Remus Berretta

Ca2+ Influx–Induced Sarcoplasmic Reticulum Ca2+ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes

Increases in Ca2+ influx through the L-type Ca2+ channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca2+ loading and the amplitude of the cytosolic Ca2+ transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca2+ influx through the LTCC cause apoptosis if the excessive influx results in SR Ca2+ overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 &bgr;2a subunit-green fluorescent protein (GFP) fusion gene (Ad&bgr;2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer &bgr;2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of &bgr;2a-VMs (20.8±1.8%) contracted spontaneously (SC-&bgr;2a-VMs), and viability was significantly correlated with the percentage of SC-&bgr;2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in &bgr;2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in &bgr;2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-&bgr;2a-VMs had higher diastolic intracellular Ca2+ (Indo-1 ratio: 1.1±0.1 versus 0.7±0.03, P<0.05) and systolic Ca2+ transients (1.89±0.27 versus 0.80±0.08) than GFP-VMs. Inhibitors of Ca2+ influx, SR Ca2+ uptake and release, mitochondrial Ca2+ uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected &bgr;2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.

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

Bone-Derived Stem Cells Repair the Heart after Myocardial Infarction Through Transdifferentiation and Paracrine Signaling Mechanisms

Rationale: Autologous bone marrow–derived or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials, but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is the key toward improving clinical outcomes. Objective: To determine the mechanism by which novel bone-derived stem cells support the injured heart. Methods and Results: Cortical bone–derived stem cells (CBSCs) and cardiac-derived stem cells were isolated from enhanced green fluorescent protein (EGFP+) transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis, and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction with injection of CBSCs (n=67), cardiac-derived stem cells (n=36), or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor), and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to cardiac-derived stem cells– or saline-treated myocardial infarction controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle, and endothelial cells could be identified in CBSC-treated, but not in cardiac-derived stem cells–treated, animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP− myocytes. Conclusions: CBSCs improve survival, cardiac function, and attenuate remodeling through the following 2 mechanisms: (1) secretion of proangiogenic factors that stimulate endogenous neovascularization, and (2) differentiation into functional adult myocytes and vascular cells.

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.

A Caveolae-Targeted L-Type Ca2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac Contractility

Rationale: The source of Ca2+ to activate pathological cardiac hypertrophy is not clearly defined. Ca2+ influx through the L-type Ca2+ channels (LTCCs) determines “contractile” Ca2+, which is not thought to be the source of “hypertrophic” Ca2+. However, some LTCCs are housed in caveolin-3 (Cav-3)–enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. Objective: To test the idea that LTCCs in Cav-3–containing signaling domains are a source of Ca2+ to activate the calcineurin–nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. Methods and Results: We developed reagents that targeted Ca2+ channel-blocking Rem proteins to Cav-3–containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca2+ influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. Conclusions: We provide proof of concept that Ca2+ influx through LTCCs within caveolae signaling domains can activate “hypertrophic” signaling, and this Ca2+ influx can be selectively blocked without reducing cardiac contractility.

Calcium Influx through Cav1.2 Is a Proximal Signal for Pathological Cardiomyocyte Hypertrophy

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca(2+)) is implicated in hypertrophic signaling pathways, the specific role of Ca(2+) influx through the L-type Ca(2+) channel (I(Ca-L)) has been controversial and is the topic of this study. To determine if and how sustained increases in I(Ca-L) induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca(2+) channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I(Ca-L), fractional shortening, peak Ca(2+) transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased I(Ca-L), cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I(Ca-L), intracellular Ca(2+), calcineurin, CaMKII, and SERCA. In conclusion, increasing I(Ca-L) is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca(2+) pools were shown to be involved.

Repair of the Injured Adult Heart Involves New Myocytes Potentially Derived From Resident Cardiac Stem Cells

Rationale: The ability of the adult heart to generate new myocytes after injury is not established. Objective: Our purpose was to determine whether the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source. Methods and Results: Cardiac injury was induced in the adult feline heart by infusing isoproterenol (ISO) for 10 days via minipumps, and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography, and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7-day minipump infusion). BrdU was infused for 7 days before euthanasia at days 10, 17, and 38 or during injury and animals euthanized at day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first 7 days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control, but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury. Conclusions: These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.

Alterations in Early Action Potential Repolarization Causes Localized Failure of Sarcoplasmic Reticulum Ca2+ Release

Depressed contractility of failing myocytes involves a decreased rate of rise of the Ca2+ transient. Synchronization of Ca2+ release from the junctional sarcoplasmic reticulum (SR) is responsible for the rapid rise of the normal Ca2+ transient. This study examined the idea that spatially and temporally dyssynchronous SR Ca2+ release slows the rise of the cytosolic Ca2+ transient in failing feline myocytes. Left ventricular hypertrophy (LVH) with and without heart failure (HF) was induced in felines by constricting the ascending aorta. Ca2+ transients were measured in ventricular myocytes using confocal line scan imaging. Ca2+ transients were induced by field stimulation, square wave voltage steps, or action potential (AP) voltage clamp. SR Ca2+ release was significantly less well spatially and temporally synchronized in field-stimulated HF versus control or LVH myocytes. Surprisingly, depolarization of HF cells to potentials where Ca2+ currents (ICa) were maximal resynchronized SR Ca2+ release. Correspondingly, decreases in the amplitude of ICa desynchronized SR Ca2+ release in control, LVH, and HF myocytes to the same extent. HF myocytes had significant loss of phase 1 AP repolarization and smaller ICa density, which should both reduce Ca2+ influx. When normal myocytes were voltage clamped with HF AP profiles SR Ca2+ release was desynchronized. SR Ca2+ release becomes dyssynchronized in failing feline ventricular myocytes because of reductions in Ca2+ influx induced in part by alterations in early repolarization of the AP. Therefore, therapies that restore normal early repolarization should improve the contractility of the failing heart.

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.