Role of apoptosis in cardiovascular disease

Apoptosis plays a key role in the pathogenesis in a variety of cardiovascular diseases due to loss of terminally differentiated cardiac myocytes. Cardiac myocytes undergoing apoptosis have been identified in tissue samples from patients suffering from myocardial infarction, diabetic cardiomyopathy, and end-stage congestive heart failure. Apoptosis is a highly regulated program of cell death and can be mediated by death receptors in the plasma membrane, as well as the mitochondria and the endoplasmic reticulum. The cell death program is activated in cardiac myocytes by various stressors including cytokines, increased oxidative stress and DNA damage. Many studies have demonstrated that inhibition of apoptosis is cardioprotective and can prevent the development of heart failure. This review provides a current overview of the evidence of apoptosis in cardiovascular diseases and discusses the molecular pathways involved in cardiac myocyte apoptosis.     

Apoptosis in heart failure: a tale of heightened expectations, unfulfilled promises and broken hearts...

Although apoptosis contributes significantly to remodeling of the fetal heart during evolution of cardiac chambers and correct routing of the great vessels, it has been believed that apoptosis does not occur in terminally differentiated adult cardiac muscle cells. However, apoptosis has recently been demonstrated in animal models of heart failure as well as in explanted hearts from patients with end-stage heart failure undergoing cardiac transplantation. Ventricular dilatation and neurohormonal activation, the hall-marks of heart failure, lead to upregulation of transctription factors, induce muscle cell hypertrophy and prepare cells for entry into the cell-division cycle. However, since terminally differentiated myocytes cannot divide, they die by apoptosis. It has been proposed that low-grade apoptosis in failing heart may be responsible for inexorable decline in left ventricular function. Better understanding of the molecular and cellular basis of apoptosis in the failing myocardium may lead to development of strategies aimed at preventing progressive myocyte loss and deterioration in left ventricular function.     

Neuregulin-1alpha and beta isoform expression in cardiac microvascular endothelial cells and function in cardiac myocytes in vitro

Neuregulins (NRGs) are a family of alternatively spliced growth factors that act through receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family in diverse tissues. The NRG-erbB signaling axis is a critical mediator of cardiac development, and growing evidence supports a role for this system in the intricate cross-talk between the microvascular endothelium and myocytes in the adult heart. The purpose of this study was first to examine the expression of splice variants of the NRG1 gene in adult rat cardiac microvascular endothelial cells and second to compare the function of these variants in cardiac myocytes. We demonstrate that cardiac microvascular endothelial cells in rat culture express multiple Type I NRG1 gene products, including both alpha and beta variants. Comparison of the activity of recombinant NRG1alpha and NRG1beta EGF-like domain proteins in cardiac myocytes shows that the beta ligand is a more potent activator of receptor phosphorylation and intracellular signaling than the alpha ligand, and only the beta ligand stimulated glucose uptake and protein synthesis in these culture conditions. Thus, cardiac microvascular endothelial cells express multiple NRG1 isotypes, but only beta-variants are biologically active on cardiac myocytes.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。     

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.     

Cardiac endothelial cells regulate reactive oxygen species-induced cardiomyocyte apoptosis through neuregulin-1beta/erbB4 signaling

Neuregulin (NRG)-1beta has a prosurvival effect on cardiac myocytes via the phosphatidylinositol-3-kinase/Akt pathway, but the physiological regulators of this system in the intact heart are unknown. In this study, we tested the hypothesis that reactive oxygen species regulate NRG/erbB signaling. We used isolated adult rat ventricular myocytes (ARVMs) or cardiac microvascular endothelial cells (CMECs) in monoculture, or together in coculture. H2O2 induced NRG-1beta release from CMECs in a concentration-dependent manner, and conditioned medium from H2O2-treated CMEC activated ARVM erbB4. NRG-1beta release occurred via proteolytic cleavage of 115-kDa transmembrane NRG-1beta and was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline. In myocyte monoculture, H2O2 induced erbB4-dependent, but NRG-independent, activation of Akt. To elucidate the bioactivity of CMEC-derived NRG-1beta on ARVMs, we examined H2O2-induced myocyte apoptosis in co-culture using an antibody to NRG-1beta. The percentages of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly higher in the anti-NRG-1beta group than in the control group. The change in apoptosis induced by anti-NRG-1beta in co-culture was similar in magnitude to the protection of myocytes by addition of recombinant NRG-1beta to ARVM monocultures. Activation of NRG/erbB paracrine signaling was also seen in the intact heart subjected to oxidative stress by ischemia-reperfusion injury. Isolated perfused mouse hearts subjected to 15 min of ischemia, followed by 30 min of reperfusion, showed complete proteolytic cleavage of 115-kDa NRG-1beta, with concomitant erbB4 phosphorylation. These results demonstrate that reactive oxygen species activate NRG-1beta/erbB4 paracrine signaling in the heart and suggest that this system is involved in cardiac adaptation to oxidative stress.     

Mechanical overload-induced apoptosis: A study in cultured neonatal ventricular myocytes and fibroblasts

Apoptosis of cardiac myocytes has been implicated in cardiac dysfunction due to chronic hemodynamic overload. Reports on the role of apoptosis in the transition from hypertrophy to decompensated heart failure are not unequivocal. In this study we analysed the direct relationship between mechanical overload and induction of apoptosis in an in vitro model of cultured heart cells. Cyclic mechanical stretch was applied to cultured neonatal rat ventricular myocytes and fibroblasts. Several indicators of apoptosis were examined, such as morphological features, caspase-3 activity and DNA fragmentation. Mechanical strain did not induce any significant change in these parameters as compared to non-stretched myocytes or fibroblasts. However, administration of staurosporine, a known inducer of apoptosis, induced massive apoptosis in myocytes as well as fibroblasts. We conclude that this in vitro cell model system lacks a direct link between mechanical stretch and apoptosis. The three-dimensional structure-function relationship of myocardial tissue in the intact heart may elicit stretch-induced molecular signaling cascades in a much more complex way than in monolayer cultures of cardiac cells.     

Mitochondrial ca(2+) signaling and cardiac apoptosis

The broad significance of apoptosis in the cardiovascular system only began to be recognized more widely recently. Apoptotic cell death is a normal component of postnatal morphogenesis of the human cardiac conduction system and may also be involved in the pathogenesis of a variety of cardiovascular diseases, including heart failure, myocardial infarction and atherosclerosis. Recently, it has become evident that mitochondria play important role in the signaling machinery of apoptotic cell death by releasing several apoptotic factors such as cytochrome c, apoptosis-inducing factor and procaspases. Furthermore, calcium signals have been identified as one of the major signals that converge on mitochondria to trigger the mitochondrion-dependent pathway of the apoptotic cell death. Calcium signals are also important in the physiological control of mitochondrial energy metabolism and it has not yet been explored how Ca(2+) turns from a signal for life to a signal for death. Since large elevations of cytosolic [Ca(2+)] ([Ca(2+)](c)) occur during each heartbeat in cardiac myocytes and these [Ca(2+)](c) signals may efficiently propagate to the mitochondria, the Ca(2+)-dependent mitochondrial pathways of apoptosis can be particularly important in the heart. This review is concerned with the role of mitochondrial Ca(2+) signaling in the control of cardiac apoptosis.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.     

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Nitric oxide and promotion of cardiac myocyte apoptosis

The removal of damaged, superfluous or energy-starved cells is essential for biological homeostasis, and occurs in every tissue type. Programmed cell death occurs through several closely regulated signal pathways, including apoptosis, in which cell components are broken down and packaged into small membrane-bound fragments that are then removed by neighbouring cells or phagocytes. This process is activated in the cardiac myocyte in response to a variety of stresses, including oxidative and nitrosative stress, and involves mitochondria-derived signals. Loss of cardiac myocytes through apoptosis has been shown to induce cardiomyopathy in a variety of gene-targeted animal models. Because cardiac myocytes have strictly limited ability to regenerate, sustained programmed cell death is likely to contribute to the development and progression of heart failure in a variety of myocardial diseases. At the same time, the cardiac myocyte possesses a number of mechanisms for defence against short-term haemodynamic and oxidative stresses. Our laboratory has recently examined the role of nitric oxide (NO) as a regulator of the programmed death of cardiac myocytes, and the potential contribution of NO and NO-dependent signalling to the loss of myocytes in heart failure. We will review the role of c-Jun N-terminal kinase in response to oxidative and nitrosative stress, and summarise evidence for its role as a cytoprotective mechanism. We will also review evidence implicating NO in the pathophysiology of heart failure, in the context of the extensive and sometimes contradictory body of research on NO and cell survival.     

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.     

Ultrastructural and immunocharacterization of undifferentiated myocardial cells in the developing mouse heart

The recent discovery of several myogenic cardiac progenitor cells in the post-natal heart suggests that some myocardial cells may remain undifferentiated during embryonic development. In this study, we examined the subcellular characteristics of the embryonic (E) mouse ventricular myocardial cells using transmission electron microscopy (TEM). At the ultrastructural level, we identified three different cell populations within the myocardial layer of the E11.5 heart. These cells were designated as undifferentiated cells (43 +/- 6%), moderately differentiated cells (43 +/- 2%) and mature cardiomyocytes (14 +/- 4%). Undifferentiated cells contained a large nucleus and sparse cytoplasm with no myofibrillar bundles. Moderately differentiated cells contained randomly arranged myofilaments in the cytoplasm. In contrast, mature cardiomyocytes contained well-developed sarcomere structures. We also confirmed the presence of similar undifferentiated cells albeit at low levels in the E16.5 ( approximately 20%) and E18.5 ( approximately 7%) myocardium. Further we used immunogold labeling technique to test whether these distinct cell populations were also positive for markers such as Nkx2.5, ISL1 and ANF. A preponderance of anti-Nkx2.5 label was found in the undifferentiated and moderately differentiated cell types. Anti-ANF label was found only in the cytoplasmic compartment of moderately differentiated and mature myocardial cells. All of the undifferentiated cells were negative for anti-ANF labeling. We did not find immuno-gold labeling with ISL1 in any of the three myocardial cell types. Based on these results, we suggest that embryonic myocardial cell differentiation is a gradual process and undifferentiated cells expressing Nkx2.5 in post-chamber myocardium may represent a progenitor cell population while cells expressing Nkx2.5 and ANF represent differentiating myocytes.