reduction of FGF23 by neutralizing antibody would modulate phosphorus balance of CKD patients. We evaluated the levels of physiological variables such as the levels of PTH, calcitriol, FGF23, Ca, and P in serum as well as urinary outputs of Ca and P using clinical data. Since a glomerular filtration rate (GFR) is a good indicator of severity of CKD, data were processed as a function of GFR. We then employed the previously developed mathematical model for mineral metabolism, and conducted numerical simulations in response to the modulation of FGF23 by neutralizing antibody.
Estimation of the relationship of the FGF23 level to other physiological variables
The FGF23 concentrations, reported in literature, considerably varied among available datasets, presumably caused by differential baseline levels or sensitivity variations among individual assays. To predict a quantitative relationship among the FGF23 level and other physiological variables, the reported FGF23 level was linearly modified:
[FGF23]AB = {[FGF23]-A}/B (1)
in which [FGF23] = reported FGF23 level, [FGF23]AB = linearly modified FGF23 level, and A and B = two correction factors. Note that these correction factors are constant and they were chosen independently for each of the physiological variables such as the serum level of PTH and the urinary output of P. The “+” and “-” values of the factor B indicate positive and negative correlations to the FGF23 level, respectively. We applied the described modification in analyzing clinical data since the observed FGF23 variation was larger than others. Without this procedure, it was difficult to estimate a quantitative relationship of its concentrations to other variables. [With the significant variation around the linear fit, it might well have been warranted to use the log transform of the modified level, LHB].
Mathematical model and prediction of effects of FGF23 antibody
We previously developed a pair of metabolism models of calcium and phosphorus with and without including the predicted action of FGF23.3,20 In this study we considered an additional state variable, GFRf, as a multiplicative term pertaining to the calcium and phosphorus renal thresholds and the kidney production of calcitriol:
GFRf = (GFR/GFR0)k (2)
in which GFR0 and GFR = glomerular filtration rates in the control state and at any given degree of renal failure, respectively, and a factor k (>0) was chosen so as to fit the clinical data as described previously.7
To predict the effects of intravenous administration of a neutralizing antibody against FGF23, we numerically examined 5 different dosages for i.v. administration at 0.003, 0.01, 0.03, 0.1 and 0.3 mg/kg (dosage levels 1–5). These dosages corresponded to a clinical trial study being proposed for a dose-escalation study of KRN23 (Kyowa Hakko Kirin Pharma Inc.). A primary outcome measure of this Phase I clinical trial is a change in a serum phosphate level, and a single dose by intravenous or subcutaneous administration is planned. The initial target is X-linked hypophosphatemia but no clinical data regarding efficacy and side effects are available. To simulate a probable injection procedure, we assumed a form of a single, smoothed-out pulse. The rise in the antibody concentration was modeled using a Gaussian type diffusion profile with a period dependent on the distribution volume and cardiac output.
Glomerular filtration rate (GFR) as an indicator in cKD patients
We plotted physiological variables of CKD patients as a function of GFR in ml/min/1.73 m2. Figure 1 illustrated the levels of PTH (pg/ml), calcitriol (pg/ml), Ca (mg/dl), and P (mg/dl) in serum as well as urinary outputs of Ca and P expressed as a fraction of the glomerular loads. The numbers in the brackets in Figure 1 were the numbers of patients. The average and SEM values were obtained in each of the sampling bins. As GFR was normal above 90, the levels of PTH and P in serum as well as the fractions of urinary Ca and P outputs were lowered. On the contrary, the level of calcitriol in serum was higher as GFR increased.
Estimation of FGF23 levels in serum in cKD patients
The relationships of the linearly modified FGF23 concentration in serum, [FGF23]AB, to the selected physiological variables in CKD patients were illustrated in Figure 2. First, a strong correlation was observed between log.e(GFR) and a negative form of log.e[FGF23]AB, indicating that the FGF23 level was sharply elevated in CKD patients with reduction in GFR. Second, an increase in [FGF23]AB was correlated to the levels of PTH, calcitriol, P in serum, and the renal threshold for P. Note that a positive correlation (i.e. B > 0) was observed for the levels of PTH and P in serum, while a negative correlation (i.e. B < 0) for the serum level of calcitriol and the renal threshold for P. Note that a majority of data points had the PTH level above 50 pg/ml, indicating a poor balance of mineral metabolism in CKD patients.
Linkage of FGF23 and P levels in serum
In all groups, a positive correction was observed between the level of P and the modified level of FGF23 in serum. Note that CKD data in Figure 2D showed the elevated P level up to 6 mg/dl, while the higher bound of the P level was ∼2 mg/dl (Tumor Induced Osteomalacia), 3.5 ∼4 mg/dl (Fibrous Dysplasia and XLH), and 4.5 mg/dl (healthy populations).
Predicted effects of the antibody specific to FGF23
Although the observed increase of FGF23 in CKD is apparently a physiological response to hyperphos-phatemia, the use of FGF23 antibody is suggested for transplanted hypophosphatemic patients of CKD with a high level of FGF23.21 In response to intravenous administration of the antibody specific to FGF23, we evaluated the predicted changes in the serum levels of PTH, calcitriol, and P as well as a normalized urinary output of P. The results were positive.
(Yokota H, Pires A, Raposa JF, Ferreira HG. 2010.)
Overview of renal Ca2+ handling
About 50% of plasma calcium (ionized and complexed form; ultrafilterable fraction, excluding the protein bound form) is freely filtered through the renal glomerulus, and 99% of the filtered calcium is actually reabsorbed along renal tubules (Table 1- see Fig below on right)). The excreted calcium in the final urine is about 200 mg per day in an adult person with an average diet. Several factors are involved in the regulation of calcium in renal tubules. PTH and activated vitamin D enhance calcium reabsorption in the thick ascending limb (TAL), distal convoluted tubule (DCT) and/or connecting tubule (CNT).
Acidosis contributes to hypercalciuria by reducing calcium reabsorption in the proximal tubule (PT) and DCT, and alkalosis vice versa3). Diuretics like thiazide and furosemide also alter calcium absorption in the renal tubules; thiazide promotes calcium reabsorption and furosemide inhibits it. Plasma calcium itself also controls renal calcium absorption through altered PTH secretion as well as via binding to the calcium sensing receptor (CaSR) in the TAL.
To facilitate Ca2+ reabsorption along renal tubules;
(i) voltage difference between the lumen and blood compartment should be favorable for Ca2+ passage, i.e., a positive voltage in the lumen;
(ii) concentration difference should be favorable for Ca2+ passage with a higher Ca2+ concentration in the lumen;
(iii) an active transporter should exist if the voltage or concentration difference is not favorable for Ca2+ reabsorption. Each renal tubular segment has a different Ca2+ concentration difference or voltage environment for its unique mechanism for calcium re-absorption.
Calcium handling along the tubules
Fifty to sixty percent of filtered calcium is absorbed in parallel with sodium and water in the PT, suggesting that the passive pathway is the main route of Ca2+ absorption in this segment. Claudin-2 is especially concentrated in the tight junction and also expressed in the basolateral membrane of the PT as the candidate for paracellular Ca2+ channel in the PT. There is no evidence that Ca2+ reabsorption occurs in the thin descending and ascending limb. In the TAL, 15% of filtered calcium is absorbed, and the passive absorption through paracellular space is known as the main mechanism (Fig. 1). Paracellin-1 (claudin-16) is exclusively expressed in the tight junction of TAL and has been known as the important magnesium channel in the TAL. Paracellin-1 mutation caused hypercalciuria and nephrocalcinosis in addition to hypomagnesemia. This finding supports that paracellin-1 is not only the main Mg2+ channel, but also works as the paracellular Ca2+ channel in the TAL. There are some evidences that active transport occurs in the TAL, but no specific channel has yet been identified). The CaSR is a member of G protein-coupled receptors and suppresses PTH secretion by sensing high plasma Ca2+ level in the parathyroid glands). In the kidney, the CaSR is most highly expressed in the TAL..
Although only 10-15% of filtered Ca2+ is absorbed in the DCT and CNT, these are the main sites in which the fine regulation of Ca2+ excretion and the major action of PTH and activated vitamin D occur. In the DCT and CNT, the luminal voltage is negative and Ca2+ concentration in the lumen is lower than that of plasma. Thus, active transport mechanism against voltage and concentration gradient should exist in these segments. Several Ca2+ transporting proteins are involved in this active transmembrane transport of Ca2+ in the DCT and CNT. Transcellular Ca2+ re-absorption can occur by three steps;
(i) entry of Ca2+ through the calcium channels (TRPV5, TRPV6) in the apical membrane,
(ii) binding of Ca2+ with calcium-binding protein (calbindin) and diffusion in the cytoplasm (which enables no significant change in the intracellular i[Ca2+], and
(iii) Ca2+ extrusion via an ATP-dependent plasma membrane Ca2+-ATPase (PMCA1b) and an Na2+/Ca2+ exchanger (NCX1) in the basolateral membrane (see Fig below on right).
In the collecting duct (CD), there is no evidence that Ca2+ reabsorption occurs even though calcium channel (TRPV6) was documented to be expressed in CD cells.
Each renal tubule has a unique environment and plays a different role in Ca2+ reabsorption.
The coordinated play of different renal tubules could maintain harmony of renal Ca2+ handling.
Transient receptor potential (TRP) channel is a super-family of ion channels permeable to monovalent and/or divalent cations with six-transmembrane domains. The mammalian TRP family consists of six subfamilies like TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin). TRPV is one of them and consists of six members in mammalians; TRPV1 to TRPV6. TRPV5 (previously known as ECaC1) and TRPV6 (ECaC2), both cloned in 1999, have characteristics distinguished from other TRPV channels; (i) constitutively active at low intracellular Ca2+ concentration, and (ii) exclusively selective for Ca2+ (PCa/PNa >100)9). TRPV5 and TRPV6 have the highest sequence homology (~730 amino acids, amino-terminal ankyrin repeats, TM5 and TM6 each forming the pore-region composed with tetramer, on human chromosome 7q34-35) (Fig. 3a). TRPV5 is exclusively expressed in the DCT and CNT in the kidney10) (Fig. 3b). On the contrary, TRPV6 is more ubiquitously distributed, especially in the intestine, and also found from the DCT to the CD in the kidney11) (Fig. 3b). Both TRPV5 and TRPV6 are located in the apical plasma membrane of the tubular epithelium, and serve as the entrance of Ca2+ from the lumen into the cytoplasm. TRPV5 knockout mice exhibited severe hypercalciuria (more than 6 times of wild type mouse) and low bone densities, but without hypocalcemia due to the compensatory elevation of activated vitamin D, clearly demonstrating that TRPV5 plays a crucial role in renal calcium reabsorption12). TRPV6 knockout mice also showed significant hypercalciuria and bone disease13). Even though TRPV5 and TRPV6 knockout mice showed congenital hypercalciuria, the mutation of the proteins has not been found in the human. Until now, TRPV5 is known
as the main entry of Ca2+ in renal tubular epithelial cells in the DCT and CNT, and TRPV6 is also known to contribute to renal Ca2+ reabsorption in the distal nephron.
Several factors (PTH, 1,25(OH)2D3, calcitonin, estrogen, i[Ca2+], acid-base status, klotho, diuretics, and im-munosuppressive drugs, etc) are involved in the regulation of both TRPV5 and TRPV610) (Table 2). Alteration of TRPV5 and TRPV6 by these factors contributes in disturbance of calcium metabolism: dyscalcemia, hypo- and hypercalciuria. 1,25(OH)2D3-depleted rats showed decreased expression of TRPV5 and calbindin-D28K mRNA and protein, and repletion of the hormone restored the expression of them.
TRPV
Transient receptor potential (TRP) channel is a super-family of ion channels permeable to monovalent and/or divalent cations with six-transmembrane domains. The mammalian TRP family consists of six subfamilies like TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin). TRPV is one of them and consists of six members in mammalians; TRPV1 to TRPV6. TRPV5 (previously known as ECaC1) and TRPV6 (ECaC2), both cloned in 1999, have characteristics distinguished from other TRPV channels;
(i) constitutively active at low intracellular Ca2+ concentration, and
(ii) exclusively selective for Ca2+ (PCa/PNa >100)9). TRPV5 and TRPV6 have the highest sequence homology (~730 amino acids, amino-terminal ankyrin repeats, TM5 and TM6 each forming the pore-region composed with tetramer, on human chromosome 7q34-35). TRPV5 is exclusively expressed in the DCT and CNT in the kidney.
On the contrary, TRPV6 is more ubiquitously distributed, especially in the intestine, and also found from the DCT to the CD in the kidney
Both TRPV5 and TRPV6 are located in the apical plasma membrane of the tubular epithelium, and serve as the entrance of Ca2+ from the lumen into the cytoplasm.
TRPV5 knockout mice exhibited severe hypercalciuria (more than 6 times of wild type mouse) and low bone densities, but without hypocalcemia due to the compensatory elevation of activated vitamin D, clearly demonstrating that TRPV5 plays a crucial role in renal calcium reabsorption. TRPV6 knockout mice also showed significant hypercalciuria and bone disease. Even though TRPV5 and TRPV6 knockout mice showed congenital hypercalciuria, the mutation of the proteins has not been found in the human. Until now, TRPV5 is known as the main entry of Ca2+ in renal tubular epithelial cells in the DCT and CNT, and TRPV6 is also known to contribute to renal Ca2+ reabsorption in the distal nephron.
Several factors (PTH, 1,25(OH)2D3, calcitonin, estrogen, i[Ca2+], acid-base status, klotho, diuretics, and im-munosuppressive drugs, etc) are involved in the regulation of both TRPV5 and TRPV6. Alteration of TRPV5 and TRPV6 by these factors contributes in disturbance of calcium metabolism: dyscalcemia, hypo- and hypercalciuria. 1,25(OH)2D3-depleted rats showed decreased expression of TRPV5 and calbindin-D28K mRNA and protein, and repletion of the hormone restored the expression of them.
Table . The regulation of calcium transporting proteins in the DCT and CNT
Factors
TRPV5 TRPV6 Calbindin- Mechanisms
D28K
PTH
+
NC
+
transcription
Vit D
+
+
+
transcription
Estrogen
+
+
+
transcription
Low Ca2+ diet
+
+
NC
transcription
Acidosis
–
ND
–
transcription
Thiazide
C
ND
C
transcription
Furosemide
+
+
+
transcription
Tacrolimus
–
ND
–
transcription
[Ca2+]
–
–
–
Channel activity
Calbindin-D28K
+
NC
Channel activity
Klotho
+
+
ND
trafficking
FGF23
FGF23, a member of the FGF family (type I trans-membrane phosphotyrosine kinase receptors), is a 30 kDa secreted protein and inactivated by cleavage into two smaller fragments (N-terminal 18 kDa fragment and C-terminal 12 kDa fragment) by a pro-convertase enzyme, furin . It was first cloned as the candidate gene for autosomal dominant hypophosphatemic rickets (ADHR). FGF23 is primarily expressed in the osteoblasts and osteocytes. Because Fgf23 knockout mice showed very similar phenotype to Klotho knockout mice including severe hyperphophatemia and osteoporosis, and gain of function mutation of Fgf23 gene was observed in ADHR patients. The main studies about the role of FGF23 in the kidney have focused on phosphate metabolism rather than calcium metabolism.
It is unknown how the FGF23:klotho complex from the DCT acts in the PT because the main action site of FGF23 in the kidney is the PT, whereas the FGF23:klotho complex is most abundant in the DCT. Both overexpression and deficiency of FGF23 cause several clinical diseases including ADHR and HFTC (hyperphosphatemic familial tumorial calcino-sis). Recently, FGF23 was suggested as a potential bio-marker for management of phosphate balance in chronic kidney disease (CKD) patients because the circulating FGF23 level was higher in CKD patients than healthy controls and the increased FGF23 level was an independent risk factor for higher mortality among dialysis patients26). FGF23 also plays some roles in the parathyroid glands and other organs like the choroid plexus, pituitary gland, and bone. However, further studies are needed to clarify the roles and the mechanisms.
Conclusion
The kidney has been known as the central organ for calcium homeostasis through fine regulation of renal calcium excretion. For the past decade, there has been big progress in the understanding of the roles of the kidney in calcium homeostasis. The identification of calcium transport proteins and the molecular approach to the regulatory mechanisms achieved a major contribution to this progress. TRPV5, TRPV6, calbindin-D28K, NCX1, and PMCA1b have been identified as the main calcium transport proteins in the distal nephron. PTH, vitamin D, i[Ca2+], CaSR, and other various conditions control renal calcium excretion through the regulation of these transport proteins. Klotho and FGF23 emerged as new players in calcium metabolism in the kidney. Thus, the role of the klotho-FGF23 axis in the regulatory mechanisms of calcium transport needs to be addressed.
Disorders of Calcium, Phosphorus and Magnesium Metabolism
Infrequently patients might present in the outpatient settings with non-specific symptoms that might be due to abnormalities of divalent cation (magnesium, calcium) or phosphorous metabolism. Several inherited disorders have been identified that result in renal or intestinal wasting of these elements. Physicians need to have a thorough understanding of the mechanism of calcium, magnesium and phosphorous metabolism and diagnoses disorders due to excess or deficiency of these elements. Prompt identification and treatment of the underlying disorders result in prevention of serious morbidity and mortality.
Maintenance of serum calcium in the extra cellular fluid space (ECF) is tightly regulated. Most calcium (around 99%) is bound and complexed in the bones. Calcium in the ECF is found in three fractions, of which 45% is in biological ionized fraction, 45% is protein bound and not filterable in the kidney and 10% is complexed with anions such as bicarbonate, citrate, phosphate, and lactate (Fig. 1 ). Most of the protein bound calcium is complexed with albumin, and a smaller amount to globulin. Each 1 g/dL of albumin binds 0.8 mg/dL (0.2 mmol/L) calcium. Hence, for each 1g/ dl decrease in serum albumin below normal value of 4.0 g/dl, one needs to add 0.8 mg/ dl to the measured serum calcium. Levels of calcium are also influenced by acid-base status, with acidosis increasing serum calcium while alkalosis decreases serum calcium levels.
Maintenance of normal calcium in ECF is dependent on fluxes of calcium between the intestine, kidneys and bone. The regulation of calcium in serum is regulated by calcium itself, through a calcium sensing receptor (Ca RG) and hormones like parathormone (PTH) and 1, 25-dihydroxyvitamin D3.
Calcium transport across the intestine occurs in two directions, absorption and secretion. The factors that influence calcium absorption in the intestine include daily amount of calcium that is ingested and 1, 25-dihydroxyvitamin D3 that binds to and activates the Vitamin D receptor (VDR) and induces the expression of calcium channel TRPV6, calbindin- D9K, and Ca2+ – ATPase. Other hormones like PTH, estrogen, prolactin and growth hormone may play a minor role in calcium absorption. Conditions that result in decreased intestinal calcium transport include high vegetable fiber and fat content of food, corticosteroid deficiency, estrogen deficiency, advanced age, gastrectomy, intestinal malabsorption, diabetes mellitus, renal failure and low Ca2+ phosphate ratio in the food.
PTH and 1, 25- dihydroxyvitamin D3 stimulate osteoclasts in bones and promote release of calcium in ECF. PTH promotes hydroxylation of 25(OH) D3 to 1, 25(OH) D3 and distal tubular calcium reabsorption.
Hypocalcaemia occurs when the loss of calcium from the ECF via renal excretion is greater than influx of Ca 2+ from intestine or bones. One of the commonest cause of low calcium is hypoalbuminemia, though the level of ionized Ca2+ is normal. The causes of hypocalcaemia is summarized in Table 1 . Acute hypocalcaemia is often seen in acute respiratory alkalosis due to hyperventilation. Idiopathic or acquired (post surgery, radiotherapy) hypoparathyroid states are usually accompanied with elevated phosphate level. Pseudo hypoparathyroidism is characterized by short neck, round face and short metacarpal and results from end-organ resistance to PTH. Chronic kidney disease and massive phosphate administration can result in hypocalcaemia with high serum phosphate levels. Familial hypocalcaemia is linked with activating mutation of Ca RG. Hypocalcaemia with low phosphate levels occur in Vitamin D deficiency, resistance to calcitriol (Type 2 vitamin D- dependent rickets) acute pancreatitis and magnesium deficiency.
Table 1 : Causes of Hypocalcemia
Idiopathic Hypoparathyroidism
Post parathyroidectomy (Hungry bones syndrome)
Pseudo-hypoparathyroidism
Familial hypocalcemia
Rapid correction of severe acidosis with dialysis
Acute respiratory and metabolic alkalosis
Acute pancreatitis
Rhabdomyolysis
Hypomagnesemia
Septic shock
Ethylene glycol toxicity
Vitamin D deficiency
Chronic kidney disease
Massive transfusion- Citrate toxicity
Hypercalcemia occurs when in influx of calcium into the ECF exceeds the efflux of calcium from intestine and kidneys. The normal calcium level ranges from 8.9- 10.1 mg/ dL. The range of serum calcium levels in mild hypercalcemia is (10.1- 12.0 mg/dL), moderate hypercalcemia (12.0 – 14.0 mg/dl) and severe hypercalcemia > 14.0 mg/ dL respectively. The various causes of hypercalcemia is depicted in Table 2. Mutation of the gene for Ca RG results in hypercalcemia in few cases.
Table 2. : Causes of hypercalcemia Parathormone Primary hyperparathyroidism
(PTH) mediated Lithium induced
Familial hypocalciuric hypercalcemia
Tertiary hyperparathyroidism
Cancer Multiple myeloma
PTHrp mediated-Breast, lung,
Exogenous Vitamin D
Dialysis patients (exogenous Vit D)
Other causes Vitamin A toxicity
Thyrotoxicosis
Paget’s disease
Adrenal insufficiency
Thiazide use
Deficiency of calcium, magnesium and phosphorous are common in general practice. A thorough understanding of pathophysiology of these elements, common dietary sources of these elements and pharmacological measures that might be necessary to correct these deficiencies could guide the physician to make an accurate diagnosis, initiate appropriate treatment and prevent future recurrences. (Ghosh AK*, Joshi SR. 2008.)
Renal Disease and the Cardiovascular System
Cardiovascular disease is a leading cause of death among patients with end stage renal failure. Animal models have played a crucial role in teasing apart the complex pathological processes involved. In addition to the anatomical and histological characteristics humans share with other species, human diseases can be reproduced in these species using pharmacological, surgical or genetic manipulation. Experimentation still provides the best evidence for disease causation, and only with this evidence can clinical science proceed to developing treatments. However, experimentation is often not possible or ethical in human subjects, and thus without these animal models the advancement in knowledge of the patho-physiology of disease would come to a standstill.
The way in which kidneys succumb to disease and the development of renal failure involves complex interactions between numerous different systems, mediated by a multitude of chemicals. Current understanding of renal disease is merely the tip of the metaphorical iceberg. The history of renal pathology is plagued by controversy, and nowhere is this more evident than in the development of cardiovascular disease in patients with chronic renal failure. Impairment of renal function increases the risk of cardiac disease to 15-20 times that of individuals with normal renal function. The result is that cardiac disease causes 40% of deaths in patients on dialysis.
This review discusses the principles of using animal models, the history of their use in the study of renal hypertension, the controversies arising from experimental models of non-hypertensive uraemic cardiomyopathy and the lessons learned from these models, and highlights important areas of future research in this field, including de novo cardiomyopathy secondary to renal transplantation.
Myocardial Interstitial Fibrosis, Cardiac Compliance and Vascular Architecture
Using subtotally nephrectomised Sprague-Dawley rats, Mall et al. showed that the increase in total heart weight demonstrated by Rambausek et al. after 21 days of uremia (as well as an increase in both right and left ventricular weight) was secondary to an increase in true interstitial volume, both cellular and non-cellular, with increased deposition of collagen. This was associated with activated interstitial cells, and a reduced capillary cross-sectional area. In 1992, this latter point was confirmed using stereological techniques to analyse perfusion-fixed hearts of subtotally nephrectomised Sprague-Dawley rats. Uremia resulted in increased blood pressure and reduced capillary length per unit myocardial volume, as well as reduced capillary luminal surface density and volume density, compared to control rats. The same group found a blood pressure-independent increase in the wall to lumen ratio of intramyocardial arteries, and in the aorta media thickness of subtotally nephrectomised rats. The intramyocardial arterial wall thickening has been found to be due to hypertrophy rather than hyperplasia, independent of blood pressure. These architectural changes were reported again in 1996. In that experiment, nephrectomised Sprague-Dawley rats were given ramipril, nifedipine or moxonidine to normalise blood pressure; these drugs had differential effects on the above architectural changes, and also acted to prevent these changes. The different changes in interstitial and capillary density in uremic cardiomyopathy have not yet been explained, but the role of growth factors such as basic fibroblast growth factor (BFGF) and vascular endothelial growth factor (VEGF) has been proposed.
Cardiac Function and Energetics in Uremia
The above experiments provided some insight into the structural changes seen in uraemic hearts. They were followed by a study using the subtotal (5/6) nephrectomy model on Wistar rats, in which the authors focused on the mechanical effects of these structural changes in vitro, thereby removing neurohormonal influences on cardiac contractility. Four weeks after surgery, isolated perfusing working heart preparations demonstrated reduced cardiac output. However, blood pressure was not controlled during the four weeks post-operatively, and could have contributed to the effects. An increased susceptibility to ischemic damage was also shown via decreased phosphocreatine content, and an increased release of inosine (a marker of ischaemic damage). These hearts failed in response to increases in calcium; the authors proposed that impaired cytosolic calcium control played a role in the relationship between renal failure and impaired cardiac function.
This in vitro experiment demonstrated the fact that impaired cardiac function was independent of circulating urea and creatinine, as the hearts were perfused with physiological saline, with no effect from the addition of urea and creatinine. The opposite has been shown in spontaneously beating mouse cardiac myocytes, in response to sera from patients on haemodialysis for chronic renal failure. Urea, creatinine, and combinations of the two reduced the cardiac inotropy and resulted in arrhythmias and asynchronies.
These experiments make a good case for uremic cardiomyopathy to be a distinct entity from hypertensive cardiac dysfunction and atherosclerotic cardiac disease secondary to the risk factors common to both heart and kidney disease. The cause of this phenomenon is still controversial, with parathyroid hormone (PTH), angiotensin II, marino-bufagenin (MBG), oxidative stress, and growth hormone.
The Role of Calcium in Uremic Cardiomyopathy
Calcium ions play a crucial role in cardiac physiology, particularly in myocardial excitation-contraction coupling. Therefore, PTH was one of the first culprits to be suspected of playing a role in the pathophysiology of uremic cardiomyopathy; this was as early as 1984. As reviewed by Rostand and Drüeke, there are numerous theories pertaining to the mechanisms whereby PTH could act as an intermediary between renal impairment and cardiomyopathy. These include
direct trophic effects on myocytes
interstitial fibroblasts,
indirect effects via anaemia or large and small vessel changes.
Rostand and Drüeke suggest an increase in blood pressure via hypercalcemia, but the effects on the heart appear to be independent of blood pressure.
Rambausek et al. noted increased cardiac calcium content in experimental rats, and that an increase in heart weight still occurred after parathyroidectomy with calcium supplementation. This was followed in the 1990s by in vitro experiments that demonstrated; an increased cytosolic calcium concentration in isolated rat myocytes in response to PTH, a reduced expression of PTH-related peptide receptor mRNA in rat hearts secondary to hyperparathyroidism due to chronic renal failure, and increased force and frequency of contraction of isolated, beating rat cardiomyocytes.
Subsequent to “chance observations” in the laboratory, Amann et al. argued for the role of PTH in the wall thickening of intramyocardial arterioles and for fibroblast activation and subsequent cardiac fibrosis. Abolishing hyperparathyroidism prevented the cardiac fibrosis and capillary changes normally seen in nephrectomised rats, which was independent of blood pressure.
The Renin-Angiotensin System (RAS) and Endothelin
Many studies have highlighted the importance of the RAS in the development of uremic cardiomyopathy. Tornig et al. showed that in nephrectomised rats, ramipril, an ACE inhibitor, prevented the increased wall thickness of the intramyocardial arterioles, as well as the expansion of nonvascular cardiac interstitial volume and the aortic wall and lumen changes, but not the reduced capillary length density. The same group subsequently repeated these observations, and demonstrated that the beneficial effects of ramipril were prevented by the use of specific bradykinin B2 receptor antagonists, suggesting a role for increased bradykinin as a mediator for the effects of ramipril.
CONCLUSIONS
Experimental models have played a crucial role in the study of the complex interplay between the heart and the kidney in chronic renal disease. In view of the numerous differences in animal and human anatomy, physiology and pathology, the results of these experiments should be interpreted with caution, but in some areas, these studies have led directly to advances in therapeutics.
(RC Grossman. 2010.)
Deficiency of the Calcium-Sensing Receptor
Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)–dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na+-K+-2Cl cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca2+ transport. (Toka HR, Al-Romaih K, Koshy JM, DiBartolo, III S, et al. 2012)
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Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
andArticleCurator: Aviva Lev-Ari, PhD, RN
Image created by Adina Hazan 06/30/2021
Voice of Justin Pearlman, MD, PhD, FACC
Catechols refer to the stress hormones that control our response to fright, flight and fight, e.g., epinephrine, also known as adrenaline. Sudden elevation of catechols increases heart rate and also the strength of heart contraction (contractility). In the short term, that provides a boost that supports special demands to run faster, work harder. Like the healthcare system, it is not sustainable in high gear. Excess catechol push causes heart failure (catechol toxicity). Race horses routinely develop pulmonary edema by the end of a race – those pretreated for that with the diuretic LASIX have an L next to their entry in the race ticket. The same issues occur as a whole-body system and at the subcellular level. Catechols increase amount and speed of the release of calcium which in turn triggers heart muscle contraction. However, the failing heart has elevated levels of calcium that impair oxygen utilization. The following discussions address the linkages between catechols and calcium traffic, including both the catechol and calcium stimulation of speed and strength, and their detrimental effects over time.
This article is Part VII in a continuation to the following article series on tightly related topics of the Calcium Release Mechanism.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Introduction to Calcium Release Mechanism in Vascular Smooth Muscle and in Cardiomyocytes
Author: Justin D Pearlman, MD, PhD, FACC PENDING
I. Cellular Contractility Capacity — Actin, Cellular Dynamics and Calcium Efflux: Emergence of the Calcium Release-related Contractile Dysfunction
Author: Justin D Pearlman, MD, PhD, FACC
II. Integration and Interpretation of Research Results in Two Labs: Mark E Anderson’s and Roger Hajjar’s Lab
Author: Justin D Pearlman, MD, PhD, FACC PENDING
Mark Anderson’s Laboratory at the University of Iowa Carver College of Medicine recently summarized the critical roles of calcium in heart failure and arrhythmia in an article in Circulation Research. That laboratory elucidated critical facts, such as the controlling role of phosphorylation of ryanodine receptors among other details of the control and impact of Ca²⁺ homeostatic and structural proteins, ion channels, and enzymes. Their review focuses on the molecular mechanisms of defective Ca²⁺ cycling in heart failure and knowledge of those pathways may translate into new innovative therapies. The highly conserved Ca2+/calmodulin-dependent protein kinase II (CaMKII)plays an essential role in cardiac myocytes. Electrichemical activation of the cariac contraction cycle triggers a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) which activates CaMKII activated through the binding of Ca2+-bound calmodulin (CaM). The activated CaMKII molecules phosphorylate many intracellular target proteins, including the sarcolemmal L-type Ca2+ channel, the ryanodine receptor, and the Ca2+ pump on the sarcoplasmic reticulum. Intersubunit autophosphorylation (positive feedback) promotes accumulation of the active CaMKII. Phosphorylated CaMKII maintains its catalytic activity until it is inactivated by constitutive phosphatase activity.
Roger J. Hajjar MD is the Director of the Cardiovascular Research Center, a cutting-edge translational research laboratory at Mt Sinai Medical Center. He is the Arthur & Janet C. Ross Professor of Medicine, Professor of Gene & Cell Medicine, Director of the Cardiology Fellowship Program, and Co-Director of the Transatlantic Cardiovascular Research Center, which combines Mount Sinai Cardiology Laboratories with those of the Universite de Paris – Madame Curie. He earned a bachelors of science degree in Biomedical Engineering at Johns Hopkins University and a medical degree from Harvard Medical School and the Harvard-MIT Division of Health Sciences and Technology. He completed his fellowship in cardiology at Massachusetts General Hospital in Boston, then became a staff cardiologist in the Heart Failure & Cardiac Transplantation Center, followed by Director of the Cardiovascular Laboratory of Integrative Physiology and Imaging, before moving to Mt. Sinai.
Roger J. Hajjar, MD and his team of investigators translate scientific findings into therapies for cardiovascular diseases. Dr. Hajjar’s team pioneered a potential gene therapy for heart failure, AAV1.SERCA2a, which can revive malfunctioning myocardium. His laboratory has completed Phase 1 and Phase 2 First-in-Man clinical trials of SERCA2a gene transfer in patients with advanced heart failure, and Phase 3 validation began in 2011. His laboratory also studies how to block signaling pathways in cardiac hypertrophy, aging, apoptosis, and diastolic failure.
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
2013
•He BJ, Anderson ME. Aldosterone and Cardiovascular Disease: the heart of the matter. Trends in Endocrinology & Metabolism 24(1):21-30, 2013. [PMID: 23040074]
•Luo M, Anderson ME, Mechanisms of altered Ca2+ handling in heart failure. Circ Res 113(6):690-708. 2013 [PMID: 23989713]
•Anderson ME. Why has it taken so long to learn what we still don’t know? Circ Res 113(7):840-2. 2013 [PMID: 24030016]
•Thomas C, Anderson ME. In memoriam: John B. Stokes, MD. Semin Nephrol. 33(3):207-8, 2013. [PMID: 23953797]
•Gyorke S, Ho HT, Anderson ME, et al. Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides. Cardiovas Res [PMID: Accepted for publication]
•Kline J, Anderson ME, et al, βIV-spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells. Proc Natl Acad Sci. [PMID: Accepted for publication]
•Maier LS, Sag C, Anderson ME, Ionizing Radiation Regulates Cardiac Ca handling via increased ROS and activated CaMKII. Bas Res in Card [PMID: Accepted for publication]
•Chen B, Guo A, Zhang C, Chen R, Zhu Y, Hong J, Kutschke W, Zimmerman K, Weiss RM, Zingman L, Anderson ME, Wehrens XH, Song LS. Critical roles of Junctophilin-2 T-tubule and excitation-contraction coupling maturation during postnatal development. Cardiovas Res 2013 Oct 1; 100(1):54-62. [PMID: 23860812] [PMC3778961]
•Purohit A, Rokita AG, Xiaoqun G, Biyi C, Koval OM, Voigt N, Neef S, Sowa T, Gao Z, Luczak E, Stefansdottir H, Behunin AC, Li N, El-Accaoui RN, Yang B, Swaminathan PD, Weiss RM, Wehrens XH, Song LS, Dobrev D, Maier LS, Anderson ME. Oxidized CaMKII Triggers Atrial Fibrillation. Circulation 2013 Sep 12 [Epub ahead of print] [PMID: 24030498]
•Yoshida-Moriguchi T, Willer T, Anderson ME, Venzke D, Whyte T, Muntoni F, Lee H, Nelson SF, Yu L, Campbell, KP. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function. Science 2013 Aug 23; 341(6148): 896-9. [PMID:23929950]
•Scott JA, Klutho PJ, El Accaoui R, Nguyen E, Venema AN, Xie L, Jiang S, Dibbern M, Scroggins S, Prasad AM, Luczak ED, Davis MK, Li W, Guan X, Backs J, Schlueter AJ, Weiss RM, Miller FJ, Anderson ME, Grumbach IM. The Multifunctional Ca2+/Calmodulin-Dependent Kinase IIδ (CaMKIIδ) Regulates Arteriogenesis in a Mouse Model of Flow-Mediated Remodeling. PLoS One 2013 Aug 8; 8(8):e71550. [PMID: 23951185] [PMC3738514]
•Scholten A, Preisinger C, Corradini E, Bourgonje VJ, Hennrick ML, van Veen TA, Swaminathan PD, Joiner ML, Vos MA, Anderson ME, Heck AJ. A Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin Dependent Protein Kinase II Regulation in the Heart. J Am Heart Assoc 2013 Aug 7; 2(4):e000318. [PMID: 23926118]
•Prasad AM, Nuno DW, Koval OM, Ketsawatsomkron P, Li W, Li H, Shen Y, Joiner ML, Kutschke W, Weiss RM, Sigmund CD, Anderson ME, Lamping KG, Grumbach IM. Differential Control of Calcium Homeostatis and Vascular Reactivity by Ca2+/Calmodulin-Dependent Kinase II. Hypertension 2013 Aug; 62(2):434-41.[PMID:23753415]
•Sanders PN, Koval OM, Jaffer OA, Prasad AM, Businga TR, Scott JA, Hayden PJ, Luczak ED, Dickey DD, Allamargot C, Olivier AK, Meyerholz DK, Robison AJ, Winder DG, Blackwell TS, Dworski R, Sammut D, Wagner BA, Buettner GR, Pope MR, Miller FJ, Dibbern ME, Haitchi HM, Mohler PJ, Howarth PH, Zabner J, Kline JN, Grumbach IM, Anderson ME. CaMKII is Essential for the Proasthmatic Effects of Oxidation. Sci Trans Med 2013 Jul 24; 5(195):195 ra97. [PMID: 23884469] Chosen as a “From the Cover” article in STM and with a commentary in JAMA. 310(9):894. doi: 10.1001/jama.2013.277035
•Wolf RM, Glynn P, Hashemi S, Zarei K, Mitchell CC, Anderson ME, Mohler PJ, Hund TJ. Atrial fibrillation and sinus node dysfunction in human ankyrin-B syndrome: A computational analysis. Am J Physiol Heart and Circ Physiol 2013 May; 304(9):H1253-66. [PMID: 23436330] [PMC3652094]
•Ather S, Wang W, Wang Q, Li N, Anderson ME, Wehrens XH. Inhibition of CaMKII Phosphorylation of RyR2 Prevents Inducible Ventricular Arrhythmias in Mice with Duchenne Muscular Dystrophy. Heart Rhythm 2013 Apr; (10)4:592-9 [PMID: 23246599] [PMC3605194]
•Yang J, Maity B, Huang J, Gao Z, Stewart A, Weiss RM, Anderson ME, Fisher RA. G- protein inactivator RGS6 mediates myocardial cell apoptosis and cardiomyopathy caused by doxorubicin. Cancer Res 2013 Mar 15; 73(6): 1662-7. [PMID: 23338613] [PMC3602152]
•Luo M, Guan X, Luczak ED, Di L, Kutschke W, Gao Z, Yang J, Glynn P , Sossalla S, Swaminathan PD, Weiss RM, Yang B, Rokita AG,5, Maier LS, Efimov I, Hund TJ, Anderson ME. Diabetes increases mortality after myocardial infarction by oxidizing CaMKII. J Clin Invest 2013 Mar 1; 123(3):1262-74. [PMID: 23426181] [ PMC3673230]
•Sierra A, Zhu Z, Sapay N, Sharotri V, Kline CF, Luczak ED, Subbotina E, Sivaprasadarao A, Snyder PM, Mohler PJ, Anderson ME, Vivaudou M, Zingman LV, Hodgson-Zingman DM. Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II. J Biol Chem 2013 Jan 18; 288(3):1568-81. [PMID: 23223335] [PMC3548467]
•Gao Z, Rasmussen TP, Li Y , Kutschke W , Koval OM, Wu Y, Wu Y, Hall DD, Joiner ML, Wu XQ, Swaminathan PD, Purohit A, Zimmerman KA, Weiss RM, Philipson K , Song LS, Hund TJ, Anderson ME. Genetic inhibition of Na+-Ca2+ exchanger current disables fight or flight sinoatrial node activity without affecting resting heart rate. Circ Res 2013 Jan 18;112(2):309-17. [PMID: 23192947][Epub: e157-e179] [PMC3562595]
•Degrande ST, Little S, Nixon DJ, Wright P, Snyder J, Dun W, Murphy N, Kilic A, Higgins R, Binkley PF, Boyden PA, Carnes CA, Anderson ME, Hund TJ, Mohler PJ. Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart. J Biol Chem 2013 Jan 11; 288(2):1032-46. [PMID: 23204520] [PMC3542989]
•He BJ, Anderson ME. Aldosterone and Cardiovascular Disease: the heart of the matter. Trends in Endocrinology & Metabolism 24(1):21-30, 2013. [PMID: 23040074]
• Luo M, Anderson ME, Mechanisms of altered Ca2+ handling in heart failure. Circ Res 113(6):690-708. 2013 [PMID: 23989713]
•Anderson ME. Why has it taken so long to learn what we still don’t know? Circ Res 113(7):840-2. 2013 [PMID: 24030016]
• Thomas C, Anderson ME. In memoriam: John B. Stokes, MD. Semin Nephrol. 33(3):207-8, 2013. [PMID: 23953797]
2012
•Wang Y and Anderson ME. Chapter 22: Intracellular Signaling Pathways in Cardiac Remodeling. Muscle: Fundamental Biology and Mechanisms of Disease. J. Hill and E. Olson (Eds), Elsevier, pp 299-308, 2012.
• Ather S, Wang W, Wang Q, Li N, Anderson ME, Wehrens XH. Inhibition of CaMKII Phosphorylation of RyR2 Prevents Inducible Ventricular Arrhythmiasin Mice with Duchenne Muscular Dystrophy. Heart Rhythm. 2012 Dec 11. doi:pii: S1547-5271(12)01450-6. 10.1016/j.hrthm.2012.12.016. PubMed PMID: 23246599.
• Sierra A, Zhu Z, Sapay N, Sharotri V, Kline CF, Luczak ED, Subbotina E, Sivaprasadarao A, Snyder PM, Mohler PJ, Anderson ME, Vivaudou M, Zingman LV, Hodgson-Zingman DM. Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II. J Biol Chem. 2012 Dec 6. [Epub ahead of print] PubMed PMID: 23223335.
• Degrande S, Nixon D, Koval O, Curran JW, Wright P, Wang Q, Kashef F, Chiang D, Li N, Wehrens XH, Anderson ME, Hund TJ, Mohler PJ. CaMKII inhibition rescues proarrhythmic phenotypes in the model of human ankyrin-B syndrome. Heart Rhythm. 2012 Dec;9(12):2034-41. doi: 10.1016/j.hrthm.2012.08.026. Epub 2012 Aug 28. PubMed PMID: 23059182.
• Degrande ST, Little S, Nixon DJ, Wright P, Snyder J, Dun W, Murphy N, Kilic A, Higgins R, Binkley PF, Boyden PA, Carnes CA, Anderson ME, Hund TJ, Mohler PJ. Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart. J Biol Chem. 2012 Nov 30. [Epub ahead of print] PubMed PMID: 23204520.
• Gao Z, Rasmussen TP, Li Y, Kutschke W, Koval OM, Wu Y, Wu Y, Hall DD, Joiner ML, Wu X, Dominic Swaminathan P, Purohit A, Zimmerman KA, Weiss RM, Philipson K, Song LS, Hund TJ, Anderson ME. Genetic Inhibition of Na+-Ca2+ Exchanger Current Disables Fight or Flight Sinoatrial Node Activity Without Affecting Resting Heart Rate. Circ Res. 2012 Nov 27. PubMed PMID: 23192947
• Joiner ML, Koval OM, Li J, He BJ, Allamargot C, Gao Z, Luczak ED, Hall DD, Fink BD, Chen B, Yang J, Moore SA, Scholz TD, Strack S, Mohler PJ, Sivitz WI, Song LS, Anderson ME. CaMKII determines mitochondrial stress responses in heart. Nature. 2012 Nov 8;491(7423):269-73. doi: 10.1038/nature11444. Epub 2012 Oct 10. PubMed PMID: 23051746; PubMed Central PMCID: PMC3471377.
• Rokita AG, Anderson ME. New therapeutic targets in cardiology: arrhythmias and Ca2+/calmodulin-dependent kinase II (CaMKII). Circulation. 2012 Oct 23;126(17):2125-39. doi: 10.1161/CIRCULATIONAHA.112.124990. Review. PubMed PMID: 23091085; PubMed Central PMCID: PMC3532717.
• Koval OM, Snyder JS, Wolf RM, Pavlovicz RE, Glynn P, Curran J, Leymaster ND, Dun W, Wright PJ, Cardona N, Qian L, Mitchell CC, Boyden PA, Binkley PF, Li C, Anderson ME, Mohler PJ, Hund TJ. Ca2+/calmodulin-dependent protein kinase II-based regulation of voltage-gated Na+ channel in cardiac disease. Circulation. 2012 Oct 23;126(17):2084-94. doi: 10.1161/CIRCULATIONAHA.112.105320. Epub 2012Sep 24. PubMed PMID: 23008441.
• Wagner S, Rokita AG, Anderson ME, Maier LS. Redox Regulation of Sodium and Calcium Handling. Antioxid Redox Signal. 2012 Oct 3. [Epub ahead of print] PubMed PMID: 22900788.
• Wu Y, Luczak ED, Lee EJ, Hidalgo C, Yang J, Gao Z, Li J, Wehrens XH, Granzier H, Anderson ME. CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban. J Mol Cell Cardiol. 2012 Sep;53(3):429-36. doi: 10.1016/j.yjmcc.2012.06.019. Epub 2012 Jul 11. PubMed PMID: 22796260.
• Majumdar S, Anderson ME, Xu CR, Yakovleva TV, Gu LC, Malefyt TR, Siahaan TJ. Methotrexate (MTX)-cIBR conjugate for targeting MTX to leukocytes: conjugate stability and in vivo efficacy in suppressing rheumatoid arthritis. J Pharm Sci. 2012 Sep;101(9):3275-91. doi: 10.1002/jps.23164. Epub 2012 Apr 26. PubMed PMID: 22539217.
• Kashef F, Li J, Wright P, Snyder J, Suliman F, Kilic A, Higgins RS, Anderson ME, Binkley PF, Hund TJ, Mohler PJ. Ankyrin-B protein in heart failure: identification of a new component of metazoan cardioprotection. J Biol Chem. 2012 Aug 31;287(36):30268-81. doi: 10.1074/jbc.M112.368415. Epub 2012 Jul 9. PubMed PMID: 22778271; PubMed Central PMCID: PMC3436279.
• Chen B, Guo A, Gao Z, Wei S, Xie YP, Chen SR, Anderson ME, Song LS. In situ confocal imaging in intact heart reveals stress-induced Ca(2+) release variability in a murine catecholaminergic polymorphic ventricular tachycardia model of type 2 ryanodine receptor(R4496C+/-) mutation. Circ Arrhythm Electrophysiol. 2012 Aug 1;5(4):841-9. doi: 10.1161/CIRCEP.111.969733. Epub 2012 Jun 21. PubMed PMID: 22722659; PubMed Central PMCID: PMC3421047.
• Swaminathan PD, Purohit A, Hund TJ, Anderson ME. Calmodulin-dependent protein kinase II: linking heart failure and arrhythmias. Circ Res. 2012 Jun 8;110(12):1661-77. doi: 10.1161/CIRCRESAHA.111.243956. Review. PubMed PMID: 22679140.
• Chen B, Li Y, Jiang S, Xie YP, Guo A, Kutschke W, Zimmerman K, Weiss RM, Miller FJ, Anderson ME, Song LS. β-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction. FASEB J. 2012 Jun;26(6):2531-7. doi: 10.1096/fj.11-199505. Epub 2012 Feb 28. PubMed PMID: 22375019; PubMed Central PMCID: PMC3360148.
• Scott JA, Xie L, Li H, Li W, He JB, Sanders PN, Carter AB, Backs J, Anderson ME, Grumbach IM. The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9. Am J Physiol Heart Circ Physiol. 2012 May 15;302(10):H1953-64. doi: 10.1152/ajpheart.00978.2011. Epub 2012 Mar 16. PubMed PMID: 22427508; PubMed Central PMCID: PMC3362103.
• Gudmundsson H, Curran J, Kashef F, Snyder JS, Smith SA, Vargas-Pinto P, Bonilla IM, Weiss RM, Anderson ME, Binkley P, Felder RB, Carnes CA, Band H, Hund TJ, Mohler PJ. Differential regulation of EHD3 in human and mammalian heart failure. J Mol Cell Cardiol. 2012 May;52(5):1183-90. doi: 10.1016/j.yjmcc.2012.02.008. Epub 2012 Mar 3. PubMed PMID: 22406195; PubMed Central PMCID: PMC3360944.
• Singh MV, Swaminathan PD, Luczak ED, Kutschke W, Weiss RM, Anderson ME. MyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction. J Mol Cell Cardiol. 2012 May;52(5):1135-44. doi: 10.1016/j.yjmcc.2012.01.021. Epub 2012 Feb 3. PubMed PMID: 22326848; PubMed Central PMCID: PMC3327770.
• Qian H, Matt L, Zhang M, Nguyen M, Patriarchi T, Koval OM, Anderson ME, He K, Lee HK, Hell JW. β2-Adrenergic receptor supports prolonged theta tetanus-induced LTP. J Neurophysiol. 2012 May;107(10):2703-12. doi: 10.1152/jn.00374.2011. Epub 2012 Feb 15. PubMed PMID: 22338020; PubMed Central PMCID: PMC3362273.
2011
• Xie YP, Chen B, Sanders P, Guo A, Li Y, Zimmerman K, Wang LC, Weiss RM, Grumbach IM, Anderson ME, Song LS. Sildenafil Prevents and Reverses Transverse-Tubule Remodeling and Ca2+ Handling Dysfunction in Right Ventricle Failure Induced by Pulmonary Artery Hypertension. Hypertension. 2011 Dec 27.[Epub ahead of print] PubMed PMID: 22203744.
•He BJ, Joiner ML, Singh MV, Luczak ED, Swaminathan PD, Koval OM, Kutschke W, Allamargot C, Yang J, Guan X, Zimmerman K, Grumbach IM, Weiss RM, Spitz DR, Sigmund CD, Blankesteijn WM, Heymans S, Mohler PJ, Anderson ME. Oxidation of CaMKII determines the cardiotoxic effects of aldosterone. Nat Med. 2011 Nov 13;17(12):1610-8. doi: 10.1038/nm.2506. PubMed PMID: 22081025.
• Zhu Z, Burnett CM, Maksymov G, Stepniak E, Sierra A, Subbotina E, Anderson ME, Coetzee WA, Hodgson-Zingman DM, Zingman LV. Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia. Biochem Biophys Res Commun. 2011 Dec 2;415(4):637-41. Epub 2011 Nov 3. PubMed PMID: 22079630; PubMed Central PMCID: PMC3230708.
•Albert CM, Chen PS, Anderson ME, Cain ME, Fishman GI, Narayan SM, Olgin JE, Spooner PM, Stevenson WG, Van Wagoner DR, Packer DL; Heart Rhythm Society Research Task Force. Full report from the first annual Heart Rhythm Society Research Forum: a vision for our research future, “dream, discover, develop, deliver”. Heart Rhythm. 2011 Dec;8(12):e1-12. Epub 2011 Nov 7. PubMed PMID: 22079558.
•Cunha SR, Hund TJ, Hashemi S, Voigt N, Li N, Wright P, Koval O, Li J, Gudmundsson H, Gumina RJ, Karck M, Schott JJ, Probst V, Le Marec H, Anderson ME, Dobrev D, Wehrens XH, Mohler PJ. Defects in ankyrin-based membrane protein targeting pathways underlie atrial fibrillation. Circulation. 2011 Sep 13;124(11):1212-22. Epub 2011 Aug 22. PubMed PMID: 21859974; PubMed Central PMCID: PMC3211046.
•Sag CM, Köhler AC, Anderson ME, Backs J, Maier LS. CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes. J Mol Cell Cardiol. 2011 Nov;51(5):749-59. Epub 2011 Jul 26. PubMed PMID: 21819992; PubMed Central PMCID: PMC3226826.
•Swaminathan PD, Purohit A, Soni S, Voigt N, Singh MV, Glukhov AV, Gao Z, He BJ, Luczak ED, Joiner ML, Kutschke W, Yang J, Donahue JK, Weiss RM, Grumbach IM, Ogawa M, Chen PS, Efimov I, Dobrev D, Mohler PJ, Hund TJ, Anderson ME. Oxidized CaMKII causes cardiac sinus node dysfunction in mice. J Clin Invest. 2011 Aug 1;121(8):3277-88. doi: 10.1172/JCI57833. Epub 2011 Jul 25. PubMed PMID: 21785215; PubMed Central PMCID: PMC3223923.
•Erickson JR, He BJ, Grumbach IM, Anderson ME. CaMKII in the cardiovascular system: sensing redox states. Physiol Rev. 2011 Jul;91(3):889-915. Review. PubMed PMID: 21742790.
•Anderson ME. Pathways for CaMKII activation in disease. Heart Rhythm. 2011 Sep;8(9):1501-3. Epub 2011 May 3. PubMed PMID: 21699838; PubMed Central PMCID: PMC3163819.
•Swaminathan PD, Anderson ME. CaMKII inhibition: breaking the cycle of electrical storm? Circulation. 2011 May 24;123(20):2183-6. Epub 2011 May 9. PubMed PMID: 21555705.
•Schulman H, Anderson ME. Ca/Calmodulin-dependent Protein Kinase II in Heart Failure. Drug Discov Today Dis Mech. 2010 Summer;7(2):e117-e122. PubMed PMID: 21503275; PubMed Central PMCID: PMC3077766.
•Zingman LV, Zhu Z, Sierra A, Stepniak E, Burnett CM, Maksymov G, Anderson ME, Coetzee WA, Hodgson-Zingman DM. Exercise-induced expression of cardiacATP-sensitive potassium channels promotes action potential shortening and energy conservation. J Mol Cell Cardiol. 2011 Jul;51(1):72-81. Epub 2011 Mar 23. PubMed PMID: 21439969; PubMed Central PMCID: PMC3103621.
•Gao Z, Singh MV, Hall DD, Koval OM, Luczak ED, Joiner ML, Chen B, Wu Y, Chaudhary AK, Martins JB, Hund TJ, Mohler PJ, Song LS, Anderson ME. Catecholamine-independent heart rate increases require Ca2+/calmodulin-dependent protein kinase II. Circ Arrhythm Electrophysiol. 2011 Jun 1;4(3):379-87. Epub 2011 Mar 15. PubMed PMID: 21406683; PubMed Central PMCID: PMC3116039.
•Singh MV, Anderson ME. Is CaMKII a link between inflammation and hypertrophy in heart? J Mol Med (Berl). 2011 Jun;89(6):537-43. Epub 2011 Jan 29. Review. PubMed PMID: 21279501.
•Anderson ME, Brown JH, Bers DM. CaMKII in myocardial hypertrophy and heart failure. J Mol Cell Cardiol. 2011 Oct;51(4):468-73. Epub 2011 Jan 27. Review. PubMed PMID: 21276796; PubMed Central PMCID: PMC3158288.
•Wagner S, Ruff HM, Weber SL, Bellmann S, Sowa T, Schulte T, Anderson ME, Grandi E, Bers DM, Backs J, Belardinelli L, Maier LS. Reactive oxygen species-activated Ca/calmodulin kinase IIδ is required for late I(Na) augmentation leading to cellular Na and Ca overload. Circ Res. 2011 Mar 4;108(5):555-65. Epub 2011 Jan 20. PubMed PMID: 21252154; PubMed Central PMCID:PMC3065330.
2010
•Hund TJ, Koval OM, Li J, Wright PJ, Qian L, Snyder JS, Gudmundsson H, Kline CF, Davidson NP, Cardona N, Rasband MN, Anderson ME, Mohler PJ. A β(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice. J Clin Invest. 2010 Oct 1;120(10):3508-19
•Yang J, Huang J, Maity B, Gao Z, Lõrca R, Gudmundsson H, Li J, Stewart A, Swaminathan PD, Ibeawuchi SR, Shepherd A, Chen CK, Kutschke W, Mohler PJ, Mohapatra DP, Anderson ME, Fisher RA. RGS6, a Modulator of Parasympathetic Activation in Heart. Circ Res. 2010 Sep 23. [Epub ahead of print]
•Li J, Kline CF, Hund TJ, Anderson ME, Mohler PJ. Ankyrin-B regulates Kir6.2 membrane expression and function in heart J Biol Chem. 2010 Sep 10;285(37):28723-30.
•Wei S, Guo A, Chen B, Kutschke W, Xie YP, Zimmerman K, Weiss RM, Anderson ME, Cheng H, Song LS. T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res. 2010 Aug 20;107(4):520-31.
•Glukhov AV, Fedorov VV, Anderson ME, Mohler PJ, Efimov IR. Functional anatomy of the murine sinus node: high-resolution optical mapping of ankyrin-B heterozygous mice.Am J Physiol Heart Circ Physiol. 2010 Aug;299(2):H482-91.
•Gudmundsson H, Hund TJ, Wright PJ, Kline CF, Snyder JS, Qian L, Koval OM, Cunha SR, George M, Rainey MA, Kashef FE, Dun W, Boyden PA, Anderson ME, Band H, Mohler PJ. EH domain proteins regulate cardiac membrane protein targeting. Circ Res. 2010 Jul 9;107(1):84-95.
•Gao Z, Chen B, Joiner ML, Wu Y, Guan X, Koval OM, Chaudhary AK, Cunha SR, Mohler PJ, Martins JB, Song LS, Anderson ME .I(f) and SR Ca(2+) release both contribute to pacemaker activity in canine sinoatrial node cells. J Mol Cell Cardiol. 2010 Jul;49(1):33-40.
•Witczak CA, Jessen N, Warro DM, Toyoda T, Fujii N, Anderson ME, Hirshman MF, Goodyear LJ. CaMKII regulates contraction- but not insulin-induced glucose uptake in mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2010 Jun;298(6):E1150-60.
•Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 beta-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4996-5000.
•Li H, Li W, Gupta AK, Mohler PJ, Anderson ME, Grumbach IM. Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy. Am J Physiol Heart Circ Physiol. 2010 Feb;298(2):H688-98.
2009
• Singh, M.V., Kapoun, A., Higgins, L., Kutschke, W., Thurman, J.M., Singh, M., Yang, J., Guan, X., Lowe, J., Weiss, R.M., Zimmerman, K., Zhang, R., Yull, F.E., Blackwell, T.S., Mohler, P.J., Anderson, M.E. Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart. J. Clin. Invest. 119(4):986-996, 2009. (Commentary in Nat Med 15:375, 2009)
• Wu Y, Gao Z, Chen B, Koval O, Singh M, Guan X, Hund T, Kutschke WJ, Sarma S, Grumbach I, Wehrens X, Mohler P, Song L, Anderson M.E. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc. Natl. Acad. Sci. 106:5972-5977, 2009. (Commentary in Sci Signaling, 2:ec130, 2009)
• Chelu M, Sarma S, Sood S, Wang S, Oort V, Jeroen R, Skapura D, Li N, Santonastasi M, Mueller F, Schotten U, Anderson ME, Valderrabano M, Dobrev D, Wehrens XHT. Calmodulin kinase II mediated sarcoplasmic reticulum calcium leak promotes atrial fibrillation. J. Clin. Invest. 119(7): 1940-1951, 2009.
• Timmins J, Ozcan L, Seimon TA, Li G, Malagelada C, Backs J, Backs T, Bassel-Duby R, Olson EN, Anderson ME, and Tabas I. Calcium/calmodulin-dependent protein kinase II links endoplasmic reticulum stress with Fas and mitochondrial apoptosis pathways.J. Clin. Invest. 119(10):2925-2941, 2009.
• Chen B, Wu Y, Mohler PJ, Anderson ME, Song L-S. Local control of Ca2+-induced Ca2+ release in mouse sinoatrial node cells. J. Mol. Cell. Cardiol. 47(5):706-715, 2009.
• Kline CF, Kurata HT, Hund TJ, Cunha SR, Koval OM, Wright PJ, Christensen M, Anderson ME, Nichols CG, Mohler PJ. Dual Role of K ATP channel C-terminal motif in membrane targeting and metabolic regulation. Proc. Natl. Acad. Sci. 106 (39):16669-74, 2009.
• Christensen MD, Dun W, Boyden PA, Anderson ME, Mohler PJ, and Hund TJ. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: A computational analysis. PLoS Comput Biol. 2009. (Accepted).
2008
•Erickson JR, Anderson ME. CaMKII and its role in cardiac arrhythmia. JCardiovasc Electrophysiol. 2008 Dec;19(12):1332-6. Epub 2008 Sep 17. PubMed PMID:18803570.
•Thiel WH, Chen B, Hund TJ, Koval OM, Purohit A, Song LS, Mohler PJ, Anderson ME. Proarrhythmic defects in Timothy syndrome require calmodulin kinase II. Circulation. 2008 Nov 25;118(22):2225-34. Epub 2008 Nov 10. PubMed PMID:19001023.
•Le Scouarnec S, Bhasin N, Vieyres C, Hund TJ, Cunha SR, Koval O, Marionneau C, Chen B, Wu Y, Demolombe S, Song LS, Le Marec H, Probst V, Schott JJ, Anderson ME, Mohler PJ. Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease. Proc Natl Acad Sci U S A. 2008 Oct7;105(40):15617-22. Epub 2008 Oct 1. PubMed PMID: 18832177; PubMed Central PMCID: PMC2563133.
•Couchonnal LF, Anderson ME. The role of calmodulin kinase II in myocardial physiology and disease. Physiology (Bethesda). 2008 Jun;23:151-9. Review. PubMed PMID: 18556468.
•Erickson JR, Joiner ML, Guan X, Kutschke W, Yang J, Oddis CV, Bartlett RK, Lowe JS, O’Donnell SE, Aykin-Burns N, Zimmerman MC, Zimmerman K, Ham AJ, Weiss RM, Spitz DR, Shea MA, Colbran RJ, Mohler PJ, Anderson ME. A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation. Cell. 2008 May 2;133(3):462-74. PubMed PMID: 18455987; PubMed Central PMCID: PMC2435269.
•Werdich AA, Lima EA, Dzhura I, Singh MV, Li J, Anderson ME, Baudenbacher FJ. Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies. Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2352-62. Epub 2008 Mar 21. PubMed PMID: 18359893.
•Mohler PJ, Anderson ME. New insights into genetic causes of sinus node disease and atrial fibrillation. J Cardiovasc Electrophysiol. 2008 May;19(5):516-8. Epub 2008 Feb 21. PubMed PMID: 18298510.
•Grueter CE, Abiria SA, Wu Y, Anderson ME, Colbran RJ. Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits. Biochemistry. 2008 Feb12;47(6):1760-7. Epub 2008 Jan 19.
PubMed PMID: 18205403; PubMed Central PMCID: PMC2814322.
•Khoo MS, Grueter CE, Eren M, Yang J, Zhang R, Bass MA, Lwin ST, Mendes LA, Vaughan DE, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition disrupts cardiomyopathic effects of enhanced green fluorescent protein. J Mol Cell Cardiol. 2008 Feb;44(2):405-10.
Epub 2007 Nov 28. PubMed PMID: 18048055; PubMed Central PMCID: PMC2695824.
•Lowe JS, Palygin O, Bhasin N, Hund TJ, Boyden PA, Shibata E, Anderson ME, Mohler PJ. Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J Cell Biol. 2008 Jan 14;180(1):173-86. Epub 2008 Jan7. PubMed PMID: 18180363; PubMed Central PMCID: PMC2213608.
2007
•Khoo MS, Grueter CE, Eren M, Yang J, Zhang R, Bass MA, Lwin ST, Mendes LA, Vaughan DE, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition disrupts cardiomyopathic effects of enhanced green fluorescent protein. J Mol Cell Cardiol. 2008 Feb;44(2):405-10.
Epub 2007 Nov 28. PubMed PMID: 18048055; PubMed Central PMCID: PMC2695824.
•Li J, Marionneau C, Koval O, Zingman L, Mohler PJ, Nerbonne JM, Anderson ME. Calmodulin kinase II inhibition enhances ischemic preconditioning by augmenting ATP-sensitive K+ current. Channels (Austin). 2007 Sep-Oct;1(5):387-94. Epub 2007 Dec 17. PubMed PMID: 18690039.
•Werdich AA, Baudenbacher F, Dzhura I, Jeyakumar LH, Kannankeril PJ, Fleischer S, LeGrone A, Milatovic D, Aschner M, Strauss AW, Anderson ME, Exil VJ. Polymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice. Am J Physiol Heart Circ Physiol. 2007
May;292(5):H2202-11. Epub 2007 Jan 5. PubMed PMID: 17209005. Anderson ME, Mohler PJ. MicroRNA may have macro effect on sudden death. Nat Med. 2007 Apr;13(4):410-1. PubMed PMID: 17415373.
•Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res. 2007 Mar 1;73(4):657-66. Epub 2006 Dec 12. Review. PubMed PMID: 17254559.
•Grimm M, El-Armouche A, Zhang R, Anderson ME, Eschenhagen T. Reduced contractile response to alpha1-adrenergic stimulation in atria from mice with chronic cardiac calmodulin kinase II inhibition. J Mol Cell Cardiol. 2007 Mar;42(3):643-52. Epub 2006 Dec 28. PubMed PMID: 17292391.
•Grueter CE, Colbran RJ, Anderson ME. CaMKII, an emerging molecular driver for calcium homeostasis, arrhythmias, and cardiac dysfunction. J Mol Med. 2007 Jan;85(1):5-14. Epub 2006 Nov 21. Review. PubMed PMID: 17119905.
2006
• Wu Y, Shintani A, Greuter C, Zhang R, Yang J, Kranias EG, Colbran RJ, Anderson ME. Calmodulin kinase II determines dynamic Ca2+ responses in heart. J Mol Cell Cardiol 2006; 40:213-23.
• Yang Y, Zhu WZ, Joiner M-L, Zhang R, Oddis CV, Hou Y, Yang J, Price EE jr, Gleaves L, Erin M, Ni G, Vaughn DE, Xiao R-P, Anderson ME. Calmodulin kinase inhibition protects against myocardial apoptosis in vivo. Am J Physiol 2006; 291:H3065-H3075.
•Kannankeril PJ, Mitchell BM, Goonasekera SA, Chelu MG, Zhang W, Sood S, Kearney DL, Danila CI, De Biasi M, Pautler RG, Roden DM, Taffet GE, Dirksen RT, Anderson ME, Hamilton SL. Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and mild cardiomyopathy. Proc Natl Acad Sci 2006; 103:12179-12184.
• Khoo MSC, Zhang R, Ni G, Greuter C, Yang Y, Zhang W, Mendes L, Olson EN, Colbran RJ, Anderson ME. Death, cardiac dysfunction and arrhythmias due to up-regulation of calmodulin kinase II in calcineurin-induced cardiomyopathy. Circulation 2006; 114:1352-1359. Published with an accompanying editorial.
• Grueter CE, Abiria SA, Dzhura I, Wu Y, Hamm A-J, Mohler PJ, Anderson ME, Colbran RJ. Molecular basis for facilitation of native Ca2+ channels by CaMKII. Mol Cell 2006; 23:641-650. Selected as a recommended citation by the Faculty of 1000 Biology.
• Li J, Shah V, Hell J, Nerbonne JM, Anderson ME. Calmodulin kinase II inhibition shortens action potential duration by up-regulation of K+ currents. Circ Res 2006; 99:1092-1099. PMID: 17038644. Published with an accompanying editorial.
•Anderson ME, Higgins, LS, Schulman H. Disease mechanisms and emerging therapies: Protein kinases and their inhibitors in myocardial disease. Nature Clin Prac 2006; 3:437-445.
III. Therapeutic Implications of Pharmacological Agents for Cardiac Contractility Dysfunction: “The Fire From Within The Biggest Ca2+ Channel Erupts and Dribbles” by Anderson, ME
Author: Justin D Pearlman, MD, PhD, FACC PENDING –
Therapeutic Implications of these physiological research discoveries
JDP: RECOMMEND SPLIT TO TWO: a. contractility b. arrhythmia
IV. Selective Research Contributions on Calcium Release-related Contractile Dysfunction
Curator: Aviva Lev-Ari, PhD, RN
Summary
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
Author: Larry H Bernstein, MD, FCAP
PENDING
V. Bibliography on Calcium Release Mechanisms in Vascular Smooth Muscle, in Cardiomyocytes and the Role in Heart Failure
Curator: Aviva Lev-Ari, PhD, RN
Anderson ME, General Hospital Iowa City and University of Iowa
Wilson S. Colucci, MD, Heart Failure Lab at BMC
William Gregory Stevenson, M.D.Heart Failure Lab at BWH
Introduction to Calcium Release Mechanism in Vascular Smooth Muscle and in Cardiomyocytes
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
I. Cellular Contractility Capacity — Actin, Cellular Dynamics and Calcium Efflux: Emergence of the Calcium Release-related Contractile Dysfunction
Author: Justin D Pearlman, MD, PhD, FACC
The pumping action of the heart is mediated by repeated cycles of the release and re-uptake of calcium stored within cardiac myocytes. Similar to skeletal muscle function, the protein complex of actinomycin creates mechanical motion when calcium interacts with the threads of the protein strand tropomyosin which are wound around an actin protein filament with the third protein troponin strung out like beads along the string. Calcium (Ca++) released from the storage space (sarcoplasmic reticulum) combines with troponin to actuate a shift in the tropomyosin threads, exposing myosin binding sites to adenosinetriphosphate (ATP, the energy source), which, in turn, consume the high-energy bond of ATP and concommitantly break and make cross-bridges resulting in shifted position (filament sliding, contraction). The spiral layers of these filaments within the heart result in a reduction of chamber size. Normally the two atrial chambers contract first, to boost the load of blood in the ventricles, then the ventricles contract, relying on one-way valves to impose a forward direction to the blood ejected from the heart.
There is barely enough ATP around to complete a single heart beat, so ATP is replenished from a higher energy storage form, phosphocreatine (PCr, aka creatinephosphate), which in turn in reconstituted during the relaxation phase of the heart (low pressure) when oxygenated blood, glucose, and fatty acids are delivered to local mitochondria to restock energy stores. Thus the contraction cycle, unlike a continual pump, provides low pressure respite after each high pressure contraction, which facilitates delivery of oxygenated nutrient blood to the heart muscle to replenish its energy for the action. When switching to a mechanical total heart replacement, it is not necessary to preserve the pulsatile pattern, which primarily serves to facilitate energizing the biologic pump.
The volume of blood ejected by the left ventricle from a single heart beat is called the stroke volume (SV). The amount of blood in the left ventricle just before the heart beat is called the end-diastolic volume (EDV), and just after, the end-systolic volume (ESV), so SV=EDV-ESV. The portion of the filled left ventricle that gets pumped forward through the aortic valve by a single heart beat is called the ejection fraction (EF). Thus EF = SV/EDV, expressed as a percentage. The cardiac output (CO) in liters/minute is simply the product of stroke volume and heart rate (HR): CO = SV x HR.
Heart failure has three clinical forms: high output failure, systolic failure and diastolic heart failure. With high output failure (elevated SV x HR), the demands of the body are elevated beyond the normal capacity of the heart to supply cardiac output. With systolic failure (low EF) the pumping action of the heart is insufficient to meet the needs of fresh blood delivery to the various organs of the body (including in particular the heart, brain, liver, and kidneys). Note that the heart does not draw any significant nutrients or oxygen from the blood in its chambers – rather, it is first in line after the oxygenated blood is pumped out through the aortic valve to tax 10% of the cardiac output via the coronary arteries. In diastolic failure, the LV resists filling (stiff LV) so the back pressure to the lungs is elevated, resulting in pulmonary congestion. Many textbooks incorrectly describe diastolic heart failure as heart failure with a normal EF; however, that would imply that diastolic heart failure (stiff LV) can be “cured” by a myocardial infarction (heart attack) so that the EF drops. Contrary to that mistaken description, the addition of reduced EF to a patient with diastolic heart failure results in combined systolic and diastolic heart failure. Inadequate delivery of blood from low EF has been called “forward failure” and pulmonary congestion from a stiff LV “backward failure” but those terms are not synonymous with systolic and diastolic failure, as low EF also contributes to congestive heart failure, and stiff LV can impede adequate filling, so each has components for forward and backward failure.
One can plot a curve relating stroke volume to the end diastolic volume, called the “Frank-Starling curve” whereby an increase in EDV is generally accommodated by an increase in SV. That adaptive feature is achieved by a stimulation of calcium-mediated increase in contractility (speed and strength of contraction) . In heart failure, the usual amounts of calcium stores are not adequate to meet the demands. Consequently, remodeling occurs, which includes reversion towards a fetal phenotype in which the sarcoplasmic reticulum stores and releases a greater amount of calcium. While this does result in some augmentation of contractility, it occurs at a cost. The higher levels of calcium can interfere with mitochondrial function and reduce the energy efficiency of oxygen replenishment of phosphocreatine and ATP. In research by the author of this section (JDP), the timing of oxygen uptake and utilization is adversely affected by this remodeling, as demonstrated by oxygen uptake sensitive dynamic cardiac MRI.
Thus strategies to genetically re-engineer cardiac function by modifying calcium uptake and release to elevate contractility at a given workload have potentially harmful consequences in terms of lowering the energy efficiency of the heart. If the blood supply of the heart is good (non-ischemic heart failure), one can expect opportunities for benefit. However, if the blood supply to the heart is limited (ischemic heart failure), such changes may be detrimental. Furthermore, the impediments to mitochondrial function may contribute to other adverse effects of remodeling, including in particular activation of fibrosis (adverse remodeling promoting worsened diastolic failure).
II. Integration and Interpretation of Research Results in Two Labs: Mark E Anderson’s and Roger Hajjar’s Lab
Author: Justin D Pearlman, MD, PhD, FACC
PENDING
III. Therapeutic Implications of Pharmacological Agents for Cardiac Contractility Dysfunction: “The Fire From Within The Biggest Ca2+ Channel Erupts and Dribbles” by Anderson, ME
Negative inotropic agents decrease myocardial contractility, and are used to decrease cardiac workload in conditions such as angina. While negative inotropism may precipitate or exacerbate heart failure, certain beta blockers (e.g. carvedilol, bisoprolol and metoprolol) have been believed to reduce morbidity and mortality in congestive heart failure. Quite recently, however, the effectiveness of beta blockers has come under renewed critical scientific scrutiny.
The above list of inotropic agents consists of agents developed to increase the contractile force of the heart and have had a long history of use. Even though they have been proved valid, they are not part of the specific advances that we are seeing that justifies a cardiology specialty in cardiac electrophysiology, the disorders, and the treatments. The developments we now witness were unknown and perhaps unexpected a quarter of a century ago. The methods required to understand the myocardiocyte were not yet developed. Our understanding is now based on a refined knowledge of the Ca(2+) release mechanism between the sarcomere and the myocyte cytoplasm, the Ca(2+) transport, the ion pores, the role of RyR2 and the phosphorylation of the Ca(2+) release mechanism. This and more will lead to far better therapeutic advances in the next few years based on earlier detection of changes preceding heart failure, and the possibility of treatments for potential life-threatening arrhythmias will be averted.
IV. Selective Research Contributions on Calcium Release-related Contractile Dysfunction
Department of Medicine, University Hospital Zurich, Switzerland.
Abstract
In epidemiological surveys and in large-scale therapeutic trials, the prognosis of patients with ischemic heart failure is worse than in patients with a non-ischemic etiology. Even heart transplant candidates may respond better to intensified therapy if they have non-ischemic heart failure. The term ‘non-ischemic heart failure’ includes various subgroups such as hypertensive heart disease, myocarditis, alcoholic cardiomyopathy and cardiac dysfunction due to rapid atrial fibrillation. Some of these causes are reversible. The therapeutic effect of essential drugs such as angiotensin-converting enzyme inhibitors, beta-blockers and diuretics does not, in general, significantly differ between ischemic and non-ischemic heart failure. However, in some trials, response to certain drugs (digoxin, tumor necrosis factor-alpha, inhibition with pentoxifylline, growth hormone and amiodarone) was found to be better in non-ischemic patients. Patients with ischemic heart failure and non-contracting ischemic viable myocardium may, on the other hand, considerably improve following revascularization. In view of prognostic and possible therapeutic differences, the etiology of heart failure should be determined routinely in all patients. http://www.ncbi.nlm.nih.gov/pubmed/12634896
Upregulation of β3-Adrenoceptors and Altered Contractile Response to Inotropic Amines in Human Failing Myocardium
From the Department of Medicine, Unit of Pharmacology and Therapeutics, University of Louvain Medical School (S.M., O.F., J.-L.B.), Brussels, Belgium; INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires (J.-N.T., C.G.) and Faculté des Sciences et Techniques (C.G.), Nantes, France; and Department of Pathology, Brigham and Women’s Hospital, and Physiology Program, Harvard School of Public Health (L.K.), Boston, Mass.
Correspondence to Jean-Luc Balligand, Department of Medicine, Unit of Pharmacology and Therapeutics, FATH 5349, University of Louvain Medical School, 53 avenue Mounier, B1200 Brussels, Belgium, e-mail Balligand@mint.ucl.ac.be; or Chantal Gauthier, INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires, 44093 Nantes, France,
Abstract
Background—Contrary to β1– and β2-adrenoceptors, β3-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of β3-adrenoceptors in nonfailing and failing human hearts.
Methods and Results—We analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6±2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2±3%). β3-Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to β1-adrenoceptor mRNA, Western blot analysis of β3-adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for Gαi-2 proteins that couple β3-adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of β3-preferential agonists was only mildly reduced.
Conclusions—Opposite changes occur in β1– and β3-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.
Increased beta-receptor density and improved hemodynamic response to catecholamine stimulation during long-term metoprolol therapy in heart failure from dilated cardiomyopathy.
Cardiology Division, Stanford University School of Medicine, CA.
Abstract
Severe heart failure is associated with a reduction in myocardial beta-adrenergic receptor density and an impaired contractile response to catecholamine stimulation. Metoprolol was administered during a 6-month period to 14 patients with dilated cardiomyopathy to examine its effects on these abnormalities. The mean daily dose of metoprolol for the group was 105 mg (range, 75-150 mg). Myocardial beta-receptor density, resting hemodynamic output, and peak left ventricular dP/dt response to dobutamine infusions were compared in 9, 14, and 7 patients, respectively, before and after 6 months of metoprolol therapy while the patients were on therapy. The second hemodynamic study was performed 1-2 hours after the morning dose of metoprolol had been given. Myocardial beta-receptor density increased from 39 +/- 7 to 80 +/- 12 fmol/mg (p less than 0.05). Resting hemodynamic output showed a rise in stroke work index from 27 +/- 4 to 43 +/- 3 g/m/m2, p less than 0.05, and ejection fraction rose from 0.26 +/- 0.03 to 0.39 +/- 0.03 after 6 months of metoprolol therapy, p less than 0.05. Before metoprolol therapy, dobutamine caused a 21 +/- 4% increase in peak positive left ventricular dP/dt; during metoprolol therapy, the same dobutamine infusion rate increased peak positive dP/dt by 74 +/- 18% (p less than 0.05). Thus, long-term metoprolol therapy is associated with an increase in myocardial beta-receptor density, significant improvement in resting hemodynamic output, and improved contractile response to catecholamine stimulation. These changes indicate a restoration of beta-adrenergic sensitivity associated with metoprolol therapy, possibly related to the observed up-regulation of beta-adrenergic receptors.
Ryanopathy: causes and manifestations of RyR2 dysfunction in heart failure
Belevych AE, Radwański PB, Carnes CA, Györke S. College of Medicine, The Ohio State University, Columbus, OH. Cardiovasc Res. 2013; 98(2):240-7. doi: 10.1093/cvr/cvt024. Epub 2013 Feb 12. PMID: 23408344 PMCID: PMC3633158 [Available on 2014/5/1] The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodeling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
Circ Res. 2005 Dec 9;97(12):1314-22. Epub 2005 Nov 3.
Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure.
Department of Medicine, University of Illinois at Chicago, IL 60612, USA.
Abstract
Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.
Editorial Comment on the above article abstract made by Anderson, ME
From the University of Iowa, Carver College of Medicine, Iowa City.
Correspondence to Mark E. Anderson, MD, PhD, University of Iowa, Carver College ofMedicine, 200 Hawkins Drive, Room E 315 GH, Iowa City, IA 53342-1081. E-mail mark-e-anderson@uiowa.edu
CaMKII Is a Pluripotent Signaling Molecule in Heart
The multifunctional Ca2+ and calmodulin (CaM)-dependent protein kinase II (CaMKII) is a serine threonine kinase that is abundant in heart where it phosphorylates Ca2+ihomeostatic proteins. It seems likely that CaMKII plays an important role in cardiac physiology because these target proteins significantly overlap with the more extensively studied serine threonine kinase, protein kinase A (PKA), which is a key arbiter of catecholamine responses in heart. However, the physiological functions of CaMKII remain poorly understood, whereas the potential role of CaMKII in signaling myocardial dysfunction and arrhythmias has become an area of intense focus. CaMKII activity and expression are upregulated in failing human hearts and in many animal models of structural heart disease.1 CaMKII inhibitory drugs can prevent cardiac arrhythmias2,3 and suppress afterdepolarizations4 that are a probable proximate focal cause of arrhythmias in heart failure. CaMKII inhibition in mice reduces left ventricular dilation and prevents disordered intracellular Ca2+ (Ca2+i) homeostasis after myocardial infarction.5 CaMKII overexpression in mouse heart causes severe cardiac hypertrophy, dysfunction, and sudden death that is heralded by increased SR Ca2+ leak6; these findings go a long way to making a case for CaMKII as a causative signal in heart disease and arrhythmias but do not identify critical molecular targets or test the potential role of CaMKII in a large non-rodent animal model. The work by Ai et al in this issue of Circulation Research makes an important contribution by demonstrating CaMKII upregulation causes increased Ca2+ leak from ryanodine receptor (RyR) Ca2+ release channels in a clinically-relevant model of structural heart disease.7
Ryanodine Receptors Are Central
Ca2+i release controls cardiac contraction, and most of the Ca2+i for contraction is released from the intracellular sarcoplasmic reticulum (SR) through ryanodine receptors (RyR). RyRs are huge proteins (565 kDa) that assemble with a fourfold symmetry to form a functional Ca2+ release channel. Approximately 90% of the RyR is not directly required to form the pore but instead protrudes into the cytoplasm where it binds numerous proteins, including PKA, CaMKII, CaM, and FK12.6 (calstabin). Cardiac contraction is initiated when Ca2+ current (ICa), through sarcolemmal L-type Ca2+ channels (LTCC), triggers RyR opening by a Ca2+-induced Ca2+ release (CICR) mechanism. LTCCs “face off” with RyRs across a highly ordered cytoplasmic cleft that delineates a kind of Ca2+furnace during each CICR-initiated heart beat (Figure). CICR has an obvious need to function reliably, so it is astounding to consider how this feed forward process is intrinsically unstable. The increased instability of CICR in heart failure is directly relevant to arrhythmias initiated by afterdepolarizations. RyRs partly rely on a collaboration of Ca2+-sensing proteins in the SR lumen to grade their opening probability and the amount of SR Ca2+ release to a given ICa stimulus. Thus the SR Ca2+ content is an important parameter for setting the inotropic state, and heart failure is generally a condition of reduced SR Ca2+ content and diminished myocardial contraction.
Ca2+-induced Ca2+ release (CICR) in health and disease. Each heart beat is initiated by cell membrane depolarization that opens Ca2+channels. The Ca2+ current (ICa) induces ryanodine receptor (RyR) opening that allows release of myofilament activating Ca2+ for contraction. In healthy CICR, RyRs close during diastole while Ca2+ is removed from the cytoplasm by uptake into the sarcoplasmic reticulum (SR). In heart failure the SR has reduced Ca2+ content so that the amount of Ca2+ released to the myofilaments is smaller than in health. RyR hyperphosphorylation by CaMKII promotes repetitive RyR openings leading to a Ca2+ leak in diastole. This leak contributes to the reduction in SR Ca2+ content and can engage the electrogenic Na+-Ca2+ exchanger to trigger afterdepolarizations and arrhythmias.
Kinases Facilitate Communication Between LTCCs and RyRs
LTCCs and RyRs form the protein machinery for initiating contraction in cardiac and skeletal muscle, but in cardiac muscle communication between these proteins occurs without a requirement for physical contact. PKA is preassociated with LTCCs and RyRs, and PKA-dependent phosphorylation increases LTCC8 and RyR9opening. The resultant increase in Ca2+i is an important reason for the positive inotropic response to cathecholamines. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by increased Ca2+I, and so catecholamine stimulation activates CaMKII in addition to PKA.5 In contrast to PKA, which is tightly linked to inotropy, CaMKII inhibition does not cause a reduction in fractional shortening during acute catecholamine stimulation in mice.5 Prolonged catecholamine exposure does reduce contractile function by uncertain mechanisms that require CaMKII.10 CaMKII colocalizes with LTCCs11 and RyRs,12 and CaMKII can also increase LTCC13 and RyR12 opening probability in cardiac myocytes. The ultrastructural environment of LTCCs and RyRs is well-suited for a Ca2+i-responsive kinase to serve as a coordinating signal between LTCCs and RyRs during CICR. The recently identified role of CaMKII in heart failure suggests the possibility that excessive CaMKII activity could cause or contribute to CICR defects present in heart failure
Heart Failure Is a Disease of Disordered Ca2+i Homeostasis
The key clinical phenotypes of contractile dysfunction and electrical instability in heart failure involve problems with Ca2+i homeostasis. Broad changes in Ca2+I-handling proteins can occur in various heart failure models, but in general heart failure is marked by a reduction in the capacity for SR Ca2+ uptake, enhanced activity of the sarcolemmal Na+-Ca2+ exchanger, and reduction in CICR-coordinated SR Ca2+ release. On the other hand, the opening probability of individual LTCCs is increased in human heart failure,14suggesting that posttranslational modifications may also be mechanistically important for understanding these Ca2+i disturbances at Ca2+ homeostatic proteins.
Is Heart Failure a Disease of Enzymatic Over-Activity?
Heart failure is marked by hyper-adrenergic tone, and beta adrenergic receptor antagonist drugs (beta blockers) are a mainstay of therapy for reducing mortality in heart failure patients. The Marks group pioneered the concept that RyRs are hyperphosphorylated by PKA in patients with heart failure and showed that successful therapies, ranging from beta blockers to left ventricular assist devices, reduce RyR phosphorylation in step with improved mechanical function. They have developed a large body of evidence in patients and in animal models that PKA phosphorylation of Ser2809 on cardiac RyRs destabilizes binding of FK12.6 to RyRs and promotes increased RyR opening that causes an insidious Ca2+ leak. This leak is potentially problematic because it can reduce SR Ca2+ content (to depress inotropy), engage pathological Ca2+-dependent transcriptional programs (to promote myocyte hypertrophy), and activate arrhythmia-initiating afterdepolarizations (to cause sudden death). Indeed, RyR hyperphosphorylation can produce arrhythmias as well as mechanical dysfunction, whereas a drug that prevents FK12.6 dissociation from RyR also reduces or prevents arrhythmias.15 Taken together these findings make a strong case that RyR hyperphosphorylation (a result of net excess kinase activity) is a central event in heart failure and sudden death.
Not all findings point to hyperphosphorylation of RyR by PKA and subsequent FK12.6 dissociation as critical determinants of heart failure16 and arrhythmias.17 For example, studies in isolated and permeabilized ventricular myocytes failed to show an increase in RyR openings, called sparks, which are monitored by photoemission of a Ca2+-sensitive fluorescent dye.18 FKBP12.6 dissociation is not universally reported to follow RyR phosphorylation by PKA.19 Furthermore, FKBP12.6 binding to RyR is not affected during catecholamine stimulation that results in arrhythmias in a mouse model of catecholamine-induced ventricular tachycardia,20,21 a genetic disorder of hypersensitive RyR Ca2+release. These findings challenge the PKA hypothesis and make room, conceptually, to consider the role of additional signals for modulating RyR activity in heart disease.
Both PKA and CaMKII may phosphorylate Ser2809, but recently CaMKII was found to exclusively phosphorylate Ser2815 and this phosphorylation caused increased RyR opening.12 However, the PKA and CaMKII responses may be mechanistically distinct because CaMKII evoked increased RyR opening in the absence of FK12.6 dissociation. These findings together with the fact that CaMKII activity is recruited under conditions of increased PKA activity suggest that CaMKII might also be important in regulating RyRs in heart failure.
The article by Ai et al shows that expression of a CaMKII splice variant that is resident in cytoplasm (CaMKIIδc) was increased, and there was enhanced phosphorylation of the recently identified CaMKII site (Ser2815) on RyR. Both Ser2815 and the PKA site (Ser2809) were hyperphosphorylated in failing hearts, but phosphorylation of the CaMKII site was greater than the PKA site. Because both Ser2809 and Ser2815 can increase RyR openings, it seemed likely that PKA and CaMKII would work together to increase Ca2+leak. Surprisingly, CaMKII inhibition but not PKA inhibition suppressed the leak. These experiments were performed with meticulous attention to matching SR Ca2+ load, a technically difficult accomplishment that is not performed by most groups evaluating SR Ca2+ release. Thus, differences in the SR intraluminal Ca2+ could not account for these findings. Although these experiments were carefully controlled, one potential limitation is that the experiments relied exclusively on CaMKII and PKA inhibitor drugs that are notorious for nonspecific actions at ion channel proteins. They also showed that the ratio of inositol tris phosphate receptors (IP3R) to RyRs was increased in failing left ventricular myocytes. IP3R are important for regulating Ca2+i in many cells types, including atrial myocytes, but their role in ventricle remains uncertain. The finding that the IP3R are increased at the expense of RyR suggests that Ca2+i release sites are fundamentally reordered in heart failure but leaves the impact of this change untested. IP3R are also a target for CaMKII, so interesting questions remain about the potential role for this channel and CaMKII in heart failure, at least in this model.
What We Learned and What We Need to Know
CaMKII activity seems to be part and parcel of the adrenergic signaling seen in structural heart disease. This work shows us that CaMKII can contribute directly to increased SR Ca2+ leak in a clinically relevant model of heart failure that is marked by arrhythmias and sudden death.22 Acute experiments with CaMKII inhibitory drugs strongly suggest that SR Ca2+ leak is principally linked to CaMKII rather than PKA activity. Excessive SR Ca2+ release can activate inward (forward mode) Na+-Ca2+ exchanger current to cause delayed afterdepolarizations and arrhythmias and CaMKII inhibition can prevent these inward Na+-Ca2+ exchanger currents.23 An important next step toward translating these findings will be to evaluate the effects of chronic CaMKII inhibition in this model to see whether it reverses cardiac dysfunction, arrhythmias, and whether chronic CaMKII inhibitor therapy can stop the RyR leak to refill the SR. It will be necessary to have improved pharmacological agents with fewer nonspecific effects to convincingly perform these experiments. These future experiments will tell us whether CaMKII inhibition is a potentially viable therapy for structural heart disease and arrhythmias in a non-genetic non-mouse model. We need to know whether CaMKII inhibition is really a highly-specific form of beta blockade that can preserve inotropic responses to catecholamines while preventing the adverse consequences of catecholamines in heart failure.5
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL070250, HL62494, and HL046681). Dr Anderson is an Established Investigator of the American Heart Association.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association
Mazur A, Roden DM, Anderson ME. Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits. Circulation. 1999; 100: 2437–2442.
Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble RW, Roden DM, Passier R, Olson EN, Colbran RJ, Anderson ME. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation. 2002; 106: 1288–1293.
Anderson ME, Braun AP, Wu Y, Lu T, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharm Exp Ther. 1998; 287: 996–1006.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nature Med. 2005; 11:409–417.
Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM Ca2+/-calmodulin-dependent protein kinase modulates cardiac RyR2 phosphorylation and SR Ca2+leak in heart failure. Circ Res. 2005; 97: 1314–1322.
Yue DT, Herzig S, Marban E. Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Nat Acad Sci U S A.1990; 87: 753–757.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): Defective regulation in failing hearts. Cell. 2000; 101:365–376.
Wehrens XH, Lehnart SE, Reiken SR, Marks AR. Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res.2004; 94: e61–e70.
Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L, Schwinger RH, Weil J, Herzig S. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation.1998; 98: 969–976.
Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, Coromilas J, Landry DW, Marks AR. Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2. Science. 2004; 304: 292–296.
Houser SR. Can novel therapies for arrhythmias caused by spontaneous sarcoplasmic reticulum Ca2+ release be developed using mouse models? Circ Res.2005; 96: 1031–1032.
Li Y, Kranias EG, Mignery GA, Bers DM. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes.Circ Res. 2002; 90: 309–316.
Xiao B, Sutherland C, Walsh MP, Chen SR. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res. 2004; 94: 487–495.
Cerrone M, Colombi B, Santoro M, di Barletta MR, Scelsi M, Villani L, Napolitano C, Priori SG. Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor. Circ Res. 2005;96: e77–e82.
Wu Y, Roden DM, Anderson ME. Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current. Circ Res. 1999; 84:906–912.
Clinical Profile Head, Department of Internal Medicine Director, Cardiovascular Research Center Professor of Internal Medicine – Cardiovascular Medicine Professor of Molecular Physiology and Biophysics
Dr. Anderson is clinically trained as a cardiac electrophysiologist. His research is focused on cellular signaling and ionic mechanisms that cause heart failure and sudden cardiac death. The multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII) is upregulated in heart disease and arrhythmias. Work in the Anderson laboratory implicates CaMKII as a signal that drives myocardial hypertrophy, apoptosis, mechanical dysfunction and electrical instability. The laboratory work ranges from molecular structure activity analysis of CaMKII to systems physiology using genetically modified mice to dissect cellular mechanisms of CaMKII signaling in heart. http://www.medicine.uiowa.edu/dept_primary_apr.aspx?appointment=Internal%20Medicine&id=andersonmar
Khoo MS, Li J, Singh MV, Yang Y, Kannankeril P, Wu Y, Grueter CE, Guan X, Oddis CV, Zhang R, Mendes L, Ni G, Madu EC, Yang J, Bass M, Gomez RJ, Wadzinski BE, Olson EN, Colbran RJ, Anderson ME. Circulation. 2006 Sep 26;114(13):1352-9. Epub 2006 Sep 18.
PMID:
16982937 [PubMed – indexed for MEDLINE] Free Article
Yang Y, Zhu WZ, Joiner ML, Zhang R, Oddis CV, Hou Y, Yang J, Price EE, Gleaves L, Eren M, Ni G, Vaughan DE, Xiao RP, Anderson ME. Am J Physiol Heart Circ Physiol. 2006 Dec;291(6):H3065-75. Epub 2006 Jul 21.
PMID:
16861697 [PubMed – indexed for MEDLINE] Free Article
Joiner ML, Koval OM, Li J, He BJ, Allamargot C, Gao Z, Luczak ED, Hall DD, Fink BD, Chen B, Yang J, Moore SA, Scholz TD, Strack S, Mohler PJ, Sivitz WI, Song LS, Anderson ME. Nature. 2012 Nov 8;491(7423):269-73. doi: 10.1038/nature11444. Epub 2012 Oct 10.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE Jr, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Nat Med. 2005 Apr;11(4):409-17. Epub 2005 Mar 27.
Werdich AA, Lima EA, Dzhura I, Singh MV, Li J, Anderson ME, Baudenbacher FJ. Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2352-62. doi: 10.1152/ajpheart.01398.2006. Epub 2008 Mar 21.
PMID:
18359893 [PubMed – indexed for MEDLINE] Free Article
Qian H, Matt L, Zhang M, Nguyen M, Patriarchi T, Koval OM, Anderson ME, He K, Lee HK, Hell JW. J Neurophysiol. 2012 May;107(10):2703-12. doi: 10.1152/jn.00374.2011. Epub 2012 Feb 15.
Publications by Prof. Wilson S. Colucci, MD on Heart Failure
Wilson S. Colucci, MD
Title
Professor
Institution
Boston University School of Medicine
Department
Medicine
Division
Cardiovascular Medicine
Address
75 E. Newton St Boston, MA 02118
Telephone
(617) 638-8706
Title
Chief – Section of Medicine, Cardiovascular Medicine
Institution
Boston University School of Medicine
Department
Medicine
Division
Cardiovascular Medicine
1.
Qin F, Siwik DA, Lancel S, Zhang J, Kuster GM, Luptak I, Wang L, Tong X, Kang YJ, Cohen RA, Colucci WS. Hydrogen Peroxide-Mediated SERCA Cysteine 674 Oxidation Contributes to Impaired Cardiac Myocyte Relaxation in Senescent Mouse Heart. J Am Heart Assoc. 2013; 2(4):e000184.
Gopal DM, Kommineni M, Ayalon N, Koelbl C, Ayalon R, Biolo A, Dember LM, Downing J, Siwik DA, Liang CS, Colucci WS. Relationship of plasma galectin-3 to renal function in patients with heart failure: effects of clinical status, pathophysiology of heart failure, and presence or absence of heart failure. J Am Heart Assoc. 2012 Oct; 1(5):e000760.
Calamaras TD, Lee C, Lan F, Ido Y, Siwik DA, Colucci WS. Post-translational Modification of Serine/Threonine Kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at Lysine Residue 97 Directly Inhibits Kinase Activity. J Biol Chem. 2012 Dec 7; 287(50):42400-6.
Kivikko M, Nieminen MS, Pollesello P, Pohjanjousi P, Colucci WS, Teerlink JR, Mebazaa A. The clinical effects of levosimendan are not attenuated by sulfonylureas. Scand Cardiovasc J. 2012 Dec; 46(6):330-8.
Qin F, Siwik DA, Luptak I, Hou X, Wang L, Higuchi A, Weisbrod RM, Ouchi N, Tu VH, Calamaras TD, Miller EJ, Verbeuren TJ, Walsh K, Cohen RA, Colucci WS. The polyphenols resveratrol and s17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice. Circulation. 2012 Apr 10; 125(14):1757-64.
Mazzini M, Tadros T, Siwik D, Joseph L, Bristow M, Qin F, Cohen R, Monahan K, Klein M, Colucci W. Primary carnitine deficiency and sudden death: in vivo evidence of myocardial lipid peroxidation and sulfonylation of sarcoendoplasmic reticulum calcium ATPase 2. Cardiology. 2011; 120(1):52-8.
Schulze PC, Biolo A, Gopal D, Shahzad K, Balog J, Fish M, Siwik D, Colucci WS. Dynamics in insulin resistance and plasma levels of adipokines in patients with acute decompensated and chronic stable heart failure. J Card Fail. 2011 Dec; 17(12):1004-11.
Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, Zsebo KM, Dittrich H, Hajjar RJ. Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation. 2011 Jul 19; 124(3):304-13.
Kivikko M, Sundberg S, Karlsson MO, Pohjanjousi P, Colucci WS. Acetylation status does not affect levosimendan’s hemodynamic effects in heart failure patients. Scand Cardiovasc J. 2011 Apr; 45(2):86-90.
Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, Shi H, Vincent J, Pocock SJ, Pitt B. Eplerenone in patients with systolic heart failure and mild symptoms. N Engl J Med. 2011 Jan 6; 364(1):11-21.
Qin F, Lennon-Edwards S, Lancel S, Biolo A, Siwik DA, Pimentel DR, Dorn GW, Kang YJ, Colucci WS. Cardiac-specific overexpression of catalase identifies hydrogen peroxide-dependent and -independent phases of myocardial remodeling and prevents the progression to overt heart failure in G(alpha)q-overexpressing transgenic mice. Circ Heart Fail. 2010 Mar; 3(2):306-13.
Dhingra R, Pencina MJ, Schrader P, Wang TJ, Levy D, Pencina K, Siwik DA, Colucci WS, Benjamin EJ, Vasan RS. Relations of matrix remodeling biomarkers to blood pressure progression and incidence of hypertension in the community. Circulation. 2009 Mar 3; 119(8):1101-7.
Biolo A, Greferath R, Siwik DA, Qin F, Valsky E, Fylaktakidou KC, Pothukanuri S, Duarte CD, Schwarz RP, Lehn JM, Nicolau C, Colucci WS. Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate. Proc Natl Acad Sci U S A. 2009 Feb 10; 106(6):1926-9.
Brooks WW, Conrad CH, Robinson KG, Colucci WS, Bing OH. L-arginine fails to prevent ventricular remodeling and heart failure in the spontaneously hypertensive rat. Am J Hypertens. 2009 Feb; 22(2):228-34.
Holubarsch CJ, Colucci WS, Meinertz T, Gaus W, Tendera M. The efficacy and safety of Crataegus extract WS 1442 in patients with heart failure: the SPICE trial. Eur J Heart Fail. 2008 Dec; 10(12):1255-63.
Olshansky B, Sabbah HN, Hauptman PJ, Colucci WS. Parasympathetic nervous system and heart failure: pathophysiology and potential implications for therapy. Circulation. 2008 Aug 19; 118(8):863-71.
Hare JM, Mangal B, Brown J, Fisher C, Freudenberger R, Colucci WS, Mann DL, Liu P, Givertz MM, Schwarz RP. Impact of oxypurinol in patients with symptomatic heart failure. Results of the OPT-CHF study. J Am Coll Cardiol. 2008 Jun 17; 51(24):2301-9.
Fonarow GC, Lukas MA, Robertson M, Colucci WS, Dargie HJ. Effects of carvedilol early after myocardial infarction: analysis of the first 30 days in Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction (CAPRICORN). Am Heart J. 2007 Oct; 154(4):637-44.
Wang TJ, Larson MG, Benjamin EJ, Siwik DA, Safa R, Guo CY, Corey D, Sundstrom J, Sawyer DB, Colucci WS, Vasan RS. Clinical and echocardiographic correlates of plasma procollagen type III amino-terminal peptide levels in the community. Am Heart J. 2007 Aug; 154(2):291-7.
Torre-Amione G, Bourge RC, Colucci WS, Greenberg B, Pratt C, Rouleau JL, Sestier F, Moyé LA, Geddes JA, Nemet AJ, Young JB. A study to assess the effects of a broad-spectrum immune modulatory therapy on mortality and morbidity in patients with chronic heart failure: the ACCLAIM trial rationale and design. Can J Cardiol. 2007 Apr; 23(5):369-76.
Shibata R, Izumiya Y, Sato K, Papanicolaou K, Kihara S, Colucci WS, Sam F, Ouchi N, Walsh K. Adiponectin protects against the development of systolic dysfunction following myocardial infarction. J Mol Cell Cardiol. 2007 Jun; 42(6):1065-74.
Givertz MM, Andreou C, Conrad CH, Colucci WS. Direct myocardial effects of levosimendan in humans with left ventricular dysfunction: alteration of force-frequency and relaxation-frequency relationships. Circulation. 2007 Mar 13; 115(10):1218-24.
Kuster GM, Siwik DA, Pimentel DR, Colucci WS. Role of reversible, thioredoxin-sensitive oxidative protein modifications in cardiac myocytes. Antioxid Redox Signal. 2006 Nov-Dec; 8(11-12):2153-9.
Arnlöv J, Evans JC, Benjamin EJ, Larson MG, Levy D, Sutherland P, Siwik DA, Wang TJ, Colucci WS, Vasan RS. Clinical and echocardiographic correlates of plasma osteopontin in the community: the Framingham Heart Study. Heart. 2006 Oct; 92(10):1514-5.
Gheorghiade M, van Veldhuisen DJ, Colucci WS. Contemporary use of digoxin in the management of cardiovascular disorders. Circulation. 2006 May 30; 113(21):2556-64.
De Luca L, Colucci WS, Nieminen MS, Massie BM, Gheorghiade M. Evidence-based use of levosimendan in different clinical settings. Eur Heart J. 2006 Aug; 27(16):1908-20.
Cohn JN, Colucci W. Cardiovascular effects of aldosterone and post-acute myocardial infarction pathophysiology. Am J Cardiol. 2006 May 22; 97(10A):4F-12F.
Izumiya Y, Shiojima I, Sato K, Sawyer DB, Colucci WS, Walsh K. Vascular endothelial growth factor blockade promotes the transition from compensatory cardiac hypertrophy to failure in response to pressure overload. Hypertension. 2006 May; 47(5):887-93.
Kotlyar E, Vita JA, Winter MR, Awtry EH, Siwik DA, Keaney JF, Sawyer DB, Cupples LA, Colucci WS, Sam F. The relationship between aldosterone, oxidative stress, and inflammation in chronic, stable human heart failure. J Card Fail. 2006 Mar; 12(2):122-7.
Ahmed A, Rich MW, Love TE, Lloyd-Jones DM, Aban IB, Colucci WS, Adams KF, Gheorghiade M. Digoxin and reduction in mortality and hospitalization in heart failure: a comprehensive post hoc analysis of the DIG trial. Eur Heart J. 2006 Jan; 27(2):178-86.
Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, Colucci WS, Walsh K. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest. 2005 Aug; 115(8):2108-18.
Sam F, Kerstetter DL, Pimental DR, Mulukutla S, Tabaee A, Bristow MR, Colucci WS, Sawyer DB. Increased reactive oxygen species production and functional alterations in antioxidant enzymes in human failing myocardium. J Card Fail. 2005 Aug; 11(6):473-80.
Rude MK, Duhaney TA, Kuster GM, Judge S, Heo J, Colucci WS, Siwik DA, Sam F. Aldosterone stimulates matrix metalloproteinases and reactive oxygen species in adult rat ventricular cardiomyocytes. Hypertension. 2005 Sep; 46(3):555-61.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A, Colucci WS, Liao R. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res. 2005 Jul 8; 97(1):52-61.
Communal C, Colucci WS. The control of cardiomyocyte apoptosis via the beta-adrenergic signaling pathways. Arch Mal Coeur Vaiss. 2005 Mar; 98(3):236-41.
Kuster GM, Pimentel DR, Adachi T, Ido Y, Brenner DA, Cohen RA, Liao R, Siwik DA, Colucci WS. Alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes is mediated via thioredoxin-1-sensitive oxidative modification of thiols on Ras. Circulation. 2005 Mar 8; 111(9):1192-8.
McMurray J, Køber L, Robertson M, Dargie H, Colucci W, Lopez-Sendon J, Remme W, Sharpe DN, Ford I. Antiarrhythmic effect of carvedilol after acute myocardial infarction: results of the Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction (CAPRICORN) trial. J Am Coll Cardiol. 2005 Feb 15; 45(4):525-30.
Bianchi P, Pimentel DR, Murphy MP, Colucci WS, Parini A. A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J. 2005 Apr; 19(6):641-3.
Kuster GM, Kotlyar E, Rude MK, Siwik DA, Liao R, Colucci WS, Sam F. Mineralocorticoid receptor inhibition ameliorates the transition to myocardial failure and decreases oxidative stress and inflammation in mice with chronic pressure overload. Circulation. 2005 Feb 1; 111(4):420-7.
Taniyama Y, Ito M, Sato K, Kuester C, Veit K, Tremp G, Liao R, Colucci WS, Ivashchenko Y, Walsh K, Shiojima I. Akt3 overexpression in the heart results in progression from adaptive to maladaptive hypertrophy. J Mol Cell Cardiol. 2005 Feb; 38(2):375-85.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Fourth Edition, Braunwald E (Series Editor). Current Medicine. 2005.
58.
Shibata R, Ouchi N, Ito M, Kihara S, Shiojima I, Pimentel DR, Kumada M, Sato K, Schiekofer S, Ohashi K, Funahashi T, Colucci WS, Walsh K. Adiponectin-mediated modulation of hypertrophic signals in the heart. Nat Med. 2004 Dec; 10(12):1384-9.
Freudenberger RS, Schwarz RP, Brown J, Moore A, Mann D, Givertz MM, Colucci WS, Hare JM. Rationale, design and organisation of an efficacy and safety study of oxypurinol added to standard therapy in patients with NYHA class III – IV congestive heart failure. Expert Opin Investig Drugs. 2004 Nov; 13(11):1509-16.
Sundström J, Evans JC, Benjamin EJ, Levy D, Larson MG, Sawyer DB, Siwik DA, Colucci WS, Wilson PW, Vasan RS. Relations of plasma total TIMP-1 levels to cardiovascular risk factors and echocardiographic measures: the Framingham heart study. Eur Heart J. 2004 Sep; 25(17):1509-16.
Ito M, Adachi T, Pimentel DR, Ido Y, Colucci WS. Statins inhibit beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes via a Rac1-dependent mechanism. Circulation. 2004 Jul 27; 110(4):412-8.
Sundström J, Evans JC, Benjamin EJ, Levy D, Larson MG, Sawyer DB, Siwik DA, Colucci WS, Sutherland P, Wilson PW, Vasan RS. Relations of plasma matrix metalloproteinase-9 to clinical cardiovascular risk factors and echocardiographic left ventricular measures: the Framingham Heart Study. Circulation. 2004 Jun 15; 109(23):2850-6.
Colucci WS. Landmark study: the Carvedilol Post-Infarct Survival Control in Left Ventricular Dysfunction Study (CAPRICORN). Am J Cardiol. 2004 May 6; 93(9A):13B-6B.
Mann DL, McMurray JJ, Packer M, Swedberg K, Borer JS, Colucci WS, Djian J, Drexler H, Feldman A, Kober L, Krum H, Liu P, Nieminen M, Tavazzi L, van Veldhuisen DJ, Waldenstrom A, Warren M, Westheim A, Zannad F, Fleming T. Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation. 2004 Apr 6; 109(13):1594-602.
Vasan RS, Evans JC, Benjamin EJ, Levy D, Larson MG, Sundstrom J, Murabito JM, Sam F, Colucci WS, Wilson PW. Relations of serum aldosterone to cardiac structure: gender-related differences in the Framingham Heart Study. Hypertension. 2004 May; 43(5):957-62.
Maytin M, Siwik DA, Ito M, Xiao L, Sawyer DB, Liao R, Colucci WS. Pressure overload-induced myocardial hypertrophy in mice does not require gp91phox. Circulation. 2004 Mar 9; 109(9):1168-71.
Sam F, Xie Z, Ooi H, Kerstetter DL, Colucci WS, Singh M, Singh K. Mice lacking osteopontin exhibit increased left ventricular dilation and reduced fibrosis after aldosterone infusion. Am J Hypertens. 2004 Feb; 17(2):188-93.
Giles TD, Chatterjee K, Cohn JN, Colucci WS, Feldman AM, Ferrans VJ, Roberts R. Definition, classification, and staging of the adult cardiomyopathies: a proposal for revision. J Card Fail. 2004 Feb; 10(1):6-8.
Siwik DA, Colucci WS. Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev. 2004 Jan; 9(1):43-51.
Sawyer DB, Colucci WS. Oxidative stress in heart failure; (Chapter 12). In: Mann DL (ed) Heart Failure: A Companion to Braunwald’s Heart Disease. Saunders. 2004; 181-92.
73.
Maytin M, Sawyer DB and Colucci WS. Role of reactive oxygen species in the regulation of cardiac myocyte phenotype. In: Pathophysiology of Cardiovascular Disease. Dhalla NS, Rupp H, Angel A and Pierce GN (eds). 51-7:Kluwer Academic Publishers . 2004.
74.
Kuramochi Y, Lim CC, Guo X, Colucci WS, Liao R, Sawyer DB. Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1beta. Am J Physiol Cell Physiol. 2004 Feb; 286(2):C222-9.
Torre-Amione G, Young JB, Colucci WS, Lewis BS, Pratt C, Cotter G, Stangl K, Elkayam U, Teerlink JR, Frey A, Rainisio M, Kobrin I. Hemodynamic and clinical effects of tezosentan, an intravenous dual endothelin receptor antagonist, in patients hospitalized for acute decompensated heart failure. J Am Coll Cardiol. 2003 Jul 2; 42(1):140-7.
Communal C, Singh M, Menon B, Xie Z, Colucci WS, Singh K. beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of beta-adrenergic receptor-stimulated apoptosis. J Cell Biochem. 2003 May 15; 89(2):381-8.
Sam F, Sawyer DB and Colucci WS. Myocardial nitric oxide in cardiac remodeling. In: Inflammation and Cardiac Diseases. Feuerstein GZ, Libby P and Mann DL (eds). Birkhäuser. 2003; 155-170.
82.
Siwik DA, Pimentel DR, Xiao L, Singh K, Sawyer DB, and Colucci WS. Adrenergic and mechanical regulation of oxidative stress in the myocardium. In: Kukin ML, Fuster V (eds). Oxidative Stress and Cardiac Failure. Armonk, NY:Futura Publishing Co., Inc.. 2003; 153-171.
83.
Ooi H, Colucci WS, Givertz MM. Endothelin mediates increased pulmonary vascular tone in patients with heart failure: demonstration by direct intrapulmonary infusion of sitaxsentan. Circulation. 2002 Sep 24; 106(13):1618-21.
Hare JM, Nguyen GC, Massaro AF, Drazen JM, Stevenson LW, Colucci WS, Fang JC, Johnson W, Givertz MM, Lucas C. Exhaled nitric oxide: a marker of pulmonary hemodynamics in heart failure. J Am Coll Cardiol. 2002 Sep 18; 40(6):1114-9.
Xiao L, Pimentel DR, Wang J, Singh K, Colucci WS, Sawyer DB. Role of reactive oxygen species and NAD(P)H oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am J Physiol Cell Physiol. 2002 Apr; 282(4):C926-34.
Communal C, Colucci WS, Remondino A, Sawyer DB, Port JD, Wichman SE, Bristow MR, Singh K. Reciprocal modulation of mitogen-activated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium. J Card Fail. 2002 Apr; 8(2):86-92.
Sawyer DB, Colucci WS. Molecular and cellular events in myocardial hypertrophy and failure. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
93.
Givertz MM, Colucci WS. Beta-Blockers. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
94.
Givertz MM, Colucci WS. Treatment of heart failure: New approaches. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Third Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 2002.
95.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Third Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 2002.
Sam F, Sawyer DB, Xie Z, Chang DL, Ngoy S, Brenner DA, Siwik DA, Singh K, Apstein CS, Colucci WS. Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction. Circ Res. 2001 Aug 17; 89(4):351-6.
Loh E, Elkayam U, Cody R, Bristow M, Jaski B, Colucci WS. A randomized multicenter study comparing the efficacy and safety of intravenous milrinone and intravenous nitroglycerin in patients with advanced heart failure. J Card Fail. 2001 Jun; 7(2):114-21.
Givertz MM, Slawsky MT, Moraes DL, McIntyre KM, Colucci WS. Noninvasive determination of pulmonary artery wedge pressure in patients with chronic heart failure. Am J Cardiol. 2001 May 15; 87(10):1213-5; A7.
Yancy CW, Fowler MB, Colucci WS, Gilbert EM, Bristow MR, Cohn JN, Lukas MA, Young ST, Packer M. Race and the response to adrenergic blockade with carvedilol in patients with chronic heart failure. N Engl J Med. 2001 May 3; 344(18):1358-65.
Fowler MB, Vera-Llonch M, Oster G, Bristow MR, Cohn JN, Colucci WS, Gilbert EM, Lukas MA, Lacey MJ, Richner R, Young ST, Packer M. Influence of carvedilol on hospitalizations in heart failure: incidence, resource utilization and costs. U.S. Carvedilol Heart Failure Study Group. J Am Coll Cardiol. 2001 May; 37(6):1692-9.
Givertz MM, Sawyer DB, Colucci WS. Antioxidants and myocardial contractility: illuminating the “Dark Side” of beta-adrenergic receptor activation? Circulation. 2001 Feb 13; 103(6):782-3.
Ooi H and Colucci WS. Pharmacological Treatment of Heart Failure; (Chapter 34). In: Hardman JG, Limbird LE and Gilman AG (eds): Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw Hill. McGraw Hill. 2001; 901-932.
113.
Colucci WS and Braunwald E. Pathophysiology of Heart Failure, (Chapter 16). In: Braunwald E (ed): Heart Disease. 6th Edition. Philadelphia:WB Saunders Co. 2001; 503-533.
114.
Colucci WS and Schoen FJ. Primary Tumors of the Heart; (Chapter 49). In: Braunwald E. (ed): Heart Disease. 6th Edition. Philadelphia:WB Saunders Co. 2001; 1807-22.
115.
Ooi H and Colucci WS. Congestive Heart Failure. In: Rakel & Bope: Conn’s Current Therapy. Philadelphia:WB Saunders Co. 2001; pp. 310-14.
116.
Colucci WS. Heart Failure. In: Essential Atlas of Heart Diseases, Second Edition, Braunwald E (Editor–in-Chief). Philadelphia:Current Medicine. 2001.
117.
Holubarsch CJ, Colucci WS, Meinertz T, Gaus W, Tendera M. Survival and prognosis: investigation of Crataegus extract WS 1442 in congestive heart failure (SPICE)–rationale, study design and study protocol. Eur J Heart Fail. 2000 Dec; 2(4):431-7.
Lim CC, Apstein CS, Colucci WS, Liao R. Impaired cell shortening and relengthening with increased pacing frequency are intrinsic to the senescent mouse cardiomyocyte. J Mol Cell Cardiol. 2000 Nov; 32(11):2075-82.
Slawsky MT, Colucci WS, Gottlieb SS, Greenberg BH, Haeusslein E, Hare J, Hutchins S, Leier CV, LeJemtel TH, Loh E, Nicklas J, Ogilby D, Singh BN, Smith W. Acute hemodynamic and clinical effects of levosimendan in patients with severe heart failure. Study Investigators. Circulation. 2000 Oct 31; 102(18):2222-7.
Moraes DL, Colucci WS, Givertz MM. Secondary pulmonary hypertension in chronic heart failure: the role of the endothelium in pathophysiology and management. Circulation. 2000 Oct 3; 102(14):1718-23.
Singh K, Communal C, Colucci WS. Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation. Basic Res Cardiol. 2000 Oct; 95(5):389-96.
Colucci WS, Elkayam U, Horton DP, Abraham WT, Bourge RC, Johnson AD, Wagoner LE, Givertz MM, Liang CS, Neibaur M, Haught WH, LeJemtel TH. Intravenous nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure. Nesiritide Study Group. N Engl J Med. 2000 Jul 27; 343(4):246-53.
Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, Amin J, Apstein CS, Colucci WS. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol. 2000 Jul; 279(1):H422-8.
Givertz MM, Colucci WS, LeJemtel TH, Gottlieb SS, Hare JM, Slawsky MT, Leier CV, Loh E, Nicklas JM, Lewis BE. Acute endothelin A receptor blockade causes selective pulmonary vasodilation in patients with chronic heart failure. Circulation. 2000 Jun 27; 101(25):2922-7.
Siwik DA, Chang DL, Colucci WS. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro. Circ Res. 2000 Jun 23; 86(12):1259-65.
Cuffe MS, Califf RM, Adams KF, Bourge RC, Colucci W, Massie B, O’Connor CM, Pina I, Quigg R, Silver M, Robinson LA, Leimberger JD, Gheorghiade M. Rationale and design of the OPTIME CHF trial: outcomes of a prospective trial of intravenous milrinone for exacerbations of chronic heart failure. Am Heart J. 2000 Jan; 139(1 Pt 1):15-22.
Sawyer DB, Colucci, WS. Myocardial Nitric Oxide in Heart Failure. In: Loscalzo J and Vita JA, (ed): Contemporary Cardiology: Nitric Oxide and the Cardiovascular System. Totowa, NJ:Humana Press Inc. 2000; pp. 309-19.
137.
Sawyer DB, Colucci WS. Role of oxidative stress, cytokines and apoptosis in myocardial dysfunction. In: Tardiff J-C and Bourassa MG, ed. Antioxidants and Cardiovascular Disease. Dordrecht:Kluwar. 2000.
138.
Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensitive G protein. Circulation. 1999 Nov 30; 100(22):2210-2.
Givertz MM, Colucci WS. Treatment of heart failure: New approaches. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Second Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1999.
143.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, Second Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 1999.
144.
Sawyer DB, Colucci WS. Molecular and cellular events in myocardial hypertrophy and failure. In: “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Second Edition, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1999.
145.
Colucci WS. The effects of norepinephrine on myocardial biology: implications for the therapy of heart failure. Clin Cardiol. 1998 Dec; 21(12 Suppl 1):I20-4.
Eberli FR, Sam F, Ngoy S, Apstein CS, Colucci WS. Left-ventricular structural and functional remodeling in the mouse after myocardial infarction: assessment with the isovolumetrically-contracting Langendorff heart. J Mol Cell Cardiol. 1998 Jul; 30(7):1443-7.
Lo MW, Toh J, Emmert SE, Ritter MA, Furtek CI, Lu H, Colucci WS, Uretsky BF, Rucinska E. Pharmacokinetics of intravenous and oral losartan in patients with heart failure. J Clin Pharmacol. 1998 Jun; 38(6):525-32.
Calderone A, Thaik CM, Takahashi N, Chang DL, Colucci WS. Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J Clin Invest. 1998 Feb 15; 101(4):812-8.
Hare JM, Givertz MM, Creager MA, Colucci WS. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta-adrenergic inotropic responsiveness. Circulation. 1998 Jan 20; 97(2):161-6.
Cohn JN, Fowler MB, Bristow MR, Colucci WS, Gilbert EM, Kinhal V, Krueger SK, Lejemtel T, Narahara KA, Packer M, Young ST, Holcslaw TL, Lukas MA. Safety and efficacy of carvedilol in severe heart failure. The U.S. Carvedilol Heart Failure Study Group. J Card Fail. 1997 Sep; 3(3):173-9.
Hare JM, Shernan SK, Body SC, Graydon E, Colucci WS, Couper GS. Influence of inhaled nitric oxide on systemic flow and ventricular filling pressure in patients receiving mechanical circulatory assistance. Circulation. 1997 May 6; 95(9):2250-3.
Cohn JN, Bristow MR, Chien KR, Colucci WS, Frazier OH, Leinwand LA, Lorell BH, Moss AJ, Sonnenblick EH, Walsh RA, Mockrin SC, Reinlib L. Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research. Circulation. 1997 Feb 18; 95(4):766-70.
Colucci WS, Braunwald E. Cardiac tumors, cardiac manifestations of systemic diseases, and traumatic cardiac injury, Chapter 241. In: Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, Kasper DL, Hauser SL, Longo DL, eds. Harrison’s Principles of Internal Medicine, 14th Edition. New York:McGraw-Hill. 1997; pp 1341-4.
161.
Colucci WS, Schoen FJ, Braunwald E. Primary tumors of the heart, Chapter 42. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co. 1997; pp 1464-77.
162.
Colucci WS, Braunwald E. Pathophysiology of heart failure, Chapter 13. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co. 1997; pp 394-420.
163.
Colucci WS. Heart Failure. In: Essential Atlas of Heart Diseases, First Edition, Braunwald E (Editor–in-Chief). Philadelphia:Current Medicine. 1997.
164.
Braunwald E, Colucci WS, Grossman W. Clinical aspects of heart failure, Chapter 15. In: Braunwald E, ed. Heart Disease, 5th Edition. Philadelphia:WB Saunders Co.. 1997; pp 445-70.
165.
Newton GE, Parker AB, Landzberg JS, Colucci WS, Parker JD. Muscarinic receptor modulation of basal and beta-adrenergic stimulated function of the failing human left ventricle. J Clin Invest. 1996 Dec 15; 98(12):2756-63.
Packer M, Colucci WS, Sackner-Bernstein JD, Liang CS, Goldscher DA, Freeman I, Kukin ML, Kinhal V, Udelson JE, Klapholz M, Gottlieb SS, Pearle D, Cody RJ, Gregory JJ, Kantrowitz NE, LeJemtel TH, Young ST, Lukas MA, Shusterman NH. Double-blind, placebo-controlled study of the effects of carvedilol in patients with moderate to severe heart failure. The PRECISE Trial. Prospective Randomized Evaluation of Carvedilol on Symptoms and Exercise. Circulation. 1996 Dec 1; 94(11):2793-9.
Givertz MM, Hare JM, Loh E, Gauthier DF, Colucci WS. Effect of bolus milrinone on hemodynamic variables and pulmonary vascular resistance in patients with severe left ventricular dysfunction: a rapid test for reversibility of pulmonary hypertension. J Am Coll Cardiol. 1996 Dec; 28(7):1775-80.
Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, Shusterman NH. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group. N Engl J Med. 1996 May 23; 334(21):1349-55.
Colucci WS. Pathophysiologic and clinical considerations in the treatment of heart failure: An overview. Chapter 8. In: Cardiovascular Therapeutics, Smith TW (Editor-in-Chief). Philadelphia:WB Saunders. 1996; pp 171-175.
177.
Stevenson LW, Colucci WS. Management of patients hospitalized with heart failure, Chapter 10. In Cardiovascular Therapeutics, Smith TW (Editor-in-Chief). Philadelphia:WB Saunders. 1996; pp 199-209.
178.
Colucci WS. Principles and practice of inotropic therapy, Chapter 126. In: Messerli FH, ed. Cardiovascular Drug Therapy, 2nd Edition. Philadelphia:WB Saunders Co. 1996; pp 1146-1150.
Calderone A, Takahashi N, Izzo NJ, Thaik CM, Colucci WS. Pressure- and volume-induced left ventricular hypertrophies are associated with distinct myocyte phenotypes and differential induction of peptide growth factor mRNAs. Circulation. 1995 Nov 1; 92(9):2385-90.
Hare JM, Loh E, Creager MA, Colucci WS. Nitric oxide inhibits the positive inotropic response to beta-adrenergic stimulation in humans with left ventricular dysfunction. Circulation. 1995 Oct 15; 92(8):2198-203.
Parker JD, Newton GE, Landzberg JS, Floras JS, Colucci WS. Functional significance of presynaptic alpha-adrenergic receptors in failing and nonfailing human left ventricle. Circulation. 1995 Oct 1; 92(7):1793-800.
Thaik CM, Calderone A, Takahashi N, Colucci WS. Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes. J Clin Invest. 1995 Aug; 96(2):1093-9.
Loh E, Barnett JV, Feldman AM, Couper GS, Vatner DE, Colucci WS, Galper JB. Decreased adenylate cyclase activity and expression of Gs alpha in human myocardium after orthotopic cardiac transplantation. Circ Res. 1995 May; 76(5):852-60.
Hare JM, Keaney JF, Balligand JL, Loscalzo J, Smith TW, Colucci WS. Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs. J Clin Invest. 1995 Jan; 95(1):360-6.
Colucci WS (Editor): Atlas of Heart Failure – Cardiac Function and Dysfunction, First Edition, Braunwald E (Series Editor). Philadelphia:Current Medicine. 1995.
189.
Colucci WS. Treatment of stable heart failure: New approaches. In “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1995.
190.
Thaik C, Colucci WS. Molecular and cellular abnormalities in hypertrophied and failing myocardium. In “Heart Failure: Cardiac Function and Dysfunction”, Colucci WS (ed): In: Atlas of Heart Diseases, Braunwald E (Editor-in-Chief). Philadelphia:Current Medicine. 1995.
191.
Colucci WS. Secondary molecular alterations in failing human myocardium. In: Molecular Interventions and Local Drug Delivery in Cardiovascular Disease, Edelman ER (ed). London:WB Saunders. 1995.
192.
Loh E, Stamler JS, Hare JM, Loscalzo J, Colucci WS. Cardiovascular effects of inhaled nitric oxide in patients with left ventricular dysfunction. Circulation. 1994 Dec; 90(6):2780-5.
Izzo NJ, Colucci WS. Regulation of alpha 1B-adrenergic receptor half-life: protein synthesis dependence and effect of norepinephrine. Am J Physiol. 1994 Mar; 266(3 Pt 1):C771-5.
Landzberg JS, Parker JD, Gauthier DF, Colucci WS. Effects of intracoronary acetylcholine and atropine on basal and dobutamine-stimulated left ventricular contractility. Circulation. 1994 Jan; 89(1):164-8.
Matoba Y, Colucci WS, Fields BN, Smith TW. The reovirus M1 gene determines the relative capacity of growth of reovirus in cultured bovine aortic endothelial cells. J Clin Invest. 1993 Dec; 92(6):2883-8.
Colucci WS, Sonnenblick EH, Adams KF, Berk M, Brozena SC, Cowley AJ, Grabicki JM, Kubo SA, LeJemtel T, Littler WA, et al. Efficacy of phosphodiesterase inhibition with milrinone in combination with converting enzyme inhibitors in patients with heart failure. The Milrinone Multicenter Trials Investigators. J Am Coll Cardiol. 1993 Oct; 22(4 Suppl A):113A-118A.
Schmidt TA, Allen PD, Colucci WS, Marsh JD, Kjeldsen K. No adaptation to digitalization as evaluated by digitalis receptor (Na,K-ATPase) quantification in explanted hearts from donors without heart disease and from digitalized recipients with end-stage heart failure. Am J Cardiol. 1993 Jan 1; 71(1):110-4.
Packer M, Narahara KA, Elkayam U, Sullivan JM, Pearle DL, Massie BM, Creager MA, and the Principal Investigators of the Reflect Study. Double-blind, placebo-controlled study of the efficacy of flosequinan in patients with chronic heart failure. J Am Coll Cardiol. 1993; 22:65-72.
201.
Colucci WS. In situ assessment of – and -Adrenergic responses in failing human myocardium. Circulation. 1993; 87(Suppl VII):63-7.
202.
Feldman AM, Bristow MR, Parmley WW, Carson PE, Pepine CJ, Gilbert EM, Strobeck JE, Hendrix GH, Powers ER, Bain RP, White BH, for the Vesnarinone Study Group. Effects of vesnarinone on morbidity and mortality in patients with heart failure. N Engl J Med. 1993; 329:149-55.
203.
Bialecki RA, Kulik TJ, Colucci WS. Stretching increases calcium influx and efflux in cultured pulmonary arterial smooth muscle cells. Am J Physiol. 1992 Nov; 263(5 Pt 1):L602-6.
Sen L, Bialecki RA, Smith E, Smith TW, Colucci WS. Cholesterol increases the L-type voltage-sensitive calcium channel current in arterial smooth muscle cells. Circ Res. 1992 Oct; 71(4):1008-14.
Willich SN, Tofler GH, Brezinski DA, Schafer AI, Muller JE, Michel T, Colucci WS. Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability. Eur Heart J. 1992 Apr; 13(4):550-5.
Parker JD, Landzberg JS, Bittl JA, Mirsky I, Colucci WS. Effects of beta-adrenergic stimulation with dobutamine on isovolumic relaxation in the normal and failing human left ventricle. Circulation. 1991 Sep; 84(3):1040-8.
Sen L, Liang BT, Colucci WS, Smith TW. Enhanced alpha 1-adrenergic responsiveness in cardiomyopathic hamster cardiac myocytes. Relation to the expression of pertussis toxin-sensitive G protein and alpha 1-adrenergic receptors. Circ Res. 1990 Nov; 67(5):1182-92.
Colucci WS. In vivo studies of myocardial beta-adrenergic receptor pharmacology in patients with congestive heart failure. Circulation. 1990 Aug; 82(2 Suppl):I44-51.
Arnold JM, Ribeiro JP, Colucci WS. Muscle blood flow during forearm exercise in patients with severe heart failure. Circulation. 1990 Aug; 82(2):465-72.
Treasure CB, Vita JA, Cox DA, Fish RD, Gordon JB, Mudge GH, Colucci WS, Sutton MG, Selwyn AP, Alexander RW, et al. Endothelium-dependent dilation of the coronary microvasculature is impaired in dilated cardiomyopathy. Circulation. 1990 Mar; 81(3):772-9.
Ribeiro JP, White HD, Hartley LH, Colucci WS. Acute increase in exercise capacity with milrinone: lack of correlation with resting hemodynamic responses. Braz J Med Biol Res. 1990; 23(11):1069-78.
Denniss AR, Colucci WS, Allen PD, Marsh JD. Distribution and function of human ventricular beta adrenergic receptors in congestive heart failure. J Mol Cell Cardiol. 1989 Jul; 21(7):651-60.
Denniss AR, Marsh JD, Quigg RJ, Gordon JB, Colucci WS. Beta-adrenergic receptor number and adenylate cyclase function in denervated transplanted and cardiomyopathic human hearts. Circulation. 1989 May; 79(5):1028-34.
Colucci WS. Observations on the intracoronary administration of milrinone and dobutamine to patients with congestive heart failure. Am J Cardiol. 1989 Jan 3; 63(2):17A-22A.
Arai Y, Saul JP, Albrecht P, Hartley LH, Lilly LS, Cohen RJ, Colucci WS. Modulation of cardiac autonomic activity during and immediately after exercise. Am J Physiol. 1989 Jan; 256(1 Pt 2):H132-41.
Colucci WS, Parker JD. Effects of beta-adrenergic agents on systolic and diastolic myocardial function in patients with and without heart failure. J Cardiovasc Pharmacol. 1989; 14 Suppl 5:S28-37.
Leatherman GF, Shook TL, Leatherman SM, Colucci WS. Use of a conductance catheter to detect increased left ventricular inotropic state by end-systolic pressure-volume analysis. Basic Res Cardiol. 1989; 84 Suppl 1:247-56.
Colucci WS, Akers M, Wise GM. Differential effects of norepinephrine and phorbol ester on alpha-1 adrenergic receptor number and surface-accessibility in DDT1 MF-2 cells. Biochem Biophys Res Commun. 1988 Oct 31; 156(2):924-30.
Colucci WS. Do positive inotropic agents adversely affect the survival of patients with chronic congestive heart failure? III. Antagonist’s viewpoint. J Am Coll Cardiol. 1988 Aug; 12(2):566-9.
Colucci WS, Denniss AR, Leatherman GF, Quigg RJ, Ludmer PL, Marsh JD, Gauthier DF. Intracoronary infusion of dobutamine to patients with and without severe congestive heart failure. Dose-response relationships, correlation with circulating catecholamines, and effect of phosphodiesterase inhibition. J Clin Invest. 1988 Apr; 81(4):1103-10.
Givertz MM, Colucci WS. Inotropic and vasoactive agents in the cardiac intensive care unit, Chapter 45. In: Brown DL, ed. Cardiac Intensive Care. Philadelphia:WB Saunders Co. 1988; pp. 545-54.
239.
Colucci WS, Leatherman GF, Ludmer PL, Gauthier DF. Beta-adrenergic inotropic responsiveness of patients with heart failure: studies with intracoronary dobutamine infusion. Circ Res. 1987 Oct; 61(4 Pt 2):I82-6.
Ribeiro JP, Knutzen A, Rocco MB, Hartley LH, Colucci WS. Periodic breathing during exercise in severe heart failure. Reversal with milrinone or cardiac transplantation. Chest. 1987 Sep; 92(3):555-6.
Ribeiro JP, White HD, Arnold JM, Hartley LH, Colucci WS. Exercise responses before and after long-term treatment with oral milrinone in patients with severe heart failure. Am J Med. 1986 Nov; 81(5):759-64.
Arnold JM, Ludmer PL, Wright RF, Ganz P, Braunwald E, Colucci WS. Role of reflex sympathetic withdrawal in the hemodynamic response to an increased inotropic state in patients with severe heart failure. J Am Coll Cardiol. 1986 Aug; 8(2):413-8.
Baim DS, Colucci WS, Monrad ES, Smith HS, Wright RF, Lanoue A, Gauthier DF, Ransil BJ, Grossman W, Braunwald E. Survival of patients with severe congestive heart failure treated with oral milrinone. J Am Coll Cardiol. 1986 Mar; 7(3):661-70.
Colucci WS, Wright RF, Jaski BE, Fifer MA, Braunwald E. Milrinone and dobutamine in severe heart failure: differing hemodynamic effects and individual patient responsiveness. Circulation. 1986 Mar; 73(3 Pt 2):III175-83.
Colucci WS, Alexander RW. Norepinephrine-induced alteration in the coupling of alpha 1-adrenergic receptor occupancy to calcium efflux in rabbit aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1986 Mar; 83(6):1743-6.
Colucci WS, Wright RF, Braunwald E. New positive inotropic agents in the treatment of congestive heart failure. Mechanisms of action and recent clinical developments. 2. N Engl J Med. 1986 Feb 6; 314(6):349-58.
Colucci WS, Wright RF, Braunwald E. New positive inotropic agents in the treatment of congestive heart failure. Mechanisms of action and recent clinical developments. 1. N Engl J Med. 1986 Jan 30; 314(5):290-9.
Ludmer PL, Wright RF, Arnold JM, Ganz P, Braunwald E, Colucci WS. Separation of the direct myocardial and vasodilator actions of milrinone administered by an intracoronary infusion technique. Circulation. 1986 Jan; 73(1):130-7.
Powers RE, Colucci WS. An increase in putative voltage dependent calcium channel number following reserpine treatment. Biochem Biophys Res Commun. 1985 Oct 30; 132(2):844-9.
White HD, Ribeiro JP, Hartley LH, Colucci WS. Immediate effects of milrinone on metabolic and sympathetic responses to exercise in severe congestive heart failure. Am J Cardiol. 1985 Jul 1; 56(1):93-8.
Fifer MA, Colucci WS, Lorell BH, Jaski BE, Barry WH. Inotropic, vascular and neuroendocrine effects of nifedipine in heart failure: comparison with nitroprusside. J Am Coll Cardiol. 1985 Mar; 5(3):731-7.
Jaski BE, Fifer MA, Wright RF, Braunwald E, Colucci WS. Positive inotropic and vasodilator actions of milrinone in patients with severe congestive heart failure. Dose-response relationships and comparison to nitroprusside. J Clin Invest. 1985 Feb; 75(2):643-9.
Colucci WS, Ludmer PL, Wright RF, Arnold JM, Ganz P, Braunwald E. Myocardial and vascular effects of intracoronary versus intravenous milrinone. Trans Assoc Am Physicians. 1985; 98:136-45.
Colucci WS, Brock TA, Atkinson WJ, Alexander RW, Gimbrone MA. Cultured vascular smooth muscle cells: an in vitro system for study of alpha-adrenergic receptor coupling and regulation. J Cardiovasc Pharmacol. 1985; 7 Suppl 6:S79-86.
Monrad ES, McKay RG, Baim DS, Colucci WS, Fifer MA, Heller GV, Royal HD, Grossman W. Improvement in indexes of diastolic performance in patients with congestive heart failure treated with milrinone. Circulation. 1984 Dec; 70(6):1030-7.
Ganz P, Gaspar J, Colucci WS, Barry WH, Mudge GH, Alexander RW. Effects of prostacyclin on coronary hemodynamics at rest and in response to cold pressor testing in patients with angina pectoris. Am J Cardiol. 1984 Jun 1; 53(11):1500-4.
Polak JF, Holman BL, Wynne J, Colucci WS. Right ventricular ejection fraction: an indicator of increased mortality in patients with congestive heart failure associated with coronary artery disease. J Am Coll Cardiol. 1983 Aug; 2(2):217-24.
Colucci WS. New developments in alpha-adrenergic receptor pharmacology: implications for the initial treatment of hypertension. Am J Cardiol. 1983 Feb 24; 51(4):639-43.
Colucci WS, Lorell BH, Schoen FJ, Warhol MJ, Grossman W. Hypertrophic obstructive cardiomyopathy due to Fabry’s disease. N Engl J Med. 1982 Oct 7; 307(15):926-8.
Colucci WS. Alpha-adrenergic receptor blockade with prazosin. Consideration of hypertension, heart failure, and potential new applications. Ann Intern Med. 1982 Jul; 97(1):67-77.
Rude RE, Grossman W, Colucci WS, Benotti JR, Carabello BA, Wynne J, Malacoff R, Braunwald E. Problems in assessment of new pharmacologic agents for the heart failure patient. Am Heart J. 1981 Sep; 102(3 Pt 2):584-90.
Colucci WS, Alexander RW, Mudge GH, Rude RE, Holman BL, Wynne J, Grossman W, Braunwald E. Acute and chronic effects of pirbuterol on left ventricular ejection fraction and clinical status in severe congestive heart failure. Am Heart J. 1981 Sep; 102(3 Pt 2):564-8.
Colucci WS, Williams GH, Braunwald E. Clinical, hemodynamic, and neuroendocrine effects of chronic prazosin therapy for congestive heart failure. Am Heart J. 1981 Sep; 102(3 Pt 2):615-21.
Colucci WS, Alexander RW, Williams GH, Rude RE, Holman BL, Konstam MA, Wynne J, Mudge GH, Braunwald E. Decreased lymphocyte beta-adrenergic-receptor density in patients with heart failure and tolerance to the beta-adrenergic agonist pirbuterol. N Engl J Med. 1981 Jul 23; 305(4):185-90.
Colucci WS, Williams GH, Alexander RW, Braunwald E. Mechanisms and implications of vasodilator tolerance in the treatment of congestive heart failure. Am J Med. 1981 Jul; 71(1):89-99.
Rude RE, Turi Z, Brown EJ, Lorell BH, Colucci WS, Mudge GH, Taylor CR, Grossman W. Acute effects of oral pirbuterol on myocardial oxygen metabolism and systemic hemodynamics in chronic congestive heart failure. Circulation. 1981 Jul; 64(1):139-45.
Dzau VJ, Colucci WS, Hollenberg NK, Williams GH. Relation of the renin-angiotensin-aldosterone system to clinical state in congestive heart failure. Circulation. 1981 Mar; 63(3):645-51.
Colucci WS, Gimbrone MA, Alexander RW. Regulation of the postsynaptic alpha-adrenergic receptor in rat mesenteric artery. Effects of chemical sympathectomy and epinephrine treatment. Circ Res. 1981 Jan; 48(1):104-11.
Colucci WS, Williams GH, Braunwald E. Increased plasma norepinephrine levels during prazosin therapy for severe congestive heart failure. Ann Intern Med. 1980 Sep; 93(3):452-3.
Dzau VJ, Colucci WS, Williams GH, Curfman G, Meggs L, Hollenberg NK. Sustained effectiveness of converting-enzyme inhibition in patients with severe congestive heart failure. N Engl J Med. 1980 Jun 19; 302(25):1373-9.
Colucci WS, Gimbrone MA, Alexander RW. Characterization of postsynaptic alpha-adrenergic receptors by [3H]-dihydroergocryptine binding in muscular arteries from the rat mesentery. Hypertension. 1980 Mar-Apr; 2(2):149-55.
Tokuda M, Kojodjojo P, Tung S, Tedrow UB, Nof E, Inada K, Koplan BA, Michaud GF, John RM, Epstein LM, Stevenson WG. Acute failure of catheter ablation for ventricular tachycardia due to structural heart disease: causes and significance. J Am Heart Assoc. 2013; 2(3):e000072.
Ng J, Barbhaiya C, Chopra N, Reichlin T, Nof E, Tadros T, Stevenson WG, John RM. Automatic external defibrillators-friend or foe? Am J Emerg Med. 2013 Aug; 31(8):1292.e1-2.
Steven D, Sultan A, Reddy V, Luker J, Altenburg M, Hoffmann B, Rostock T, Servatius H, Stevenson WG, Willems S, Michaud GF. Benefit of pulmonary vein isolation guided by loss of pace capture on the ablation line: results from a prospective 2-center randomized trial. J Am Coll Cardiol. 2013 Jul 2; 62(1):44-50.
Nof E, Stevenson WG, Epstein LM, Tedrow UB, Koplan BA. Catheter Ablation of Atrial Arrhythmias After Cardiac Transplantation: Findings at EP Study Utility of 3-D Mapping and Outcomes. J Cardiovasc Electrophysiol. 2013 May; 24(5):498-502.
Tracy CM, Epstein AE, Darbar D, Dimarco JP, Dunbar SB, Mark Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD, Epstein AE, Dimarco JP, Ellenbogen KA, Mark Estes NA, Freedman RA, Gettes LS, Marc Gillinov A, Gregoratos G, Hammill SC, Hayes DL, Hlatky MA, Kristin Newby L, Page RL, Schoenfeld MH, Silka MJ, Warner Stevenson L, Sweeney MO, Anderson JL, Jacobs AK, Halperin JL, Albert NM, Creager MA, Demets D, Ettinger SM, Guyton RA, Hochman JS, Kushner FG, Ohman EM, Stevenson W, Yancy CW. 2012 ACCF/AHA/HRS focused update of the 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Thorac Cardiovasc Surg. 2012 Dec; 144(6):e127-45.
John RM, Tedrow UB, Koplan BA, Albert CM, Epstein LM, Sweeney MO, Miller AL, Michaud GF, Stevenson WG. Ventricular arrhythmias and sudden cardiac death. Lancet. 2012 Oct 27; 380(9852):1520-9.
Tracy CM, Epstein AE, Darbar D, DiMarco JP, Dunbar SB, Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD, Ellenbogen KA, Freedman RA, Gettes LS, Gillinov AM, Gregoratos G, Hayes DL, Page RL, Stevenson LW, Sweeney MO. 2012 ACCF/AHA/HRS focused update of the 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2012 Oct 2; 126(14):1784-800.
Tracy CM, Epstein AE, Darbar D, Dimarco JP, Dunbar SB, Estes NA, Ferguson TB, Hammill SC, Karasik PE, Link MS, Marine JE, Schoenfeld MH, Shanker AJ, Silka MJ, Stevenson LW, Stevenson WG, Varosy PD. 2012 ACCF/AHA/HRS Focused Update of the 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2012 Oct; 9(10):1737-53.
Wissner E, Stevenson WG, Kuck KH. Catheter ablation of ventricular tachycardia in ischaemic and non-ischaemic cardiomyopathy: where are we today? A clinical review. Eur Heart J. 2012 Jun; 33(12):1440-50.
Vollmann D, Stevenson WG, Lüthje L, Sohns C, John RM, Zabel M, Michaud GF. Misleading long post-pacing interval after entrainment of typical atrial flutter from the cavotricuspid isthmus. J Am Coll Cardiol. 2012 Feb 28; 59(9):819-24.
Albert CM, Chen PS, Anderson ME, Cain ME, Fishman GI, Narayan SM, Olgin JE, Spooner PM, Stevenson WG, Van Wagoner DR, Packer DL. Full report from the first annual Heart Rhythm Society Research Forum: a vision for our research future, “dream, discover, develop, deliver”. Heart Rhythm. 2011 Dec; 8(12):e1-12.
Wijnmaalen AP, Roberts-Thomson KC, Steven D, Klautz RJ, Willems S, Schalij MJ, Stevenson WG, Zeppenfeld K. Catheter ablation of ventricular tachycardia after left ventricular reconstructive surgery for ischemic cardiomyopathy. Heart Rhythm. 2012 Jan; 9(1):10-7.
Stevenson WG, Couper GS. A surgical option for ventricular tachycardia caused by nonischemic cardiomyopathy. Circ Arrhythm Electrophysiol. 2011 Aug; 4(4):429-31.
Bohnen M, Stevenson WG, Tedrow UB, Michaud GF, John RM, Epstein LM, Albert CM, Koplan BA. Incidence and predictors of major complications from contemporary catheter ablation to treat cardiac arrhythmias. Heart Rhythm. 2011 Nov; 8(11):1661-6.
Bohnen M, Shea JB, Michaud GF, John R, Stevenson WG, Epstein LM, Tedrow UB, Albert C, Koplan BA. Quality of life with atrial fibrillation: do the spouses suffer as much as the patients? Pacing Clin Electrophysiol. 2011 Jul; 34(7):804-9.
Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Huezey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Smith SC, Priori SG, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Jacobs AK, Anderson JL, Albert N, Buller CE, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Tarkington LG, Yancy CW. 2011 ACCF/AHA/HRS focused updates incorporated into the ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2011 Mar 15; 123(10):e269-367.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Heuzey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Jacobs AK, Anderson JL, Albert N, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on Dabigatran): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2011 Mar 15; 123(10):1144-50.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on dabigatran): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2011 Mar 15; 57(11):1330-7.
Wann LS, Curtis AB, Ellenbogen KA, Estes NA, Ezekowitz MD, Jackman WM, January CT, Lowe JE, Page RL, Slotwiner DJ, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA, Halperin JL, Kay GN, Le Heuzey JY, Lowe JE, Olsson SB, Prystowsky EN, Tamargo JL, Wann LS, Jacobs AK, Anderson JL, Albert N, Creager MA, Ettinger SM, Guyton RA, Halperin JL, Hochman JS, Kushner FG, Ohman EM, Stevenson WG, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on dabigatran). A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2011 Mar; 8(3):e1-8.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson SB, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann LS, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2011 Jan 11; 57(2):223-42.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson S, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann LS, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Heart Rhythm. 2011 Jan; 8(1):157-76.
Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA, Page RL, Ezekowitz MD, Slotwiner DJ, Jackman WM, Stevenson WG, Tracy CM, Fuster V, Rydén LE, Cannom DS, Le Heuzey JY, Crijns HJ, Lowe JE, Curtis AB, Olsson S, Ellenbogen KA, Prystowsky EN, Halperin JL, Tamargo JL, Kay GN, Wann L, Jacobs AK, Anderson JL, Albert N, Hochman JS, Buller CE, Kushner FG, Creager MA, Ohman EM, Ettinger SM, Stevenson WG, Guyton RA, Tarkington LG, Halperin JL, Yancy CW. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (updating the 2006 guideline): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2011 Jan 4; 123(1):104-23.
Sacher F, Roberts-Thomson K, Maury P, Tedrow U, Nault I, Steven D, Hocini M, Koplan B, Leroux L, Derval N, Seiler J, Wright MJ, Epstein L, Haissaguerre M, Jais P, Stevenson WG. Epicardial ventricular tachycardia ablation a multicenter safety study. J Am Coll Cardiol. 2010 May 25; 55(21):2366-72.
Steven D, Seiler J, Roberts-Thomson KC, Inada K, Stevenson WG. Mapping of atrial tachycardias after catheter ablation for atrial fibrillation: use of bi-atrial activation patterns to facilitate recognition of origin. Heart Rhythm. 2010 May; 7(5):664-72.
Roberts-Thomson KC, Seiler J, Steven D, Inada K, Michaud GF, John RM, Koplan BA, Epstein LM, Stevenson WG, Tedrow UB. Percutaneous access of the epicardial space for mapping ventricular and supraventricular arrhythmias in patients with and without prior cardiac surgery. J Cardiovasc Electrophysiol. 2010 Apr; 21(4):406-11.
Steven D, Reddy VY, Inada K, Roberts-Thomson KC, Seiler J, Stevenson WG, Michaud GF. Loss of pace capture on the ablation line: a new marker for complete radiofrequency lesions to achieve pulmonary vein isolation. Heart Rhythm. 2010 Mar; 7(3):323-30.
Aliot EM, Stevenson WG, Almendral-Garrote JM, Bogun F, Calkins CH, Delacretaz E, Della Bella P, Hindricks G, Jaïs P, Josephson ME, Kautzner J, Kay GN, Kuck KH, Lerman BB, Marchlinski F, Reddy V, Schalij MJ, Schilling R, Soejima K, Wilber D. EHRA/HRS Expert Consensus on Catheter Ablation of Ventricular Arrhythmias: developed in a partnership with the European Heart Rhythm Association (EHRA), a Registered Branch of the European Society of Cardiology (ESC), and the Heart Rhythm Society (HRS); in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA). Heart Rhythm. 2009 Jun; 6(6):886-933.
Aliot EM, Stevenson WG, Almendral-Garrote JM, Bogun F, Calkins CH, Delacretaz E, Bella PD, Hindricks G, Jaïs P, Josephson ME, Kautzner J, Kay GN, Kuck KH, Lerman BB, Marchlinski F, Reddy V, Schalij MJ, Schilling R, Soejima K, Wilber D. EHRA/HRS Expert Consensus on Catheter Ablation of Ventricular Arrhythmias: developed in a partnership with the European Heart Rhythm Association (EHRA), a Registered Branch of the European Society of Cardiology (ESC), and the Heart Rhythm Society (HRS); in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA). Europace. 2009 Jun; 11(6):771-817.
Lee JC, Steven D, Roberts-Thomson KC, Raymond JM, Stevenson WG, Tedrow UB. Atrial tachycardias adjacent to the phrenic nerve: recognition, potential problems, and solutions. Heart Rhythm. 2009 Aug; 6(8):1186-91.
Roberts-Thomson KC, Seiler J, Steven D, Inada K, John R, Michaud G, Stevenson WG. Short AV response to atrial extrastimuli during narrow complex tachycardia: what is the mechanism? J Cardiovasc Electrophysiol. 2009 Aug; 20(8):946-8.
Eckart RE, Field ME, Hruczkowski TW, Forman DE, Dorbala S, Di Carli MF, Albert CE, Maisel WH, Epstein LM, Stevenson WG. Association of electrocardiographic morphology of exercise-induced ventricular arrhythmia with mortality. Ann Intern Med. 2008 Oct 7; 149(7):451-60, W82.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/american College of Cardiology Foundation/heart Rhythm Society scientific statement on noninvasive risk stratification techniques for identifying patients at risk for sudden cardiac death: a scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. Heart Rhythm. 2008 Oct; 5(10):e1-21.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/American College of Cardiology Foundation/Heart Rhythm Society scientific statement on noninvasive risk stratification techniques for identifying patients at risk for sudden cardiac death: a scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. Circulation. 2008 Sep 30; 118(14):1497-1518.
Goldberger JJ, Cain ME, Hohnloser SH, Kadish AH, Knight BP, Lauer MS, Maron BJ, Page RL, Passman RS, Siscovick D, Stevenson WG, Zipes DP. American Heart Association/American College of Cardiology Foundation/Heart Rhythm Society Scientific Statement on Noninvasive Risk Stratification Techniques for Identifying Patients at Risk for Sudden Cardiac Death. A scientific statement from the American Heart Association Council on Clinical Cardiology Committee on Electrocardiography and Arrhythmias and Council on Epidemiology and Prevention. J Am Coll Cardiol. 2008 Sep 30; 52(14):1179-99.
Seiler J, Roberts-Thomson KC, Raymond JM, Vest J, Delacretaz E, Stevenson WG. Steam pops during irrigated radiofrequency ablation: feasibility of impedance monitoring for prevention. Heart Rhythm. 2008 Oct; 5(10):1411-6.
Roy D, Talajic M, Nattel S, Wyse DG, Dorian P, Lee KL, Bourassa MG, Arnold JM, Buxton AE, Camm AJ, Connolly SJ, Dubuc M, Ducharme A, Guerra PG, Hohnloser SH, Lambert J, Le Heuzey JY, O’Hara G, Pedersen OD, Rouleau JL, Singh BN, Stevenson LW, Stevenson WG, Thibault B, Waldo AL. Rhythm control versus rate control for atrial fibrillation and heart failure. N Engl J Med. 2008 Jun 19; 358(25):2667-77.
Roberts-Thomson KC, Seiler J, Raymond JM, Stevenson WG. Exercise induced tachycardia with atrioventricular dissociation: what is the mechanism? Heart Rhythm. 2009 Mar; 6(3):426-8.
Zeppenfeld K, Schalij MJ, Bartelings MM, Tedrow UB, Koplan BA, Soejima K, Stevenson WG. Catheter ablation of ventricular tachycardia after repair of congenital heart disease: electroanatomic identification of the critical right ventricular isthmus. Circulation. 2007 Nov 13; 116(20):2241-52.
Sacher F, Sobieszczyk P, Tedrow U, Eisenhauer AC, Field ME, Selwyn A, Raymond JM, Koplan B, Epstein LM, Stevenson WG. Transcoronary ethanol ventricular tachycardia ablation in the modern electrophysiology era. Heart Rhythm. 2008 Jan; 5(1):62-8.
Sacher F, Jais P, Stephenson K, O’Neill MD, Hocini M, Clementy J, Stevenson WG, Haissaguerre M. Phrenic nerve injury after catheter ablation of atrial fibrillation. Indian Pacing Electrophysiol J. 2007; 7(1):1-6.
Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, Popma JJ, Stevenson W. The cardiovascular disease continuum validated: clinical evidence of improved patient outcomes: part I: Pathophysiology and clinical trial evidence (risk factors through stable coronary artery disease). Circulation. 2006 Dec 19; 114(25):2850-70.
Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, Popma JJ, Stevenson W. The cardiovascular disease continuum validated: clinical evidence of improved patient outcomes: part II: Clinical trial evidence (acute coronary syndromes through renal disease) and future directions. Circulation. 2006 Dec 19; 114(25):2871-91.
Veenhuyzen GD, Hruczkowski T, Dhir SK, Stevenson WG. Another way to prove the presence and participation of an accessory pathway in supraventricular tachycardia? J Cardiovasc Electrophysiol. 2006 Oct; 17(10):1147-9.
Yan AT, Shayne AJ, Brown KA, Gupta SN, Chan CW, Luu TM, Di Carli MF, Reynolds HG, Stevenson WG, Kwong RY. Characterization of the peri-infarct zone by contrast-enhanced cardiac magnetic resonance imaging is a powerful predictor of post-myocardial infarction mortality. Circulation. 2006 Jul 4; 114(1):32-9.
Sapp JL, Cooper JM, Zei P, Stevenson WG. Large radiofrequency ablation lesions can be created with a retractable infusion-needle catheter. J Cardiovasc Electrophysiol. 2006 Jun; 17(6):657-61.
Tedrow UB, Kramer DB, Stevenson LW, Stevenson WG, Baughman KL, Epstein LM, Lewis EF. Relation of right ventricular peak systolic pressure to major adverse events in patients undergoing cardiac resynchronization therapy. Am J Cardiol. 2006 Jun 15; 97(12):1737-40.
Parkash R, Stevenson WG, Epstein LM, Maisel WH. Predicting early mortality after implantable defibrillator implantation: a clinical risk score for optimal patient selection. Am Heart J. 2006 Feb; 151(2):397-403.
Nazarian S, Maisel WH, Miles JS, Tsang S, Stevenson LW, Stevenson WG. Impact of implantable cardioverter defibrillators on survival and recurrent hospitalization in advanced heart failure. Am Heart J. 2005 Nov; 150(5):955-60.
Intini A, Goldstein RN, Jia P, Ramanathan C, Ryu K, Giannattasio B, Gilkeson R, Stambler BS, Brugada P, Stevenson WG, Rudy Y, Waldo AL. Electrocardiographic imaging (ECGI), a novel diagnostic modality used for mapping of focal left ventricular tachycardia in a young athlete. Heart Rhythm. 2005 Nov; 2(11):1250-2.
Parkash R, Maisel WH, Toca FM, Stevenson WG. Atrial fibrillation in heart failure: high mortality risk even if ventricular function is preserved. Am Heart J. 2005 Oct; 150(4):701-6.
Stevenson WG, Chaitman BR, Ellenbogen KA, Epstein AE, Gross WL, Hayes DL, Strickberger SA, Sweeney MO. Clinical assessment and management of patients with implanted cardioverter-defibrillators presenting to nonelectrophysiologists. Circulation. 2004 Dec 21; 110(25):3866-9.
Tedrow U, Maisel WH, Epstein LM, Soejima K, Stevenson WG. Feasibility of adjusting paced left ventricular activation by manipulating stimulus strength. J Am Coll Cardiol. 2004 Dec 7; 44(11):2249-52.
Curtis AB, Abraham WT, Chen PS, Ellenbogen KA, Epstein AE, Friedman PA, Hohnloser SH, Kanter RJ, Stevenson WG. Highlights of Heart Rhythm 2004, the Annual Scientific Sessions of the Heart Rhythm Society: May 19 to 22, 2004, in San Francisco, California. J Am Coll Cardiol. 2004 Oct 19; 44(8):1550-6.
Brunckhorst CB, Delacretaz E, Soejima K, Maisel WH, Friedman PL, Stevenson WG. Identification of the ventricular tachycardia isthmus after infarction by pace mapping. Circulation. 2004 Aug 10; 110(6):652-9.
Sapp JL, Soejima K, Cooper JM, Epstein LM, Stevenson WG. Ablation lesion size correlates with pacing threshold: a physiological basis for use of pacing to assess ablation lesions. Pacing Clin Electrophysiol. 2004 Jul; 27(7):933-7.
Sapp JL, Cooper JM, Soejima K, Sorrell T, Lopera G, Satti SD, Koplan BA, Epstein LM, Edelman E, Rogers C, Stevenson WG. Deep myocardial ablation lesions can be created with a retractable needle-tipped catheter. Pacing Clin Electrophysiol. 2004 May; 27(5):594-9.
Lopera G, Stevenson WG, Soejima K, Maisel WH, Koplan B, Sapp JL, Satti SD, Epstein LM. Identification and ablation of three types of ventricular tachycardia involving the his-purkinje system in patients with heart disease. J Cardiovasc Electrophysiol. 2004 Jan; 15(1):52-8.
Blomström-Lundqvist C, Scheinman MM, Aliot EM, Alpert JS, Calkins H, Camm AJ, Campbell WB, Haines DE, Kuck KH, Lerman BB, Miller DD, Shaeffer CW, Stevenson WG, Tomaselli GF, Antman EM, Smith SC, Alpert JS, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Hiratzka LF, Hunt SA, Jacobs AK, Russell RO, Priori SG, Blanc JJ, Budaj A, Burgos EF, Cowie M, Deckers JW, Garcia MA, Klein WW, Lekakis J, Lindahl B, Mazzotta G, Morais JC, Oto A, Smiseth O, Trappe HJ. ACC/AHA/ESC guidelines for the management of patients with supraventricular arrhythmias–executive summary. a report of the American college of cardiology/American heart association task force on practice guidelines and the European society of cardiology committee for practice guidelines (writing committee to develop guidelines for the management of patients with supraventricular arrhythmias) developed in collaboration with NASPE-Heart Rhythm Society. J Am Coll Cardiol. 2003 Oct 15; 42(8):1493-531.
Blomström-Lundqvist C, Scheinman MM, Aliot EM, Alpert JS, Calkins H, Camm AJ, Campbell WB, Haines DE, Kuck KH, Lerman BB, Miller DD, Shaeffer CW, Stevenson WG, Tomaselli GF, Antman EM, Smith SC, Alpert JS, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Hiratzka LF, Hunt SA, Jacobs AK, Russell RO, Priori SG, Blanc JJ, Budaj A, Burgos EF, Cowie M, Deckers JW, Garcia MA, Klein WW, Lekakis J, Lindahl B, Mazzotta G, Morais JC, Oto A, Smiseth O, Trappe HJ. ACC/AHA/ESC guidelines for the management of patients with supraventricular arrhythmias–executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the European Society of Cardiology Committee for Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Supraventricular Arrhythmias). Circulation. 2003 Oct 14; 108(15):1871-909.
Brunckhorst CB, Stevenson WG, Soejima K, Maisel WH, Delacretaz E, Friedman PL, Ben-Haim SA. Relationship of slow conduction detected by pace-mapping to ventricular tachycardia re-entry circuit sites after infarction. J Am Coll Cardiol. 2003 Mar 5; 41(5):802-9.
Koplan BA, Stevenson WG, Epstein LM, Aranki SF, Maisel WH. Development and validation of a simple risk score to predict the need for permanent pacing after cardiac valve surgery. J Am Coll Cardiol. 2003 Mar 5; 41(5):795-801.
Stevenson WG, Epstein LM. Predicting sudden death risk for heart failure patients in the implantable cardioverter-defibrillator age. Circulation. 2003 Feb 4; 107(4):514-6.
Khan HH, Maisel WH, Ho C, Suzuki M, Soejima K, Solomon S, Stevenson WG. Effect of radiofrequency catheter ablation of ventricular tachycardia on left ventricular function in patients with prior myocardial infarction. J Interv Card Electrophysiol. 2002 Dec; 7(3):243-7.
Fenelon G, Stambler BS, Huvelle E, Brugada P, Stevenson WG. Left ventricular dysfunction is associated with prolonged average ventricular fibrillation cycle length in patients with implantable cardioverter defibrillators. J Interv Card Electrophysiol. 2002 Dec; 7(3):249-54.
Soejima K, Stevenson WG. Ventricular tachycardia associated with myocardial infarct scar: a spectrum of therapies for a single patient. Circulation. 2002 Jul 9; 106(2):176-9.
Friedman RA, Walsh EP, Silka MJ, Calkins H, Stevenson WG, Rhodes LA, Deal BJ, Wolff GS, Demaso DR, Hanisch D, Van Hare GF. NASPE Expert Consensus Conference: Radiofrequency catheter ablation in children with and without congenital heart disease. Report of the writing committee. North American Society of Pacing and Electrophysiology. Pacing Clin Electrophysiol. 2002 Jun; 25(6):1000-17.
Soejima K, Suzuki M, Maisel WH, Brunckhorst CB, Delacretaz E, Blier L, Tung S, Khan H, Stevenson WG. Catheter ablation in patients with multiple and unstable ventricular tachycardias after myocardial infarction: short ablation lines guided by reentry circuit isthmuses and sinus rhythm mapping. Circulation. 2001 Aug 7; 104(6):664-9.
Soejima K, Stevenson WG, Maisel WH, Delacretaz E, Brunckhorst CB, Ellison KE, Friedman PL. The N + 1 difference: a new measure for entrainment mapping. J Am Coll Cardiol. 2001 Apr; 37(5):1386-94.
Delacretaz E, Soejima K, Gottipaty VK, Brunckhorst CB, Friedman PL, Stevenson WG. Single catheter determination of local electrogram prematurity using simultaneous unipolar and bipolar recordings to replace the surface ECG as a timing reference. Pacing Clin Electrophysiol. 2001 Apr; 24(4 Pt 1):441-9.
Soejima K, Stevenson WG, Delacretaz E, Brunckhorst CB, Maisel WH, Friedman PL. Identification of left atrial origin of ectopic tachycardia during right atrial mapping: analysis of double potentials at the posteromedial right atrium. J Cardiovasc Electrophysiol. 2000 Sep; 11(9):975-80.
Maisel WH, Stevenson WG. Sudden death and the electrophysiological effects of angiotensin-converting enzyme inhibitors. J Card Fail. 2000 Jun; 6(2):80-2.
Delacretaz E, Stevenson WG, Ellison KE, Maisel WH, Friedman PL. Mapping and radiofrequency catheter ablation of the three types of sustained monomorphic ventricular tachycardia in nonischemic heart disease. J Cardiovasc Electrophysiol. 2000 Jan; 11(1):11-7.
Delacretaz E, Soejima K, Stevenson WG, Friedman PL. Short ventriculoatrial intervals during orthodromic atrioventricular reciprocating tachycardia: what is the mechanism? J Cardiovasc Electrophysiol. 2000 Jan; 11(1):121-4.
Soejima K, Delacretaz E, Stevenson WG, Friedman PL. DDD-pacing-induced cardiomyopathy following AV node ablation for persistent atrial tachycardia. J Interv Card Electrophysiol. 1999 Dec; 3(4):321-3.
Kocovic DZ, Harada T, Friedman PL, Stevenson WG. Characteristics of electrograms recorded at reentry circuit sites and bystanders during ventricular tachycardia after myocardial infarction. J Am Coll Cardiol. 1999 Aug; 34(2):381-8.
Delacretaz E, Stevenson WG, Winters GL, Mitchell RN, Stewart S, Lynch K, Friedman PL. Ablation of ventricular tachycardia with a saline-cooled radiofrequency catheter: anatomic and histologic characteristics of the lesions in humans. J Cardiovasc Electrophysiol. 1999 Jun; 10(6):860-5.
Ellison KE, Friedman PL, Ganz LI, Stevenson WG. Entrainment mapping and radiofrequency catheter ablation of ventricular tachycardia in right ventricular dysplasia. J Am Coll Cardiol. 1998 Sep; 32(3):724-8.
Lefroy DC, Ellison KE, Friedman PL, Stevenson WG. Arrhythmia of the month: shortening of ventriculoatrial conduction time during radiofrequency catheter ablation of a concealed accessory pathway. J Cardiovasc Electrophysiol. 1998 Apr; 9(4):445-7.
Ganz LI, Couper GS, Friedman PL, Stevenson WG, Ellison K. Use of telemetered permanent pacemaker intracardiac electrograms to diagnose ventricular tachycardia. Am J Cardiol. 1997 Dec 1; 80(11):1511-3.
Ellison KE, Stevenson WG, Couper GS, Friedman PL. Ablation of ventricular tachycardia due to a postinfarct ventricular septal defect: identification and transection of a broad reentry loop. J Cardiovasc Electrophysiol. 1997 Oct; 8(10):1163-6.
Harada T, Stevenson WG, Kocovic DZ, Friedman PL. Catheter ablation of ventricular tachycardia after myocardial infarction: relation of endocardial sinus rhythm late potentials to the reentry circuit. J Am Coll Cardiol. 1997 Oct; 30(4):1015-23.
Maisel WH, Kuntz KM, Reimold SC, Lee TH, Antman EM, Friedman PL, Stevenson WG. Risk of initiating antiarrhythmic drug therapy for atrial fibrillation in patients admitted to a university hospital. Ann Intern Med. 1997 Aug 15; 127(4):281-4.
Hadjis TA, Harada T, Stevenson WG, Friedman PL. Effect of recording site on postpacing interval measurement during catheter mapping and entrainment of postinfarction ventricular tachycardia. J Cardiovasc Electrophysiol. 1997 Apr; 8(4):398-404.
Stevenson WG, Ridker PM. Should survivors of myocardial infarction with low ejection fraction be routinely referred to arrhythmia specialists? JAMA. 1996 Aug 14; 276(6):481-5.
Stevenson WG, Stevenson LW, Middlekauff HR, Fonarow GC, Hamilton MA, Woo MA, Saxon LA, Natterson PD, Steimle A, Walden JA, et al. Improving survival for patients with advanced heart failure: a study of 737 consecutive patients. J Am Coll Cardiol. 1995 Nov 15; 26(6):1417-23.
Stevenson WG, Sager PT, Natterson PD, Saxon LA, Middlekauff HR, Wiener I. Relation of pace mapping QRS configuration and conduction delay to ventricular tachycardia reentry circuits in human infarct scars. J Am Coll Cardiol. 1995 Aug; 26(2):481-8.
Metabolic analysis has been widely used in laboratory research applications. One of the main uses in this field was the metabolic phenotyping of mouse models of cardiovascular diseases; this approach was pioneered by the group of Julian Griffin mainly using models of Duchenne muscular distrophy where they were able to show different metabolic profiles associated with the expression of dystrophin and utrophin in heart muscle. In a later work the same group applied the FANCY approach (Functional Analysis by Co-responses in Yeast) to mouse models of cardiac diseases and showed that although the background strain of mice was an important source of metabolic variation, multivariate statistics were able to separate each disease model from the control strain.
Since the beginning of the 21st century the term ‘personalized medicine’ has continuously gained popularity and is now considered an essential trait of present and future medicine. For personalized medicine to be successful, it is necessary to properly identify subjects at increased risk of developing a disease, which patients will respond to a given therapy or how a disease will evolve in each case. In other words it is important to genotype and or phenotype the individual patient so that its individual response to disease and treatment can be predicted.
Sabatine et al. in 2005 showed that it was possible to apply metabolomic analysis in a carefully characterized cohort of patients undergoing exercise stress testing and to differentiate between patients that developed inducible ischemia from the ones that did not. This work was done by analyzing serum samples obtained before, during and after stress testing by high performance liquid chromatography coupled to mass spectroscopy; ischaemic patients had higher circulating levels of metabolites belonging to the citric acid pathway. Ischemic patients had relatively higher lactate levels than non ischemic suggesting an underlying ischemic process although it could not be directly related to myocardial ischemia.
It has been known for a time that patients with heart failure (HF) have an altered heart metabolism and that metabolic modulation (shifting the main substrate from free fatty acids to glucose) improved VO2max, left ventricular ejection fraction, symptoms, resting and peak stress myocardial function, and skeletal muscle energetics. Metabolic modulation as a tool to treat patients with heart failure has attracted interest but the metabolomic analysis has not followed suit until recently when Kang et al. 2011 showed by profiling urine by NMR spectroscopy that it was possible to detect changes between HF patients and controls. It could be interesting to evaluate possible changes in the urine metabolic profile of patients treated with drugs targeting heart metabolism for example, perhexiline or trimetazidine. In conclusion, the future of metabolomics is now. It is clear that metabolomics can be applied to various cardiovascular related diseases, although its clinical value in different settings remains to be determined; this is the next big challenge in the field.
Nitric oxide plays wide variety of roles in cardiovascular system and acts as a central point for signal transduction pathway in endothelium. NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances. The endothelial NOS (eNOS) is expressed in endothelial cells, the inducible NOS (iNOS) is expressed in macrophages and neuronal NOS (nNOS) is expressed in certain neurons and skeletal muscle. Although the basic mechanism of action for NO production is the same for all three NOS isoforms, yet deficiencies of each one of them manifest differently or with varying severity in the body e.g. eNOS deficiency might lead to hypertension, more severe form of vascular injury to cerebral ischaemia and more severe form of atherosclerosis induced by hypercholesterolemic diet whereas nNOS deficiency might show less severe form of vascular injury to cerebral ischaemia and absence of iNOS might lead to reduced hypotension in septic shock.
Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to
reduction in eNOS production
reduction in eNOS enzymatic activity
reduced bioavailability of NO
eNOS production is increased by physiological sheer stress on endothelial cells resulting from normal flow of blood along the arterial walls. Alterations in fluid sheer stress patterns e.g due to arterial constriction has been shown to have detrimental effect on eNOS production in endothelial cells. eNOS production is decreased by LDL, angiotensin II and TNF alpha. eNOS is tightly coupled enzyme and its activity can be significantly reduced by reduction in availability of cofactors and substrates, and by competitive inhibitors such as ADMA. Furthermore, uncoupling of eNOS can result in increased production of reactive species of both oxygen (superoxide) and nitrogen (peroxinitrite), which inturn can further reduce eNOS bioavailability. A range of therapeutic targets aim at increasing bioavailability of eNOS and they are summarised here.
Increased production of ROS and peroxinitrite is associated with endothelial dysfunction. Coronary heart disease risk factors may increase NOS mediated ROS formation and peroxinitrite formation. Such risk factors are associated with decreased NO production levels in the vasculature. However, recent data suggests that reduction in bioavailable NO levels in the arteries could be due to increased local oxidative stress rather than reduction in basal NO production.
Oxidation dependent mechanisms have been implicated in endothelial dysfunction. Oxidized low density lipo-proteins (oxLDL) play an important role in early endothelial dysfunction and hence early atherosclerosis (see figure below)
oxLDL can uncouple eNOS and reduced uptake of L-ariginine that can lead to production of superoxide radical oxygen. OxLDL can interfere with NO production and lead to altered NO signalling in the vascular endothelium. In addition, different arteries can be affected differently by these physiological changes e.g. oxLDL affects carotid artery and not the basilar artery thereby implying that intracranial arteries might be protected from endothelium-mediated oxidative injury and hence atherosclerosis. And finally NO can modulate oxidation mediated apoptotic signals in the vessel wall. Hence atherosclerosis can result from the derangement of fine imbalance between NO bioavailability and local oxidative stress.
Therapeutic targets:
There are various pathways being targeted to modulate the bioavailability of eNOS and NO such as
Recoupling of eNOS to cofactors and substrates
Modulation of eNOS activity by genomic and non genomic mechanisms e.g. by statins, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers , KLF2 modulators
The suppression of inflammatory signalling pathways by PPAR-α activation
Modulation of caeviolin mediated endocytosis and thus dissociation of eNOS from caevolin
The above list is not exhaustive, but this posthere summarises recent developments in therapeutic targets in NO /eNOS regulation.
A new proof-of-concept study shows that plasma concentrations of precursor fragments of the neuropeptide enkephalin (proenkephalin A, or PENK-A) are elevated in patients with acute stroke compared with those with TIA and nonischemic events.
Researchers are making efforts to investigate neuropeptides in patients presenting with symptoms of acute cerebrovascular disease.
Although the mature neuropeptides are degraded within minutes, their precursor fragments are much more stable and represent neuropeptide synthesis in stoichiometric relations. “They are therefore well suited as biomarkers and may be suitable for measurement in clinical settings,” said Dr. Doehner.
The precursor neuropeptides proenkephalin A (PENK-A) and protachykinin (PTA) are markers of blood-brain barrier integrity and have been recently discussed in vascular dementia and neuroinflammatory disorders.
{Ernst A., Kohrle J., Bergmann A.; Proenkephalin A 119—159, a stable proenkephalin. A precursor fragment identified in human circulation, Peptides 27 2006 1835-1840
Ernst A., Suhr J., Kohrle J., Bergmann A.; Detection of stable N-terminal protachykinin A immunoreactivity in human plasma and cerebrospinal fluid, Peptides 29 2008 1201-1206}
Researchers are making efforts to use these precursor fragments as markers to distinguish an ischemic stroke from a transient ischemic attack (TIA) or an intracerebral hemorrhage.
The authors strongly hope that it may help to advance the use of biomarkers in the clinical evaluation of stroke patients.
Despite the limitations, elevated PENK-A levels correlated with stroke severity and with brain lesion size, and they predicted mortality and more functional disability.
“There is clearly an unmet need to establish biomarker-guided prognostic and functional evaluations for patients with stroke, said the lead author Wolfram Doehner, MD, PhD, from the Center for Stroke Research, in Berlin, Germany
The new report was published in Journal of the American College of Cardiology.
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