Naser Jaleel

Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure

Loss of cardiac myocytes in heart failure is thought to occur largely through an apoptotic process. Here we show that heart failure can also be precipitated through myocyte necrosis associated with Ca2+ overload. Inducible transgenic mice with enhanced sarcolemmal L-type Ca2+ channel (LTCC) activity showed progressive myocyte necrosis that led to pump dysfunction and premature death, effects that were dramatically enhanced by acute stimulation of beta-adrenergic receptors. Enhanced Ca2+ influx-induced cellular necrosis and cardiomyopathy was prevented with either LTCC blockers or beta-adrenergic receptor antagonists, demonstrating a proximal relationship among beta-adrenergic receptor function, Ca2+ handling, and heart failure progression through necrotic cell loss. Mechanistically, loss of cyclophilin D, a regulator of the mitochondrial permeability transition pore that underpins necrosis, blocked Ca2+ influx-induced necrosis of myocytes, heart failure, and isoproterenol-induced premature death. In contrast, overexpression of the antiapoptotic factor Bcl-2 was ineffective in mitigating heart failure and death associated with excess Ca2+ influx and acute beta-adrenergic receptor stimulation. This paradigm of mitochondrial- and necrosis-dependent heart failure was also observed in other mouse models of disease, which supports the concept that heart failure is a pleiotropic disorder that involves not only apoptosis, but also necrotic loss of myocytes in association with dysregulated Ca2+ handling and beta-adrenergic receptor signaling.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

Background— Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results— Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 &mgr;m) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions— Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se.

Adolescent Feline Heart Contains a Population of Small, Proliferative Ventricular Myocytes With Immature Physiological Properties

Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit+ (stem cell receptor) CS/PCs were isolated from 11- and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16INK4a immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca2+ transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; P<0.05), and p16INK4a negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca2+ transients were prolonged in SMMs versus LBMs and Ca2+ release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca2+ current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period

CaMKII Negatively Regulates Calcineurin–NFAT Signaling in Cardiac Myocytes

Rationale: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca2+ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca2+ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca2+ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. Objective: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Methods and Results: Green fluorescent protein–tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII&dgr;c were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9±1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca2+ increased Cn-dependent NFAT translocation (to 71.7±7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. Conclusion: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

Ca2+ Influx Through T- and L-Type Ca2+ Channels Have Different Effects on Myocyte Contractility and Induce Unique Cardiac Phenotypes

T-type Ca2+ channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca2+ influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the &agr;1G-TTCC was used to increase Ca2+ influx through TTCCs. &agr;1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca2+ influx through the L-type Ca2+ channel (LTCC) secondary to overexpression of its &bgr;2a subunit. Although &agr;1G and &bgr;2a myocytes had similar increases in Ca2+ influx, &agr;1G myocytes had smaller increases in contraction magnitude, and, unlike &bgr;2a myocytes, there were no increases in sarcoplasmic reticulum Ca2+ loading. Ca2+ influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca2+ release. &agr;1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca2+ homeostasis. Immunostaining suggested that the majority of &agr;1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca2+ influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.

Increasing Cardiac Contractility After Myocardial Infarction Exacerbates Cardiac Injury and Pump Dysfunction

Rationale: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. Objectives: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca2+-handling defects could avert post-MI cardiac pump dysfunction. Methods and Results: MI was produced in mice with inducible, cardiac-specific expression of the &bgr;2a subunit of the L-type Ca2+ channel. Myocyte and cardiac function were compared in control and &bgr;2a animals before and after MI. &bgr;2a myocytes had increased Ca2+ current; sarcoplasmic reticulum Ca2+ load, contraction and Ca2+ transients (versus controls), and &bgr;2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in &bgr;2a versus control hearts after MI. &bgr;2a animals also had poorer survival after MI. Myocytes isolated from &bgr;2a hearts after MI did not develop depressed Ca2+ handling, and Ca2+ current, contractions, and Ca2+ transients were still above control levels (before MI). Conclusions: Maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

Cardiac G-Protein–Coupled Receptor Kinase 2 Ablation Induces a Novel Ca2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of &bgr;-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca2+ content because of increased sodium-Ca2+ exchanger activity and inhibited SR Ca2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca2+ handling phenotype is explained by a higher fractional SR Ca2+ release induced by increased L-type Ca2+ channel currents. After &bgr;-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca2+ transients, which were not mediated through cardiac L-type Ca2+ channels but through an increased SR Ca2+. Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca2+ handling phenotype. The SR Ca2+ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca2+ channel current density and no increase in sodium-Ca2+ exchanger in GRK2KO myocytes. An L-type Ca2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca2+ handling phenotype resisting adverse remodeling after MI.

c-Kit+ Bone Marrow Stem Cells Differentiate into Functional Cardiac Myocytes

The utility of bone marrow cells (BMCs) to regenerate cardiac myocytes is controversial. The present study examined the capacity of different types of BMCs to generate functional cardiac myocytes. Isolated c‐kit+ BMCs (BMSCs), c‐kit+ and crude BMCs from the adult feline femur were membrane stained with PKH26 dye or infected with a control enhanced green fluorescence protein transcript (EGFP)‐adenovirus prior to co‐culture upon neonatal rat ventricular myocytes (NRVM). Co‐cultured cells were immuno‐stained for c‐kit, α‐tropomyosin, α‐actinin, connexin 43 (C×43) and Ki67 and analyzed with confocal microscopy. Electrophysiology of BMSC derived myocytes were compared to NRVMs within the same culture dish. Gap junction function was analyzed by fluorescence recovery after photo‐bleaching (FRAP). BMCs proliferated and differentiated into cardiac myocytes during the first 48 hours of co‐culturing. These newly formed cardiac myocytes were able to contract spontaneously or synchronously with neighboring NRVMs. The myogenic rate of c‐kit+ BMSCs was significantly greater than c‐kit+ and crude BMCs (41.2 ± 2.1, 6.1 ± 1.2, and 17.1 ± 1.5%, respectively). The newly formed cardiac myocytes exhibited an immature electrophysiological phenotype until they became electrically coupled to NRVMs through functional gap junctions. BMSCs did not become functional myocytes in the absence of NRVMs. In conclusion, c‐kit+ BMSCs have the ability to transdifferentiate into functional cardiac myocytes.

Cardiac GRK2 Ablation Induces a Novel Ca2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of &bgr;-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca2+ content because of increased sodium-Ca2+ exchanger activity and inhibited SR Ca2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca2+ handling phenotype is explained by a higher fractional SR Ca2+ release induced by increased L-type Ca2+ channel currents. After &bgr;-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca2+ transients, which were not mediated through cardiac L-type Ca2+ channels but through an increased SR Ca2+. Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca2+ handling phenotype. The SR Ca2+ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca2+ channel current density and no increase in sodium-Ca2+ exchanger in GRK2KO myocytes. An L-type Ca2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca2+ handling phenotype resisting adverse remodeling after MI.

Cardiac G-Protein–Coupled Receptor Kinase 2 Ablation Induces a Novel Ca2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial InfarctionCLINICAL PERSPECTIVE

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of &bgr;-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca2+ content because of increased sodium-Ca2+ exchanger activity and inhibited SR Ca2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca2+ handling phenotype is explained by a higher fractional SR Ca2+ release induced by increased L-type Ca2+ channel currents. After &bgr;-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca2+ transients, which were not mediated through cardiac L-type Ca2+ channels but through an increased SR Ca2+. Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca2+ handling phenotype. The SR Ca2+ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca2+ channel current density and no increase in sodium-Ca2+ exchanger in GRK2KO myocytes. An L-type Ca2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca2+ handling phenotype resisting adverse remodeling after MI.