3.1. Cell death in myocardial infarction

The most significant type of myocardial infarction is ST-segment elevation myocardial infarction (the term is based on its electrocardiographic features). Infarction itself refers to cell death in a tissue, regardless of the underlying death process. ST-segment elevation myocardial infarction results from the acute thrombotic occlusion of a coronary artery, the immediate consequence of which is ischemia that eventually leads to cell death in the segment of myocardium supplied by the artery. The prompt re-establishment of adequate blood flow in the coronary artery limits myocardial damage.58,59,60,61 Accordingly, optimal therapy is acute reperfusion, currently best accomplished through angioplasty/stenting.

Ischemia subjects the myocardium to deficits of oxygen, nutrients, and survival factors and to surpluses of waste products resulting in intracellular acidosis. Although the overall benefit of reperfusion in saving myocardium is firmly established, reperfusion itself may trigger a set of detrimental processes collectively termed reperfusion injury.59 While somewhat controversial, these include rapid re-activation of the electron transport chain and generation of reactive oxygen species, mitochondrial and cytoplasmic Ca2+ overload, overly rapid resolution of intracellular acidosis that can paradoxically trigger opening of the mitochondrial permeability transition pore (MPTP; discussed later in this chapter), and inflammation.59 Current work is directed toward reducing these potential toxicities to further enhance the benefit of reperfusion.

Human myocardial infarction can be modeled in animals by permanent surgical occlusion of the left coronary artery or by an extended period of left coronary artery occlusion followed by reperfusion, known as ischemia-reperfusion. These models simulate the clinical courses of untreated myocardial infarction and myocardial infarction treated with reperfusion, respectively.

Permanent coronary occlusion and ischemiareperfusion both result in large levels of apoptotic,62,63 necrotic,63,64,65,66 and possibly autophagic67,68 cell death. The creation of a definitive temporal and spatial map of the magnitudes and kinetics of each type of cell death in these models has been hampered by differences in markers and methodologies used in the various studies. Nevertheless, it can be stated that apoptosis, even as measured by late markers such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or DNA laddering, is evident within a few hours of permanent coronary artery occlusion and

even sooner after ischemia-reperfusion.69 Necrosis, as evaluated by electron microscopy and histology, occurs within hours of permanent coronary occlusion.63,70 Autophagy (not autophagic cell death), as evaluated by LC3 (microtubule-associated protein 1 light chain 3) conversion or dots, can be detected less than 1 hour into reperfusion after ischemia.67

In addition to timing, location is also an important consideration. In a brain undergoing stroke, ischemia induces necrosis in the central infarct zone and apoptosis in surrounding penumbra.71 In contrast, the infarcting heart does not exhibit a grossly demarcated penumbra. Moreover, the infarct zone in the heart appears to involve both apoptosis and necrosis, although the relative magnitudes of each and their contributions to infarct size and cardiac dysfunction are poorly understood. The peri-infarct zone (the region immediately surrounding the infarct) and noninfarcted myocardium also undergo apoptosis after myocardial infarction with the magnitude decreasing progressively with the distance from the infarct zone.72,73 The extent to which necrosis takes place in these zones has not been precisely defined.

As will be discussed later, the deaths of cardiac myocytes play critical roles in the structural and functional changes in the heart during and after myocardial infarction. It should be noted, however, that other myocardial cell types (fibroblasts, endothelial cells, smooth muscle cells, neural cells, and others) also die at significant rates during infarction.70,74 In fact, myocytes may actually be relatively resistant to apoptosis because of low Apaf-1 (apoptotic protease activating factor-1) levels that may increase X-linked IAP–mediated inhibition of apoptosis in these cells.75,76 It is possible that the loss of these non-myocytes, which has not been studied in detail, also affects cardiac damage and dysfunction during infarction.

3.1.1. Apoptosis in myocardial infarction

The discussion here focuses on experimental perturbations of the central apoptosis pathways in intact animals that have revealed causal connections between cardiac myocyte apoptosis and subsequent infarct size and cardiac function. As described, these studies show that apoptosis plays a critical role in the genesis of a myocardial infarction and the resulting cardiac dysfunction. In the whole-animal studies cited below, cardiac myocyte apoptosis is often measured by TUNEL (in conjunction with immunostaining for a cardiac myocyte-specific marker) and infarction (total cell death) with 2,3,5- triphenyl tetrazolium chloride (a substrate for mitochondrial reductases).

Mice deficient in Fas (lpr [lymphoproliferation] on MRL/MpJ background, used before the onset of autoimmune manifestations) exhibit 64% less cardiac myocyte apoptosis and 62% smaller infarcts after ischemiareperfusion in vivo compared with wild-type mice.77 Similar reductions in apoptosis are observed after ex vivo ischemia-reperfusion of isolated, buffer-perfused hearts obtained from lpr mice. Moreover, the reperfusion phase of ischemia-reperfusion induced the secretion of Fas ligand and tumor necrosis factor-α (TNF- α) from isolated, buffer-perfused hearts, suggesting a paracrine loop.78 These experiments indicate a role for Fas-mediated apoptosis in ischemia-reperfusion. The recent recognition that death receptors can also participate in necrosis signaling79,80 raises the possibility that the rescue in infarct size in lpr mice may represent decreases in both apoptosis and necrosis.

The influence of TNF-α signaling on myocardial infarction is more complex because of effects on cardiac myocyte viability (survival, apoptosis, necrosis), contractility, and inflammation. An added intricacy relates to the existence of two receptors, TNF-α receptor subtypes 1 and 2 (TNFR1 and TNFR2). Deletion of either TNFR1 or TNFR2 does not alter infarct size after permanent coronary occlusion in vivo or ischemia-reperfusion in isolated, buffer-perfused hearts.81,82 However, concomitant deletion of both receptors increases infarct size 39% and 40%, respectively, in these models. Conversely, low-level cardiac myocyte-specific transgenic expression of TNF-α or TRAF2 (TNF receptor-associated factor 2) on a wild-type background decreases infarct size and improves function after ischemia-reperfusion in isolated, buffer-perfused hearts.82 These data suggest that both TNFR1 and TNFR2 transduce survival signals in the context of prolonged myocardial ischemia with or without reperfusion. Paradoxically, other studies report that pharmacological antagonism of TNF-α, which would be predicted to decrease signaling through both TNF receptors, reduces infarct size.83,84,85 Moreover, in some models of heart failure (as opposed to myocardial infarction), TNFR1 appears to transduce pathogenic signals, whereas TNFR2 is protective.86,87 The reasons for these discrepant findings are unclear but may reflect differences in models or the pleiotropic effects of TNF signaling in the heart.

Given the great importance of nutrients/energy for normal cardiac function, it is not surprising that the intrinsic apoptosis pathway also plays an important role in cardiac myocyte apoptosis and myocardial infarction. Deletion of the multidomain proapoptotic protein Bax results in a 35% reduction in infarct size after permanent coronary occlusion in vivo88 and 49% reduction in

isolated, buffer-perfused hearts subjected to ischemiareperfusion.89 Decreases in cardiac myocyte apoptosis and cardiac dysfunction parallel these reductions in infarct size. Similarly, mice lacking the multidomain proapoptotic protein Bak exhibit 36% smaller infarcts after ischemia-reperfusion in vivo (Whelan, Jha, and Kitsis, unpublished data). Thus, in distinction to some cell culture models that reveal a high degree of redundancy between Bax and Bak,90 full cardiac myocyte killing during infarction in vivo appears dependent on both Bax and Bak. The importance of the intrinsic pathway in myocardial ischemia-reperfusion is confirmed by transgenic over-expression of the antiapoptotic protein Bcl-2 in the heart. Infarct size after ischemia-reperfusion in vivo is reduced 64%91 and 48%92 in two independent mouse lines.

The actions of the various multidomain Bcl-2 proteins are mediated through a variety of mechanisms. These include their effects on mitochondrial apoptogen release, ER Ca2+ handling,93,94 and possibly the unfolded protein response.95 Which of these mechanisms predominate with respect to their effects on myocardial infarction is not clear.

A multitude of BH3 [B cell leukemia/lymphoma- 2 (Bcl-2) homology domain 3]-only proteins activate Bax and Bak directly96 or indirectly.97 Deletion of the BH3-only protein Bid [BH3-interacting domain death agonist], which links extrinsic and intrinsic pathways, decreases infarct size by 53% after ischemia-reperfusion

Notably, Bid activation during ischemiareperfusion may reflect cleavage by calpains as well as caspase-8.99 Loss of p53-upregulated modulator of apoptosis (PUMA) also decreases infarct size by 50% in isolated, buffer-perfused hearts subjected to ischemiareperfusion.100

It is clear from the preceding discussion that both the extrinsic and intrinsic pathways mediate cardiac myocyte death during myocardial infarction. Although most apoptosis inhibitors target either the intrinsic (e.g., Bcl-2) or extrinsic (e.g., FLIP) pathways, ARC (apoptosis repressor with a CARD [caspase recruitment domain]) antagonizes both pathways through inhibition of death-inducing signaling complex (DISC) formation and Bax activation and by promoting p53 nuclear hyperexport.101,102 The effects of ARC deletion on infarct size are unresolved, as one group observed an increase in infarct size that was not seen in an independently generated null mouse (Donath et al.103; Ji, Jha, and Kitsis, unpublished data). The reasons for this discrepancy are unclear but may include genetic compensation and technical variation in the experiments. In addition, differences between ARC knockout and

wild-type animals may be further blunted because of dramatic reperfusion-induced decreases in ARC protein levels resulting from degradation in the ubiquitinproteasomal pathway.104,105 Consistent with this, even low-level (three-fold) cardiac-specific transgenic overexpression of ARC is adequate to reduce infarct size by approximately 40% after ischemia-reperfusion in vivo.106

The intrinsic pathway is also modulated at the postmitochondrial level. IAPs bind and inhibit already activated effector caspases107 and may interact with procaspase-9 to interfere with apoptosome assembly.108 In addition, IAPs can ubiquitinate effector caspases, targeting them for degradation in the proteasome.109,110 Cardiac-specific transgenic over-expression (two-fold) of cIAP2 reduces infarct size by 32% after ischemiareperfusion in vivo.111 Although caspase-3/7 activity was not measured in this study, TUNEL staining was shown to be decreased. Apart from their actions in the postmitochondrial apoptosis pathway, however, cIAP1 and cIAP2 also inhibit necrosis and apoptosis pathways induced by death receptor signals. These effects are mediated by non-canonical K63-polyubiquitination of receptor interacting protein 1 (RIP1) by cIAP1 or cIAP2 (in conjunction with TRAF2), leading ultimately to NF-κB (nuclear factor κ light-chain enhancer of activated B cells) activation and transcription of survival genes.112,113,114,115,116,117 In light of this complexity, the antiapoptotic/necrotic mechanisms that mediate reduction of infarct size by cIAP2 remain undefined. The pleiotropic actions of the IAPs, however, suggest that they may be useful in novel therapies to limit infarct size.

Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and Omi/HtrA2 (Omi/High temperature requirement protein A2) are mitochondrial apoptogens that neutralize IAPs through direct binding, resulting in the displacement of active caspases.118,119 In addition, Omi/HtrA2 degrades IAPs irreversibly through its serine protease activity.120 UCF-101 [(5-[5-(2-nitrophenyl) furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid)] is a small-molecule inhibitor of the Omi/HtrA2 serine protease activity. UCF-101 reduces infarct size by 47%121 and 30%122 after ischemia-reperfusion in vivo in mice and rats, respectively. This was accompanied by attenuation of IAP loss and decreases in caspase activity, apoptosis, and cardiac dysfunction. The efficacy of UCF101 is notable in light of potential redundancy from Smac/DIABLO. A possible explanation may relate to the fact that UCF-101 antagonizes IAP degradation, an irreversible event that can be carried out by Omi/HtrA2 but not Smac/DIABLO. Another possible explanation for the efficacy of UCF-101 is that, by maintaining IAP levels, it

promotes the death receptor survival pathway, discussed previously. Despite these open mechanistic questions, there is significant interest in the clinical potential of this compound.

Polycaspase inhibitors have been shown to decrease infarct size by 21% to 52% in mice, rats, and rabbits after ischemia-reperfusion in vivo.123,124,125,126 The mechanisms responsible for these effects are incompletely understood, however. For example, it is unclear whether amelioration of cardiac damage is mostly due to antagonism of upstream or downstream caspases. In a similar vein, it is not known whether downstream caspase inhibition lessens mitochondrial dysfunction or mitochondrial necrotic events.127

Another level of complexity involves several striated muscle-specific caspase-3 substrates. The contractile proteins α-cardiac actin and troponin T are cleaved by caspase-3 in cardiac myocytes, and experimental cleavage of α-cardiac actin decreases contractile function in skinned fibers.128 Although these caspase-3 cleavage events may simply be part of the cell death pathway, they raise the possibility that caspases can also cause contractile dysfunction apart from inducing cell death.

3.1.2. Necrosis in myocardial infarction

The extensive studies in the preceding section establish that cardiac myocyte apoptosis is important in ischemia- reperfusion-induced myocardial cell death; however, myocardial infarction has traditionally been thought of as involving predominantly necrosis. Although necrosis is often envisioned as an unregulated processes, work over the past decade in worms and mice has demonstrated that at least some instances are highly controlled. This regulation involves two pathways, one mediated by cell surface death receptors79,80,116,117 and the other by events at the inner mitochondrial membrane65,66,129,130,131,132,133 Most investigations involving myocardial ischemia-reperfusion have focused on the mitochondrial necrosis pathway.

In this context, Ca2+ overload and oxidative stress trigger opening of the MPTP, a channel in the inner mitochondrial membrane.129 Ischemia leads to intracellular acidosis, causing increases in intracellular Na+ via the Na+/H+ exchanger. Increases in intracellular Ca2+ subsequently result via the Na+/Ca2+ exchanger and through Ca2+-induced Ca2+ release from the ER/sarcoplasmic reticulum. Meanwhile, reperfusion induces oxidative stress and rapid alkalinization, both resulting in MPTP opening.134 MPTP opening has two major consequences: (1) loss of inner mitochondrial membrane potential resulting in cessation of