Thomas R. Cimato

Regional Myocardial Sympathetic Denervation Predicts the Risk of Sudden Cardiac Arrest in Ischemic Cardiomyopathy

OBJECTIVES The PAREPET (Prediction of ARrhythmic Events with Positron Emission Tomography) study sought to test the hypothesis that quantifying inhomogeneity in myocardial sympathetic innervation could identify patients at highest risk for sudden cardiac arrest (SCA). BACKGROUND Left ventricular ejection fraction (LVEF) is the only parameter identifying patients at risk of SCA who benefit from an implantable cardiac defibrillator (ICD). METHODS We prospectively enrolled 204 subjects with ischemic cardiomyopathy (LVEF ≤35%) eligible for primary prevention ICDs. Positron emission tomography (PET) was used to quantify myocardial sympathetic denervation ((11)C-meta-hydroxyephedrine [(11)C-HED]), perfusion ((13)N-ammonia) and viability (insulin-stimulated (18)F-2-deoxyglucose). The primary endpoint was SCA defined as arrhythmic death or ICD discharge for ventricular fibrillation or ventricular tachycardia >240 beats/min. RESULTS After 4.1 years follow-up, cause-specific SCA was 16.2%. Infarct volume (22 ± 7% vs. 19 ± 9% of left ventricle [LV]) and LVEF (24 ± 8% vs. 28 ± 9%) were not predictors of SCA. In contrast, patients developing SCA had greater amounts of sympathetic denervation (33 ± 10% vs. 26 ± 11% of LV; p = 0.001) reflecting viable, denervated myocardium. The lower tertiles of sympathetic denervation had SCA rates of 1.2%/year and 2.2%/year, whereas the highest tertile had a rate of 6.7%/year. Multivariate predictors of SCA were PET sympathetic denervation, left ventricular end-diastolic volume index, creatinine, and no angiotensin inhibition. With optimized cut-points, the absence of all 4 risk factors identified low risk (44% of cohort; SCA <1%/year); whereas ≥2 factors identified high risk (20% of cohort; SCA ∼12%/year). CONCLUSIONS In ischemic cardiomyopathy, sympathetic denervation assessed using (11)C-HED PET predicts cause-specific mortality from SCA independently of LVEF and infarct volume. This may provide an improved approach for the identification of patients most likely to benefit from an ICD. (Prediction of ARrhythmic Events With Positron Emission Tomography [PAREPET]; NCT01400334).

Protein methylation: a signal event in post-translational modification

Despite the data discussed here, little is known about the precise mechanisms that regulate protein methylation in eukaryotic cells. Ligand-stimulated regulation of a specific methyltransferase has not been directly demonstrated. In the case of the interferon receptor, histone methylating activity is constitutive and was not altered by treating intact cells for 5 min with interferon β. PRMT1 activity is qualitatively and quantitatively regulated by GST–TIS21 and GST–BTG1, two similar immediate-early gene fusion proteins, but these effects have only been demonstrated in vitro. The evidence for the involvement of protein methylation in differentiation is indirect, since these studies either rely on methylation inhibitors or measure the changes in methylation associated with unidentified, albeit specific, cellular proteins. The effects of methylation on the function of these proteins are also unknown. Thus, further efforts are required to establish the identities of both the enzymes and the substrates involved in regulated protein methylation in any given cellular model. For example, the nonreceptor agonist, elevated glucose, was recently shown to increase the incorporation of base-labile methyl groups (indicative of carboxyl methylation) into anti-CDC42 and anti-Rap1 immunoprecipitates from intact cells[24xKowluru, A. et al. J. Clin. Invest. 1996; 98: 540–555CrossrefSee all References][24]. Additional experiments indicate that carboxyl methylation of G proteins in this system is a requisite step in nutrient-induced insulin secretion. Similar approaches aimed at the identification of specific substrates altered by receptor-mediated events and the identification of the protein methyltransferase and methylesterase involved will further focus the molecular analysis of the role of regulated protein methylation in distinct cellular functions.The abundance of important signaling molecules that are methylated (including PP2A, G proteins, the Ras family of proteins, transcription factors and RNA processing proteins) and recent studies coupling growth factor receptors with protein methylation ensure that the role of protein methylation in cellular signaling events will receive a renewed and deserved interest in the near future.

Recipient selection in cardiac transplantation: contraindications and risk factors for mortality

Currently the only acknowledged, definitive treatment for refractory heart failure is heart transplantation (HTx). During the past 10 years, selection criteria for heart transplant recipients have been developed that use an analysis of risk factors associated with mortality, which were identified by consensus opinion and by single-center and multi-center database review. A number of other studies also have been designed to evaluate specific risk factors for transplant such as advanced age, diabetes, and sex. This review identifies variables that continue to provoke controversy during the candidate selection process or variables that have changed from absolute to relative contraindications for HTx. Clinicians may use the data summarized in this review as a guide to making decisions about patient candidacy for HTx. One could conclude from this analysis that a more formalized and objective scale to select patients and to assess risk of death after HTx is necessary. Moreover, as alternative therapies to HTx become reality, a better instrument for triaging patients to one form of therapy or another may be necessary.

Nerve growth factor‐mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1

Nerve growth factor (NGF)‐specific signal transduction leads to changes in protein methylation during neuronal differentiation of PC12 cells (Cimato et al. [1997] J. Cell Biol. 138:1089–1103). In the present work, we demonstrate that, among NGF‐regulated proteins, arginine methylation is more prevalent than carboxylmethylation. Type I protein arginine methyltransferase (PRMT) activity produces asymmetric dimethylation of the terminal guanidinonitrogen of arginines in substrate proteins, particularly glycine and arginine‐rich (GAR) segments of proteins. Several GAR peptides were used to assay for methyltransferase activity and to compete with endogenous cellular proteins for the PRMT activity in PC12 cell extracts. Peptides derived from fibrillarin and nucleolin, as well as a synthetic GAR peptide containing a repetitive GRG motif, are each extremely effective at blocking in vitro methylation of the NGF‐regulated PC12 cell methylated proteins. Myelin basic protein, a substrate for type II PRMT, selectively inhibits a 45 kDa protein but is a much less effective inhibitor of total methylation at an equimolar concentration. In addition, the fibrillarin‐ and nucleolin‐derived peptides were used to detect elevated PRMT activity in homogenates of NGF‐treated PC12 cells. Finally, immunoprecipitation of PRMT1 from PC12 cells provides the first demonstration of an NGF‐activated methyltransferase and implicates PRMT1 in NGF signal transduction.

Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells

BACKGROUND Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity and resulting variability and potentially anomalous behavior of AP parameters. OBJECTIVE To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes. METHODS An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp. RESULTS Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes. CONCLUSIONS The electronic expression of I(K1) is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

p21Cip1 modulates arterial wound repair through the stromal cell–derived factor-1/CXCR4 axis in mice

Cyclin-dependent kinase inhibitors, including p21Cip1, are implicated in cell turnover and are active players in cardiovascular wound repair. Here, we show that p21Cip1 orchestrates the complex interactions between local vascular and circulating immune cells during vascular wound repair. In response to femoral artery mechanical injury, mice with homozygous deletion of p21Cip1 displayed accelerated proliferation of VSMCs and increased immune cell infiltration. BM transplantation experiments indicated that local p21Cip1 plays a pivotal role in restraining excessive proliferation during vascular wound repair. Increased local vascular stromal cell-derived factor-1 (SDF-1) levels were observed after femoral artery injury in p21+/+ and p21-/- mice, although this was significantly greater in p21-/- animals. In addition, disruption of SDF-1/CXCR4 signaling inhibited the proliferative response during vascular remodeling in both p21+/+ and p21-/- mice. We provide evidence that the JAK/STAT signaling pathway is an important regulator of vascular SDF-1 levels and that p21Cip1 inhibits STAT3 binding to the STAT-binding site within the murine SDF-1 promoter. Collectively, these results suggest that p21Cip1 activity is essential for the regulation of cell proliferation and inflammation after arterial injury in local vascular cells and that the SDF-1/CXCR4 signaling system is a key mediator of vascular proliferation in response to injury.

Pravastatin Improves Function in Hibernating Myocardium by Mobilizing CD133+ and cKit+ Bone Marrow Progenitor Cells and Promoting Myocytes to Reenter the Growth Phase of the Cardiac Cell Cycle

3-Hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors have been reported to increase circulating bone marrow progenitor cells and variably improve global function in heart failure. The potential role of improved perfusion versus direct effects of statins on cardiac myocytes has not been established. We chronically instrumented swine with a left anterior descending artery (LAD) stenosis to produce chronic hibernating myocardium with regional contractile dysfunction in the absence of heart failure. Hemodynamics, function, perfusion, and histopathology were assessed in pigs treated for 5 weeks with pravastatin (n=12) versus untreated controls (n=10). Regional LAD wall thickening was depressed under baseline conditions (LAD 3.7±0.3 versus 6.6±0.3 in remote regions, P<0.01). It remained unchanged in untreated animals but increased from 3.8±0.6 to 5.2±0.5 mm after pravastatin (P<0.01). There was no increase in myocardial perfusion at rest or during vasodilation. Pravastatin mobilized circulating CD133+/cKit+ bone marrow progenitor cells and increased myocardial tissue levels (LAD CD133+ cells from 140±33 to 884±167 cells/106 myocyte nuclei and cKit+ cells from 223±49 to 953±123 cells/106 myocyte nuclei). Pravastatin increased myocytes in mitosis (phospho–histone-H3; 9±5 to 43±7 nuclei/106 myocyte nuclei, P<0.05) and the growth phase of the cell cycle (Ki67; 410±82 to 1261±235 nuclei/106 myocyte nuclei, P<0.05) in diseased but not normal hearts. As a result, pravastatin increased LAD myocyte nuclear density from 830±41 to 1027±55 nuclei/mm2 (P<0.05). These data indicate that, in the absence of impaired endothelial function and heart failure, dysfunctional hibernating myocardium improves after pravastatin. This effect is independent of myocardial perfusion and related to mobilization of CD133+/cKit+ bone marrow progenitor cells which stimulate myocyte proliferation resulting in quantitative increases in myocyte nuclear density.

Neuropilin-1 Identifies Endothelial Precursors in Human and Murine Embryonic Stem Cells Before CD34 Expression

Background— In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells. Methods and Results— We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2+NRP-1+ cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. Conclusions— The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.

Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells Improves Ventricular Function and Stimulates Endogenous Myocyte Regeneration throughout the Heart in Swine with Hibernating Myocardium

Background Cardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation. Methods and Results Swine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×106 icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs. Conclusions Global icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.

Statins enhance clonal growth of late outgrowth endothelial progenitors and increase myocardial capillary density in the chronically ischemic heart.

Background Coronary artery disease and ischemic heart disease are leading causes of heart failure and death. Reduced blood flow to heart tissue leads to decreased heart function and symptoms of heart failure. Therapies to improve heart function in chronic coronary artery disease are important to identify. HMG-CoA reductase inhibitors (statins) are an important therapy for prevention of coronary artery disease, but also have non-cholesterol lowering effects. Our prior work showed that pravastatin improves contractile function in the chronically ischemic heart in pigs. Endothelial progenitor cells are a potential source of new blood vessels in ischemic tissues. While statins are known to increase the number of early outgrowth endothelial progenitor cells, their effects on late outgrowth endothelial progenitor cells (LOEPCs) and capillary density in ischemic heart tissue are not known. We hypothesized that statins exert positive effects on the mobilization and growth of late outgrowth EPCs, and capillary density in ischemic heart tissue. Methodology/Principal Findings We determined the effects of statins on the mobilization and growth of late outgrowth endothelial progenitor cells from pigs. We also determined the density of capillaries in myocardial tissue in pigs with chronic myocardial ischemia with or without treatment with pravastatin. Pravastatin therapy resulted in greater than two-fold increase in CD31+ LOEPCs versus untreated animals. Addition of pravastatin or simvastatin to blood mononuclear cells increased the number of LOEPCs greater than three fold in culture. Finally, in animals with chronic myocardial ischemia, pravastatin increased capillary density 46%. Conclusions Statins promote the derivation, mobilization, and clonal growth of LOEPCs. Pravastatin therapy in vivo increases myocardial capillary density in chronically ischemic myocardium, providing an in vivo correlate for the effects of statins on LOEPC growth in vitro. Our findings provide evidence that statin therapy can increase the density of capillaries in the chronically ischemic heart.