adenosine diphosphate→adenosine triphosphate synthesis and (2) influx of water down its osmotic gradient into the matrix, resulting in massive mitochondrial swelling and even rupture of the outer mitochondrial membrane.129 The resulting release of apoptogens through a Bax/Bak-independent mechanism may then engage the downstream apoptotic machinery and further augment necrotic killing.

The biochemical composition of MPTP is not known. It is clear, however, that the peptidyl-prolyl cis-trans isomerase cylophilin D in the mitochondrial matrix regulates MPTP.129 Deletion of ppif (encoding cyclophilin D) in the mouse confers marked resistance to necrosis without affecting apoptosis.65,66,131 In two independent knockout mice, myocardial infarct size was reduced 40% and 75% after ischemia-reperfusion in vivo.65,66 These data show that necrosis is an important death program during ischemia-reperfusion and that the mitochondrial pathway plays a critical role.

The role of the death receptor necrosis pathway in myocardial ischemia-reperfusion is less defined. However, administration of necrostatin-1, a small-molecule inhibitor of the kinase activity of RIP1,135 reduces infarct size 40% to 67% after ischemia-reperfusion in vivo.136,137 Thus the death receptor necrosis pathway also appears to be involved. Interestingly, necrostatin-1 had no additional effect in the setting of cyclophilin D deletion, suggesting that the death receptor and mitochondrial necrosis pathways intersect. Although reactive oxygen species resulting from catabolic pathways activated by RIP3138 may provide a link between these two pathways, our current understanding of these connections is rudimentary at the mechanistic level.

3.1.3. Autophagy in myocardial infarction

Macroautophagy helps cells survive periods of starvation and stress in organisms ranging from yeast to humans.139 In a number of systems, the question has been raised regarding whether autophagy, under some circumstances, can beget cell death. In fact, there may be evidence to this effect in some systems.140 However, the testing of this hypothesis has suffered because of (1) a conceptual gap regarding what death machinery carries out autophagic cell death; (2) an unclear understanding of what trigger converts a survival process to a death process; and (3) most importantly, the absence of markers for autophagic cell death as opposed to autophagy itself. Despite these limitations, there are several cardiac studies that deserve consideration.

Autophagy is induced during both permanent coronary occlusion and ischemia-perfusion in vivo, but

with important differences in mechanism and significance.67,68 5 Adenosine monophosphate-activated protein kinase (AMPK), an inducer of autophagy, is activated during permanent coronary occlusion, but not during ischemia-reperfusion. On the other hand, the abundance of Beclin-1, also a mediator of autophagy, increases during ischemia-reperfusion. Transgenic over-expression of a dominant negative AMPK during permanent coronary occlusion in vivo inhibits induction of autophagy and results in increased infarct size.68 These data suggest that autophagy plays a protective role in persistent myocardial ischemia without reperfusion, which is consistent with its role in starvation. Of note, however, potential effects of AMPK on apoptosis and metabolism may cloud this interpretation. This result contrasts with what is observed during ischemia-reperfusion. Deletion of a single Beclin-1 allele decreases autophagy associated with decreased infarct size after ischemia-reperfusion in vivo.67 This suggests that, in contrast to the protective role of autophagy in permanent coronary occlusion, autophagy appears to play a pathogenic role in ischemia-reperfusion.

3.2. Cell death in heart failure

In the failing heart, the myocardium is too weak to pump blood efficiently to meet the metabolic needs of the various tissues. Heart failure is a final common outcome for many serious cardiac diseases. A common etiology is prior myocardial infarction(s), which acutely destroy segments of functional myocardium (as discussed previously) and cause the remaining noninfarcted myocardium to fail gradually. Additional causes include hypertension, valvular heart disease, and cardiomyopathies, among others. In each of these cases, heart failure develops gradually – over months to several years – such that the final phenotype cannot be distinguished by etiology.

Heart failure is complex at the physiologic, cellular, and molecular levels.141 There are multiple abnormalities in cell signaling (including activation of a variety of stress pathways), Ca2+ handling and excita- tion-contraction coupling, cytoskeleton, contractile proteins, energetics, and extracellular matrix. Many of these processes culminate in cell death. However, it is important to keep in mind that heart failure may also ensue because of cardiac myocyte dysfunction without cell death. Thus cell death is an important factor in heart failure, but not the only one.

Although the sequence of events in the development of heart failure varies with the underlying etiology, the process often starts with a mechanical “load” being

imposed on the heart. For example, in hypertension, the left ventricle (major pumping chamber) is subjected to increased pressure. It responds by undergoing hypertrophic growth, in which the walls of ventricle becomes thicker as a result of the individual cardiac myocytes becoming larger (but not more numerous). After a sustained period of pressure overload, the heart transitions from hypertrophy to failure. Microscopically, this is characterized by cardiac myocytes becoming elongated rather than wider. Macroscopically, the left ventricular chamber becomes larger in volume and the thick, hypertrophic walls become thinned. Functionally, the contractions of the heart weaken. Symptoms of heart failure develop, including tiredness, shortness of breath especially on exertion, and swelling of the legs and feet. This is a lethal disease, with patients dying either from “pump failure” or arrhythmias (sudden cardiac death).

As noted previously, myocardial infarction is characterized by a large burst of cardiac myocyte apoptosis that lasts only for hours. In contrast, the frequency of cardiac myocyte apoptosis in the failing heart is very low (0.08%– 0.25%), but still 10to 100-fold higher than that in normal controls (0.001%–0.002%).142,143,144 The frequencies of necrosis and autophagic cell death are not known with any degree of reliability. We will review studies that assess whether these death programs play a causal role in heart failure.

3.2.1. Apoptosis in heart failure

Can rates of cardiac myocyte apoptosis as low as 0.08% to 0.25% (0.001%–0.002% in controls) contribute significantly to human heart failure142,143,144? To address this issue experimentally, transgenic mice were created with cardiac-specific expression of a human caspase-8 allele145,146 that exhibits low levels of constitutive activation.147 These mice develop lethal heart failure at 3 to 9 weeks of age (Figure 26-4). In contrast, mice expressing equivalent levels of a similar, but enzymatically dead, caspase-8 allele had normal hearts. Mice with heart failure exhibited rates of cardiac myocyte apoptosis of 0.023%, 15-fold higher than those of controls (0.016%). These apoptotic rates of 0.023%, however, are 4 to 10 times lower than those in patients with heart failure, suggesting that a low (but relatively elevated) level of cardiac myocyte apoptosis is sufficient to cause, over time, lethal heart failure. To test the dependency of heart failure on caspase activation and apoptosis in this model, a polycaspase inhibitor was administered before the development of heart failure. Caspase inhibition markedly attenuated development of abnormalities of cardiac structure and function. These data indicate that the modest rates

of cardiac myocyte apoptosis observed in heart failure can contribute to the pathogenesis of this syndrome.

Signals from several cell surface receptors (type 1 angiotensin II receptor, α1-adrenergic receptor, endothelin receptor) modulate cardiac hypertrophy and failure via Gαq. Transgenic expression of Gαq bypasses the need for these receptors in the induction of cardiac hypertrophy and failure.148 Analysis of hearts from Gαq transgenic mice revealed increases in transcripts encoding Nix/Bnip3L (Nip3 [19-kD interacting protein-3]-like protein X/Bcl-2/adenovirus E1B 19 kD interacting protein 3-like), a BH3-only–like Bcl-2 protein. Expression of Nix/Bnip3L in transgenic mice was sufficient to cause extensive cardiac myocyte apoptosis and lethality.149 A subset of Gαq female mice developed overwhelming heart failure with pregnancy.150 Cardiac myocyte apoptosis, cardiac dysfunction, and mortality is attenuated in these mice by caspase inhibition or concurrent transgenic expression of sNix, a dominant negative splice variant of Nix/Bnip3L.151 These data link pathways that mediate cardiac myocyte hypertrophy and apoptosis and demonstrate a role for apoptosis in heart failure.

The preceding two studies used models in which apoptosis had been induced to demonstrate that inhibition of apoptosis could improve heart failure. The next study tests whether apoptosis inhibition will ameliorate heart failure induced by clinically relevant pathophysiologic stresses. Ischemia-reperfusion was used to cause myocardial infarction, which, as previously noted, can precipitate failure of the noninfarcted myocardium. Bnip3 (Bcl-2/adenovirus E1B 19 kD interacting protein 3) is a BH3-only–like protein in the same subfamily as Nix/Bnip3L (discussed previously). Of note, Bnip3 is induced in cardiac myocytes by hypoxia. Deletion of Bnip3 in the mouse does not affect infarct size after ischemia-reperfusion in vivo – possibly because of inadequate time for its induction during infarction. In contrast, absence of Bnip3 significantly reduces cardiac myocyte apoptosis in the peri-infarct zone and noninfarcted myocardium and attenuates pathological myocardial remodeling. These data demonstrate that cardiac myocyte apoptosis is a causal component of postinfarct heart failure.

In addition to the induction of apoptotic mediators, heart failure may also be triggered by the loss of survival mechanisms. One such survival pathway is mediated by gp130, a subunit of the receptors that bind several prosurvival cytokines of the interleukin- 6 family, some of which also induce cardiac hypertrophy. Cardiac-specific deletion of gp130 in the mouse results in no baseline abnormalities. The imposition of

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hemodynamic overload from aortic constriction, however, precipitates extensive cardiac myocyte apoptosis and severe heart failure.152 The physiologic role of gp130 appears to be phosphorylation of STAT3 (signal transducer and activator of transcription 3) during times of stress, which in turn activates transcription of Bcl-xL.

3.2.2. Necrosis in heart failure

Rates of necrosis are increased in human heart failure,142,153 but the significance of necrotic death in the pathogenesis of this syndrome is not known. Diastolic Ca2+ overload is a characteristic of heart failure, and

increased mitochondrial Ca2+ can trigger MPTP opening and necrosis. Accordingly, to assess the pathogenic significance of necrosis in heart failure, transgenic mice were generated with cardiac-specific, inducible overexpression of the β2a subunit of the L-type Ca2+ channel.154 Ca2+ overload-induced necrosis in the hearts of these mice elicited heart failure, which was rescued by deletion of cyclophilin D but not by over-expression of Bcl-2. Cyclophilin D absence also ameliorated heart failure in a model of doxorubicin-induced cardiomyopathy. Thus, in addition to the previously discussed role of apoptosis in heart failure, these studies suggest that cardiac myocyte necrosis is also a pathogenic component.