Associate editor: F. BrunnerModulation of apoptosis by nitric oxide: implications in myocardial ischemia and heart failure
Introduction
Cellular death plays a central role in homeostasis of an organ and disease processes. One form of cell death whereby the cell undergoes an ordered disassembly followed by death and phagocytosis is known as apoptosis (Kerr et al., 1972). Morphological features of apoptosis include cell shrinkage, chromatin condensation, membrane blebbing, and preservation of organelle ultrastructural integrity. The other form of cell death is necrosis. Cells die from necrosis as a result of gross and overt cellular injury causing depletion of cellular energy. Necrosis is characterized by cell swelling, membrane lysis, and release of the intracellular contents, leading to an inflammatory response, with edema and damage to the surrounding cells (Yaoita et al., 2000).
Two major signaling mechanisms of apoptosis have been well described; the death receptor pathway and the mitochondrial death pathway (Joza et al., 2002). Accordingly, the death receptor pathway involves activation of cell surface “death receptors”, which belong to a specialized subset of the tumor necrosis factor receptor (TNFR) superfamily (Ashkenazi & Dixit, 1998). Upon activation, intracellular adapter proteins are recruited by these receptors, which subsequently stimulate caspase-8 autoproteolytic activation and initiate a well-defined caspase cascade leading to apoptotic cell death. Alternatively, increases in mitochondrial permeability lead to cytochrome c release and activation of caspase-9, which initiates the downstream caspases resulting in apoptosis (Joza et al., 2002).
Apoptosis plays a crucial role in development and homeostasis of multicellular organisms. For instance, synaptogenesis, morphogenesis, tissue turn over and resolution of an immune response all require apoptotic cell death (Ellis et al., 1991). While failure to undergo apoptosis after sustaining of severe DNA damage may lead to carcinogenesis, unscheduled apoptosis is an essential component of disease processes such as Alzheimer's and Parkinson's (Nijhawan et al., 2000). Emerging evidence also demonstrated that apoptosis contributes to the pathogenesis of many cardiovascular diseases (Bennett, 2002). As adult heart muscle has very limited ability to regenerate, loss of cardiomyocytes due to apoptosis may play an important role in myocardial ischemia and development of heart failure (Nadal-Ginard et al., 2003).
Nitric oxide (NO) is produced from the guanidino group of l-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS). There are at least 3 distinct NOS isoforms, derived from separate genes: neuronal NOS (nNOS, or NOS1), inducible NOS (iNOS, or NOS2), and endothelial NOS (eNOS, or NOS3). The 3 isoforms are similar in structure, utilizing l-arginine, oxygen and NADPH as substrates and requiring cofactors such as calmodulin and tetrahydrobiopterin (Stuehr, 1999, Alderton et al., 2001). While eNOS and nNOS are Ca2+-dependent enzymes, iNOS is usually induced by cytokines and its enzyme activity is Ca2+-independent. Strictly speaking, eNOS and nNOS are not “constitutive” as the expression of eNOS, for example, is up-regulated by shear stress, exercise, pregnancy and down-regulated by pro-inflammatory cytokines (Kelly et al., 1996). While activation of nNOS and eNOS has physiological and homeostatic effects, induction of iNOS and high levels of NO production are often, but not always, associated with pathological conditions (Bolli, 2001, Jugdutt, 2002).
The eNOS isoform, which was originally characterized in large conduit vessel endothelium, is expressed within the heart in the endocardium and in the endothelium of the coronary vasculature, including capillary and venular endothelium. It is also expressed in cardiac myocytes and in specialized cardiac conduction system, including sinoatrial and atrioventricular nodal tissues (Balligand & Cannon, 1997). Under normal physiological conditions, NO released from eNOS in the heart has several major roles including coronary vasodilation, regulation of platelet and neutrophil functions, and tonic inhibition of mitochondrial O2 consumption (Boveris et al., 2000). NO may also play a role in muscarinic-cholinergic inhibition of β-adrenergic-stimulated chronotropy, inotropy, atrioventricular nodal conduction, and cardiac myocyte L-type Ca2+ currents (Massion et al., 2003).
A number of cellular constituents of cardiac muscle, including the endothelium and smooth muscle of the cardiac microvasculature, the endocardial endothelium, tissue macrophages, and cardiac myocytes, are capable of expressing iNOS in response to LPS and and/or cytokine stimulation (Muller et al., 2000). Myocardial iNOS induction has been demonstrated to cause contractile dysfunction in various preparations including isolated myocytes, perfused working hearts, and in vivo animal preparations. The physiological outcomes of iNOS induction are not limited to a reversible decline in myocyte contractile function. Some beneficial effects of NO from iNOS are apparent. For example, NO generated by iNOS has been shown to suppress viral replication and infection (Lowenstein et al., 1996). In this regard, Coxsackievirus replicates to higher titers, where viral clearance is hindered in iNOS−/− mice as compared to wild-type controls (Zaragoza et al., 1998). More importantly, myocarditis induced by Coxsackievirus is much more severe in infected iNOS−/− mice than the wild-type mice (Zaragoza et al., 1998), suggesting iNOS is crucial for the host response to Coxsackievirus.
NO has both pro- or anti-apoptotic effects depending on its concentration, source of production and pathological conditions. Transient production of NO by eNOS and nNOS as a result of increases in intracellular Ca2+ due to receptor activation or phosphorylation of eNOS by AKT/PKB has been attributed to physiological NO effects (McCabe et al., 2000). On the other hand, overproduction of NO by iNOS can be injurious to host and foreign cells alike (Li & Forstermann, 2000). The pro- and anti-apoptotic effects of NO closely tie into the delicate regulation of cell cycle checkpoint control and the normal function of mitochondria that controls the cellular redox state. The purpose of this review is to summarize the molecular mechanisms of NO in the regulation of apoptosis. Furthermore, the potential role that NO plays in cardiomyocyte apoptosis during myocardial ischemia/reperfusion (I/R) injury and the development of heart failure will also be discussed.
Section snippets
Effects of nitric oxide on death receptors
Death receptors belong to the TNF receptor gene superfamily of single-pass transmembrane receptor proteins, which contain conserved intracellular death domains and extracellular cysteine-rich domains. These latter domains allow the formation of external disulfide bridges during arrangement of activated death receptor homotrimers. Intracellular death domains permit for engagement of the receptor with intracellular proteins that also contain death domains and set into motion the apoptotic process
Anti-apoptotic mechanisms of nitric oxide
In contrast to its pro-apoptotic effects, NO also has prominent anti-apoptotic effects (Kolb, 2000). These effects could conceivably be attributed to cGMP/PKG-mediated Bcl-2 expression, linking the NO signaling pathway with inhibition of mitochondrial permeability. However, in most cases, protection from NO against apoptosis is clearly independent of cGMP. Although NO can up-regulate cell protective proteins such as heme oxygenase-1 and metallothionein (Spahl et al., 2003, Pae et al., 2004),
Apoptosis in myocardial ischemia and reperfusion
Cardiomyocytes apoptosis occurs during myocardial ischemia in animals and humans. In this regard, apoptosis has been shown in the infarcted myocardium in rats following coronary artery occlusion. Cardiomyocyte apoptosis started at 2 hr and peaked at 4.5 hr following coronary artery occlusion in vivo. Specifically, 2 hr after myocardial infarction, apoptotic cell death involved 2.8 million cells and necrotic cell death only 90,000 myocytes in the rat heart (Kajstura et al., 1996). Furthermore,
Apoptosis in heart failure
Chronic heart failure is the final clinical presentation for a variety of cardiovascular diseases, including myocardial infarction, hypertension, and cardiomyopathy, as a result of persistent stress to the myocardium and progressive cardiac remodeling. Recent studies from patients and animal models of heart failure have demonstrated that cardiomyocyte apoptosis is observed during development of heart failure. In this regard, apoptotic myocytes have been documented in patients with heart failure
Therapeutic implications
Caspases are a group of cysteine proteases that play a crucial role in initiating and executing apoptosis. It is therefore indubitable that caspases are potential targets for anti-apoptotic treatment. Indeed, in rats that underwent 30 min of myocardial ischemia followed by 24 hr of reperfusion, a broad-spectrum caspase inhibitor, benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone, reduced both infarct size and cardiomyocyte apoptosis, with significant hemodynamic improvement in vivo (
Conclusions
Apoptosis, or programmed cell death, is a highly regulated and evolutionarily conserved mechanism of cell death. The mechanism of apoptosis is multifactorial and complex. NO is an important regulator of apoptosis within the mammalian system, capable of both inducing and preventing apoptosis, depending upon the environmental milieu. This bifunctional capacity is well illustrated within the heart. A variety of cell types, including endothelium, vascular smooth muscle, and cardiomyocytes, can
Acknowledgments
Supported by grants awarded to Dr. Qingping Feng from the Canadian Institutes of Health Research (MOP-64395) and the Heart and Stroke Foundation of Ontario (T-4923). Dr. Feng is a recipient of Premier's Research Excellence Award (PREA) from the Province of Ontario, Canada.
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