Clinical Implications of Apoptosis in Ischemic Myocardium

Clinical Implications of Apoptosis in Ischemic Myocardium Tiziano M. Scarabelli, MD, PhD, Richard Knight, MD, PhD, Anastasis Stephanou, PhD, Paul Townsend, PhD, Carol Chen-Scarabelli, MS, Kevin Lawrence, PhD, Roberta Gottlieb, MD, David Latchman, PhD, Jagat Narula, MD Abstract: Apoptosis, a genetically programmed form of cell death, contributes to myocyte cell loss in a variety of cardiac pathologies, including cardiac failure and those related to ischemia/reperfusion injury. The apoptotic program is complex, involving both pro- and anti-apoptotic proteins, and apoptosis occurs when the equilibrium between these opposing factors is perturbed. Some of these factors are intrinsic to the apoptotic pathway, such as the pro- and anti-apoptotic members of the Bcl2 family. Other, extrinsic, cellular factors can also modify the outcome of the response to an apoptotic stimulus. In this review, we have focused on some of these extrinsic factors, such as STAT-1 as a pro-apoptotic agent and the urocortins and Bag-1 as anti-apoptotic factors, since these may be potential therapeutic targets. In addition, we discuss the profound cytoprotective effects of the antibiotic, minocycline. (Curr Probl Cardiol 2006;31:181-264.)

Overview of Apoptosis Cell Death Overview. All cells, including those in the heart, can die by a variety of mechanisms. Although it is often implied that these mechanisms are mutually exclusive, in practice, considerable overlap exists between The authors have no conflicts of interest to disclose. Curr Probl Cardiol 2006;31:181-264. 0146-2806/$ – see front matter doi:10.1016/j.cpcardiol.2005.11.002

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them, and individual dying cells may exhibit features of more than one mechanism over time (reviewed in 1). Indeed, writing of the distinction between apoptosis and necrosis, Farber2 was moved to comment “There is no field of basic cell biology and cell pathology that is more confusing and more unintelligible.” Therefore, although we will describe the different cell death mechanisms separately for ease of exposition, these qualifications must always be considered. Moreover, although we may now use different descriptive terms, the morphology of the death processes that we recognize today was originally described by nineteenth century microscopists (reviewed in 3). Thus, what we now call apoptosis was first described in the epithelial cells of atretic ovarian follicles by Flemming in 1885. Apoptosis remained a morphological concept following its rediscovery and renaming in 1971,4 until its molecular mechanism was identified by Horwitz’s lab in caenorhabditis elegans (reviewed in 5), and the realization that the single apoptotic regulatory genes in the nematode had multigene family orthologues in mammals. In this portion of the review, we will cite relevant reviews wherever possible, rather than original articles, to avoid the bibliography becoming excessive.

Apoptosis Introduction. Morphologically, apoptosis is characterized by cell shrinkage, condensation, and margination of the chromatin and budding of the plasma membrane (Fig 1). Cellular organelles and nuclear and cytoplasmic material become surrounded by intact plasma membrane, and these apoptotic bodies are phagocytosed by professional phagocytes or, less efficiently, by neighboring cells.4 If, however, phagocytosis is delayed, for reasons not well understood, the apoptotic fragments undergo necrosis. In apoptosis, as a result of the rapid vesiculation and phagocytosis, no intracellular material is released into the extracellular environment, and consequently, no widespread local inflammatory response is generated. In contrast, rupture of the plasma membrane during necrosis results in leakage of intracellular contents, which induce a strong inflammatory reaction (Fig 1). Therefore, lesions where apoptosis is the predominant mode of cell death will be smaller than those in which death is necrotic. Classically, apoptosis can be initiated either by ligation of a death receptor or by mitochondrial injury (Fig 2). 182

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FIG 1. Apoptotic versus necrotic morphology.

FIG 2. The mitochondria-initiated and the death receptor-mediated apoptotic pathways. (Color version of figure is available online).

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Both cause activation of a cascade of proteolytic enzymes—the caspases—which cleave a large number of substrates essential for the maintenance of cellular integrity, resulting in death of the cell (reviewed in 6). Francis Blankenberg: Membrane changes in apoptosis. Coincident with caspase activation, there is a rapid rearrangement of the plasma membrane phospholipid structure (Fig 1). The membrane changes result in the sudden selective expression of phosphatidylserine (PS), a negatively charged aminophospholipid, which is normally restricted to the inner leaflet of the lipid bilayer, on the cell surface. This is accompanied by a random scrambling of all other choline and aminophospholipids across the plasma membrane, effectively abolishing the normal phospholipid asymmetry. This scrambling is associated with an increase in membrane lipid fluidity.i The exposure of PS on the cell surface and increased membrane fluidity allow regions of the plasma membrane of apoptotic cells to protrude (membrane blebbing) and break off (forming small vesicles called “apoptotic bodies,” see Fig 1). Apoptotic bodies can be found in the circulation, especially if apoptosis involves the endothelium. Recent studies by Mallat and coworkersii identified a marked increase in circulating apoptotic bodies in patients with acute myocardial infarction and unstable angina, suggesting a major role for apoptosis in the genesis of these syndromes. In addition to shedding apoptotic bodies, apoptotic vesicles/lipid droplets also appear within the cytoplasm of the cell undergoing apoptosis and are visible histologically and via MR spectroscopy. Recent studies have shown that 1H MR spectroscopy can be used to track a range of these small molecules and membrane components that change during the course of apoptosis.iii Increases in membrane fluidity in apoptotic cells have been reported in vitro.iv The accumulation of cytoplasmic polyunsaturated lipid containing droplets (0.2 to 2.0 ␮m in diameter) has been observed following severe myocardial ischemia.v Reeves and coworkersvi also noted increases in myocardial lipid with postischemic dysfunction (“myocardial stunning”).

The Death Receptor Pathway Death receptors are ubiquitously expressed and are characterized by the presence of an intracellular “death domain,” which, on ligation of the receptor, transduces the apoptotic signal (reviewed in 7,8). Six death receptors have been identified, including CD95 (also known as APO-1, Fas), tumor necrosis factor alpha (TNF␣) TNFR1, and DR3-6, and all are expressed in the heart.9,10 Their corresponding ligands, CD95 ligand (CD95L), TNF␣, and TNF-related apoptosis-inducing ligand (TRAIL), are also expressed in the heart. Although the precise mechanisms of apoptosis induction following ligation of death receptors varies between the different receptors, there are common features. In general, receptor ligation results in the recruitment of adaptor molecules to the death 184

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FIG 3. The death receptor-mediated signaling pathway. (Color version of figure is available online).

domain, which, in turn, recruits the enzymatically inactive procaspase-8 (also known as FLICE). The resulting complex is known as the deathinducing signaling complex (DISC). The recruitment of procaspase-8 to the DISC results in its oligomerization and activation through selfcleavage, and enzymatically active caspase-8 then cleaves downstream caspases, such as caspases-3, -6, and -7 (reviewed in 11) (Fig 3). As an example, CD95L is a 40-kDa transmembrane molecule that is expressed as both membrane-bound and soluble form (reviewed in 12). CD95L engages its cognate receptor, CD95, resulting in recruitment of Fas-associated death domain (FADD) through homotypic interactions mediated by the death domain of CD95,13 and subsequent recruitment and activation of pocaspase-8. There are also natural inhibitors of caspase-8 activation following death receptor activation, known as FLICE-like inhibitor proteins (FLIP). There are both viral and cellular (c) FLIPs, and cFLIP is expressed as both short and long forms (FLIPS and FLIPL) derived by alternative splicing.14 cFLIPs are enzymatically inactive splice variants of procaspase-8, which compete with procaspase-8 for binding to FADD, although FLIPS and FLIPL inhibit procaspase-8 activation by different mechanisms.15 cFLIP is expressed in cardiac myocytes, and its expression has, for example, been shown to be downregulated in allografted apoptotic cardiac myocytes.16 Curr Probl Cardiol, March 2006

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An additional anti-apoptotic pathway can be activated following ligation of TNFR and TRAIL receptors (reviewed in 17,18). In the case of TNFR ligation, two complexes are formed. Complex I contains TNFR and a number of adaptor molecules, together with receptor interacting protein (RIP) but not procaspase-8, while complex I forms in the cytosol and lacks TNFR and RIP but does contain procaspase-8.17 RIP degrades I␬B␣, a protein that retains NF-␬B in an inactive form in the cytosol, thus allowing NF-␬B to translocate to the nucleus and effect transcription of NF-␬B responsive genes.19 Active NF-␬B antagonizes TNFR-mediated pro-apoptotic signaling, since TNF␣-induced apoptosis is increased in the absence of NF-␬B activity, and enforced activation of NF-␬B protects against TNF␣-induced apoptosis.20,21 Therefore, the outcome of death receptor ligation depends on the relative degree of activation of pro- and anti-apoptotic signaling pathways, though the determinants influencing these antagonistic effects are not fully understood.

The Mitochondrial Pathway A wide range of apoptotic stimuli converge on the mitochondria and cause the release of a number of apoptotic factors present in the mitochondrial intermembrane space (reviewed in 22). As well as agents that damage the mitochondria directly, the mitochondrial pathway can also be activated following death receptor ligation, where active caspase-8 cleaves the BH3 only protein (see below), Bid, whose cleavage product, tBid, migrates to the mitochondria and damages the mitochondrial membrane (reviewed in 23). Cytochrome-c, a component of the electron transport chain, is normally localized on the outside of the inner mitochondrial membrane, and its release into the cytosol is generally the earliest and most critical initiating factor for mitochondrial-mediated apoptosis24 (Fig 1). In the cytosol, cytochrome-c binds, in the presence of ATP, to apoptosis protease activating factor (Apaf-1). Procaspase-9 is recruited to, and activated, in this complex, called the apoptosome (reviewed in 25,26) (Fig 4). Enzymatically active caspase-9 then cleaves and activates effector caspases such as caspases-3, -6, and -7. Cytochrome-c is released either through channels created by integration of Bax and Bak (see below) in the mitochondrial membrane or following opening of the mitochondrial permeability transition pore (MTP). This channel is composed of proteins of both inner and outer mitochondrial membranes together with proteins of the intermembrane space, and its opening results in influx of ions, such as calcium, causing swelling of the mitochondria. This swelling produces 186

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FIG 4. The mitochondrial signaling pathway and the apoptosome formation. (Color version of figure is available online).

breaks in the outer membrane, although the inner membrane remains intact, and cytochrome-c escapes through these outer membrane breaks.27

Cytoplasmic Contraction in Apoptosis A classic feature of apoptosis is the loss of cytoplasmic volume associated with the breakdown of normal cytoskeletal elements and membrane bleb formation. Contraction of the cytoplasm appears to be an essential feature of apoptosis and the packaging of cytoplasm and organelles into apoptotic bodies.

The Bcl2 Family The integrity of mitochondrial membranes is largely under the control of members of the Bcl2 family. Bcl2 (B-cell lymphoma 2) was originally identified as a gene linked to an immunoglobulin locus as a result of chromosomal translocation in follicular lymphoma. Currently, at least 20 members of the Bcl2 family have been identified, all of which share at least one Bcl2 homology (BH) domain (reviewed in 28). The family may be divided into three groups, one of which contains five anti-apoptotic proteins, Bcl-xL, Bcl-w, A1, Mcl1, and Bcl2 itself. Two further subfamilies are pro-apoptotic proteins; the Bax family has BH1-3 domains similar to those in Bcl2, whereas the other pro-apoptotic proteins have only the BH3 domain (Fig 5). Anti-apoptotic Bcl2 proteins have a C-terminal region that targets them to the outer mitochondrial membrane as well as to the endoplasmic Curr Probl Cardiol, March 2006

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FIG 5. Bcl2 proteins and the modulation of apoptotic cell death. (Color version of figure is available online).

reticulum and the nuclear membrane. Bcl2 is an integral mitochondrial membrane protein, even in the absence of any cellular insult, whereas other protective Bcl2 proteins only become associated with mitochondria following cellular injury. There is some debate as to whether the sole action of protective Bcl2 proteins is to prevent mitochondrial release of pro-apoptotic proteins such as cytochrome-c, or whether they may also influence caspase activation directly. The pro-apoptotic Bcl2 proteins Bax and Bak are thought to act mainly on the mitochondrial membrane. In healthy cells, they are cytoplasmic, but change conformation, migrate to the mitochondria, and oligomerize following an apoptotic signal. Oligomers of Bax/Bak promote permeabilization of the outer mitochondrial membrane, which allows release of death-promoting factors such as cytochrome-c. However, the evidence that Bax/Bak directly interacts with and opens the MTP is controversial. The activity of BH3-only proteins is normally inhibited by a variety of mechanisms, including sequestration by binding to other proteins and transcriptional regulation. They appear to function as cellular sensors, becoming activated after intracellular damage and developmental signals. BH3 proteins do not, however, induce apoptosis directly, but require other pro-apoptotic Bcl2 proteins such as Bax and Bak (reviewed in 29). Therefore, the outcome of a cellular insult depends, at least in part, on 188

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FIG 6. An overview of the signaling pathway of apoptotic cell death.

the balance between anti- and pro-apoptotic Bcl2 family proteins. However, the mechanism of their interaction remains unclear, although it would appear physiologically not to involve physical interaction between, say, Bcl2 and Bax.

More Checks and Balances As in the death receptor mediated pathway, there are a number of intrinsic proteins, which inhibit or augment mitochondrial-mediated apoptosis (Fig 6). Francis Blankenberg: Mitochondrial Changes with Ischemic Stress. In addition to the cytoplasmic contraction early in the apoptotic cascade, there is also significant decrease in volume and number of cardiac mitochondria (programmed death of a mitochondrion, “mitoptosis”), which ultimately fragment into lipid containing multilamellar vesicles.vii,viii,ix Cardiac mitochondria occupy 38 to 40% of the entire cytoplasmic volume of the myocyte.x In terms of the absolute number there is between 1000 and 2000 mitochondria per myocyte. Heart function is intimately associated with the biochemical health, ultrastructural integrity, and the absolute number of mitochondria within the myocardium.xi,xii,xiii Mitochondria also play critical roles in programmed cell death following ischemic stimuli, as mentioned by Dr. Knight’s excellent review.xiv,xv

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FIG 7. A: XIAP-mediated inhibition of caspases 3 and 9. B: Smac/DIABLO removal of XIAP-mediated inhibition of caspase 9. (Color version of figure is available online).

The Inhibitor of Apoptosis Proteins (IAP). The IAPs (reviewed in 30) are a family of seven proteins that are characterized by the presence of at least one baculovirus IAP repeat (BIR) motif, reflecting their original identification in baculovirus. Some BIR-containing proteins are inhibitors of the activation of caspases (Fig 7A). Caspase inhibition is largely dependent on the BIR domains, and individual BIR domains appear to be selective for particular caspases. Thus BIR2 and the region between BIR1 and BIR2 are important for the inhibition of the effector caspases-3 and -7, although some studies have claimed that the inter-BIR region is sufficient for caspase-3 inhibition. In contrast, BIR3 is important for inhibition of the initiator caspases, caspase-9. Thus, since individual IAPs contain only either BIR1, BIR2, or 190

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BIR1-3, they differ in their inhibitory specificity for different caspases. In addition to their inhibition of caspase activation, IAPs can also ubiquitinate caspases, thus promoting their proteosomal degradation. Inhibitors of IAPs. Currently, three proteins are known that bind and suppress IAPs—XIF1 (XIAP associated factor 1), second mitochondrial activator of caspases (Smac, also known as DIABLO), and Omi (also known as HtrA2). XIF1 is a nuclear protein, while its target, XIAP, is predominantly cytosolic. However, it has been proposed that XIF1 promotes nuclear relocalization of XIAP and that this sequestration inhibits XIAP activity. In contrast, Smac (reviewed in 31) is a mitochondrial protein. Mitochondrial injury results in the removal of the mitochondrial localization signal and release of Smac into the cytoplasm either together with, or after, cytochrome-c release (Fig 7B). It has been suggested that, whereas the small cytochrome-c (c 12 kDa) can exit the mitochondria through Bax/Bak-formed pores, the larger (c 100 kDa) Smac can only be released from mitochondria following opening of the MTP. Smac binds to five IAPs (XIAP, cIAP1, cIAP2, survivin, and livin) and inhibits their activity. The mechanism of Smac binding and inhibition of IAPs is not fully clarified, but it appears that one Smac dimer interacts strongly with the BIR2 and BIR3 domains of XIAP, thus displacing XIAP from caspase-7 and -9. However, Smac/IAP interaction with IAPs need not necessarily inhibit IAP-mediated caspase inhibition, since IAPs can also ubiquitinate Smac, thus targeting it for proteosomal degradation. The, presumably, multiple factors that determine the biological outcome of this complex set of interactions and modifications remain to be determined. Omi/HtrA2 is a trimeric serine protease which is also present in the mitochondrial inner membrane space and which also translocates to the cytoplasm following mitochondrial damage. Like Smac, Omi competes with caspase-3, -7, and -9 for IAP binding, and therefore, promotes caspase activation. However, its action is confined to XIAP, cIAP1, and cIAP2, and it preferentially binds to the BIR2 domain of XIAP. IAPs are also cleaved by the serine protease activity, and Omi can induce apoptosis by proteolysis independently of its effects on IAPs. It is unknown whether Omi can be ubiquitinated and inactivated by IAPs.

Other Pro-Apoptotic Mitochondrial Proteins Apoptosis is characterized by chromatin condensation and DNA cleavage into both high and low molecular weight fragments. Two other Curr Probl Cardiol, March 2006

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mitochondrial proteins—apoptosis inducing factor (AIF) and endonuclease G (EndoG)—are also released after mitochondrial injury and may well contribute to this feature of apoptosis. There is some debate as to whether the release of these two factors can occur after opening of the MTP or requires further proteolysis of the mitochondria by activated caspases. AIF is a conundrum. It is a flavoprotein with NADH oxidase activity, and therefore, has the potential to exert anti-apoptotic effects. However, AIF also clearly degrades mitochondrial and nuclear DNA independently of caspase activity, although the mechanism by which it alters chromatin structure, and produces DNA fragmentation and apoptosis, is unclear, since it lacks intrinsic nuclease activity. Nevertheless, AIF mutants that are no longer able to bind to DNA, and mutants lacking the C-terminal sequence, lack chromatin condensing activity. Translocation of AIF from the cytosol to the nucleus is inhibited by heat shock protein (hsp) 70, and this may be one mechanism for the anti-apoptotic effects of hsp70. Endo G (reviewed in 31) was originally identified in mitochondrial supernatants as a factor that produces caspase-independent DNA fragmentation in purified nuclei, although its main function appears to be maintenance of the integrity of mitochondrial DNA. The major nucleases responsible for DNA fragmentation in apoptosis in intact cells are caspase-activated DNase (CAD) and acid lysosomal DNase II (reviewed in 32), since CAD- and DNase-II-deficient mice show little DNA degradation following apoptotic stimuli. Therefore, although it remains possible that Endo G can cooperate with these and other DNases, its precise role in DNA fragmentation remains to be established.

Caspases Caspases (reviewed in 33) are the proteolytic executioners of the apoptotic process. Fourteen have been identified in mammals, at least seven of which are involved in apoptosis. They may conveniently be divided into initiator and effector enzymes, and all are expressed as inactive precursor zymogens, which must be proteolytically processed to generate the active enzymes. The initiator caspases, such as caspase-2, -8, -9, and -10, are characterized by long N-terminal regions that contain one or more adaptor domains (death effector domain, DED, or caspase recruitment domain, CARD), which are absent in the effector enzymes. As described above, activation of initiator caspases takes place in a multiprotein complex, such as the apoptosome for caspase-9 and the DISC for caspase-8. Active initiator caspases then sequentially activate downstream effector 192

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caspases, such as caspase-3, -6, and -7 by cleavage at internal Asp residues. Effector caspases are expressed as homodimers and their activation involves intrachain cleavage that generates fragments of c.10 and c.20 kDa still in a dimeric form. Active effector caspases recognize a 4-amino-acid motif in their substrates, P4-P3-P2-P1, and cleave after the C-terminal (P1) Asp. When last reviewed,34 more than 280 caspase substrates had been identified. Many of these are structural and regulatory proteins, which are inactivated by caspase cleavage, resulting in the classic apoptotic morphology. In a minority, however, caspase cleavage results in a gain of function, and although the consequences of this are not well understood, in some cleaved proteins the active fragment may serve to amplify the apoptotic process.

Other Forms of Cell Death To revert to a theme raised at the beginning of this section, pure apoptosis, as observed in in vitro models, may not be the only cell death mechanism operative in complex in vivo pathologies, such as those involved in ischemia/reperfusion injury and cardiac failure. Here, for the sake of completion, we will briefly review other modes of cell death.

Autophagy Autophagy involves the formation of a membrane surrounding normal or damaged organelles, and the digestion of the resulting vesicles by lysosomes (reviewed in 35). It appears to be a physiological mechanism of cell survival during periods of temporary starvation, and has, for example, been observed in mice between birth and suckling.36 As such, it may also occur during brief periods of ischemia, though its contribution, for example, to preconditioning has not been studied. Classic autophagocytic vesicles and autophagy have been observed in rabbit hearts rendered hypoxic for 20 to 40 minutes and then reperfused,37 and this is associated with functional myocyte recovery. Surprisingly, perhaps, the subsequent cardiology literature has paid little attention to autophagy as a cell protective mechanism in the ischemic myocardium, and this could be a promising area for further research. If the starvation period is prolonged, cells undergoing autophagy proceed to apoptosis, and thus autophagy may be both a protective process as well as a precursor to cell death depending on the intensity and duration of the insult. Francis Blankenberg: Mononuclear Clearance of Apoptotic Cells. When monocytes arrive at the sites of tissue injury, they identify damaged or Curr Probl Cardiol, March 2006

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FIG 8. Oncosis, apoptosis, and necrosis, as final stage of both death processes. (Color version of figure is available online).

senescent myocytes, cells which selectively express high levels of PS early after activation of the caspase cascade, through a PS-specific recognition membrane protein receptor located on their cell surface.xvi This system is the primary mechanism clearance of apoptotic myocytes, mononuclear cells, granulocytes, and all other types of cells that have outlived their useful function.xvii In general inflammatory cells, including granulocytes, lymphocytes, and macrophages, undergo apoptosis when they have completed their assigned tasks at sites of tissue injury. Monocytes and macrophages also serve to accelerate the apoptosis of bystander (unwanted) granulocytes at sites of myocardial apoptosis via the selective release of a variety of soluble proteins in concert with soluble Fas ligand, a peptide which binds to the Fas death receptor expressed on granulocytes and other types of inflammatory cells.xviii

Oncosis In contrast to apoptosis, which is characterized by cell shrinkage, oncosis (reviewed in 1,38) is characterized by swelling, of both organelles and the whole cell (Fig 8). 194

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It is the major mechanism of cell death in ischemic injury, and the balance between oncosis and apoptosis appears to be largely determined by intracellular ATP content. If ATP content has fallen to below 80% or so of normal levels, death occurs by oncosis; better ATP retention results in apoptosis. Like apoptosis, oncosis is also a proteolytic process, but involving calpains rather than caspases.

Necrosis There is a consensus that necrosis refers not to a mechanism of cell death but is a description of an end-stage dead cell. Autophagy, oncosis, and apoptosis can therefore all result in necrosis. Francis Blankenberg: PARP-Mediated Cell Death. Cell swelling can also be due to a metabolic cell death initiated by massive activation of a normally quiescent nuclear enzyme called poly-ADP-ribose polymerase (PARP or PARS).xix PARP is generally activated in the later stages of apoptosis and helps induce fragmentation of a cell’s DNA. Direct DNA damage by ionizing radiation, or radical ion formation by agents such as doxorubicin or HII,xx however, in some circumstances can induce massive unregulated activation of PARP and sequestration of NAD⫹, its substrate that in normal conditions is used to repair relatively minor DNA strand breaks. If massive enough, this activation can totally deplete a cell’s reserve of NAD⫹, and subsequently, all intracellular stores of ATP (the normal mechanism by which NAD⫹ is replenished). The total loss of ATP-derived energy effectively stops the apoptotic cascade, an ATP-energy-dependent process resulting in apparent necrotic cell death.

Molecular Mechanisms of Endogenous Cardioprotection In addition to the various proteins inherent in the apoptotic process that can influence the balance between death or survival of the cell, a number of other proteins that are not integral components of the apoptotic pathway can also influence outcome. These include the signal transducers and activators of transcription (STATs), the Bcl-2 Associated athanoGene 1 (Bag-1) proteins, the heat shock proteins, and the urocortins. Here, we will focus on the death-modulating role of the STATs and BAG-1 proteins.

Role of STAT-1 and STAT-3 in the Ischemic Myocardium Introduction. Protection of the ischemic myocardium against tissue injury continues to elude basic investigators and clinicians and is therefore still a major objective for the identification of effective Curr Probl Cardiol, March 2006

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strategies for the treatment of ischemic heart disease. The limitations of current therapies largely arise from our limited understanding of the molecular events that modulate the severity of myocardial damage during ischemia/reperfusion (I/R) injury. However, over the past decade, it has become clear that the ischemic myocardium initiates a number of complex signaling pathways that either mediate an adaptive stressinduced protective response or, if the insult is more severe, activate the cell death pathway that leads to loss of myocytes and compromised cardiac function. Cell death in cardiac myocytes can occur by necrosis or apoptosis, although the relative contribution of the two mechanisms to total myocyte loss remains controversial. The following section will focus on the transcriptional modulation of the apoptotic process that also plays an important role in the initiation of cell death. Within the p53 family members, p53 and p73 have been well described as major players in promoting apoptosis following various stressful stimuli.38 Most studies on p53 and p73 have focused on models of DNA damage-induced cell death and very little is known about these pro-apoptotic transcription factors in the heart exposed to I/R injury.

STATs STAT factors are a family of cytoplasmic transcription factors that mediate intracellular signaling initiated at cytokine cell-surface receptors and transmitted to the nucleus. STATs are activated by phosphorylation on conserved tyrosine and serine residues by the Janus kinases (JAKs) and mitogen-activated protein kinase (MAPK) families, respectively, which allow the STATs to dimerize and translocate to the nucleus and thereby regulate gene expression (Fig 9). The C-terminal domains of STAT proteins all contain a transcriptional transactivation domain (TAD), plus the phosphorylation site for JAKs and MAPK, which are essential for maximal STAT function. At present, seven different STAT family members have been characterized and found to encode by distinct genes (STAT-1, STAT-2, STAT-3, STAT-4, STAT-5a, STAT-5b, and STAT-6). Different STATs are activated by a distinct group of cytokines. For example, interferon␥ is a potent activator of STAT-1, while the interleukin-6 (IL-6) family members, including IL-6, leukemia inhibitory factor (LIF), and cardiotropin-1 (CT-1), primarily activate STAT-3.39,40 The overall structure among the STAT proteins (especially STAT-1 and STAT-3) is quite conserved within the coiled-coiled domain (residues 114 to 317), the DNA binding domain (residues 320 and 490), the linker domain (residues 490 and 580), and the SH2 domain (residues 580 and 680). The carboxy-terminal TAD (residues 680 to 750 in STAT-1 and 196

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FIG 9. STA1 activation signaling pathway. (Color version of figure is available online).

STAT-3) is also highly conserved. In contrast, the amino-terminal domain is less conserved among the STATs, suggesting that this part of the protein may be involved in mediating specific responses. Both the coiled-coiled and the SH2 domains are involved in protein–protein interaction. Curr Probl Cardiol, March 2006

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STAT signaling has also been shown to be negatively regulated by two groups of proteins. One group was identified following the discovery that cytokines that activated STATs were also shown to induce the expression of suppressors of cytokine signaling (SOCs) or STAT-induced STAT inhibitors (SSIs).41 These SOCs proteins were shown to bind to the active receptors, which abrogated binding of JAKs and therefore inhibited activation of STAT signaling.42 Another group of negative STAT activators was identified as nuclear factors that were able to bind to phosphorylated STATs and were named PIAS (protein inhibitors of activated STATs).43 PIAS1 was shown to specifically inhibit STAT-1 activation,44 whereas PIAS3 was a specific inhibitor of STAT-3.45 Thus, PIAS1, in addition to inhibiting STAT-1 activation, may also have other roles in modulating p53 function.

STAT Signaling in the Heart Activation of the STAT pathway was first documented in rat cardiac myocytes following exposure to LIF, which resulted in STAT-3 activation.46 It was subsequently demonstrated in the intact heart following pressure overload-induced hypertrophy, leading to the activation of STAT-1 and STAT-3.47 Activation of the JAK STAT pathway has recently been shown to play a role in ischemic preconditioning in the intact heart. Inhibition of STAT-1 and STAT-3 phosphorylation with the JAK inhibitor AG-490 blocked preconditioning-induced cardioprotection.48 Furthermore, the abolition of classical preconditioning effects was observed in STAT-3-deficient mice.49 Activation of STAT-1 and STAT-3 has also been demonstrated in the isolated intact heart following I/R injury.50-52 Pretreatment with the JAK-inhibitor reduced STAT-3 phosphorylation and enhanced apoptosis following I/R. Conversely, in cultured cardiac myocytes treated with CT-1, which activates the STAT-3 pathway, enhanced cell survival following exposure to simulated I/R injury and reduction in the level of apoptotic cell death were observed.53,54 Furthermore, STAT-3-deficient mice were shown to be more susceptible to cardiac injury and sensitive to developing heart failure following various stresses to the myocardium.55,56 Following I/R injury, larger infarct sizes and a greater number of apoptotic cardiac myocytes were noted in STAT-3-deficient mice compared to wild-type mice.55,56 Thus, these studies demonstrate that STAT-3 may be an anti-apoptotic signaling factor in the heart, with the ability to protect the myocardium following ischemic injury. In contrast to STAT-3, STAT-1 plays a role in enhancing apoptotic cell death in cardiac myocytes, following simulated I/R injury, by inducing 198

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the expression of the pro-apoptotic caspase-1, Fas, and FasL genes leading to increased cardiac cell death.50-52 Moreover, inhibition of STAT-1, using an antisense approach, prevented the augmentation of caspase-1, Fas, and FasL gene activity in cardiac myocytes exposed to simulated I/R and protected cardiac cells from I/R-induced cell death.50,51 In addition, it was also shown that STAT-1 inhibited the promoters of genes encoding the anti-apoptotic Bcl-2 and Bcl-x proteins.57 Hence, STAT-1 activation appears to induce apoptosis in cardiac myocytes by activating pro-apoptotic genes, as well as repressing anti-apoptotic genes. The mechanism of STAT-1 activity in cardiac myocytes exposed to simulated I/R has been previously investigated. Earlier studies demonstrated that both the tyrosine 701 and the serine 727 sites of STAT-1 were phosphorylated in cultured cardiac myocytes, as well as in the isolated intact heart exposed to I/R.50,51 However, studies using STAT-1 mutant constructs demonstrated that the induction of Fas and FasL, as well as enhanced apoptosis in cardiac myocytes exposed to simulated I/R, required the phosphorylation of STAT-1 on serine 727 but not on tyrosine 701.51 The phosphorylation of serine 727 of STAT-1 appears to be accomplished by p38 MAPK activation during I/R, since it can be blocked by both the chemical inhibitor SB203580 and a dominantnegative form of MKK6, the upstream activator of p38 MAPK.51 Although phosphorylation of tyrosine 701 was originally thought to be essential for STAT-1 function, recent studies have shown that some genes can be induced by STAT-1 in a tyrosine 701 independent manner. In addition, serine 727 phosphorylation has also been reported to be critical for the activity of the C-terminal STAT-1 transactivation domain (TD) to bind to other coactivator molecules such as MCM5 and BRCA1.58,59 Recently, a novel protein–protein interaction between STAT-1 and tumor suppressor p53 transcription factor has been described.60 This association enhances the activity of pro-apoptotic genes in a manner that is dependent on the p53 binding site in their promoters.60 Similarly, STAT-1, together with p53, was able to enhance the level of apoptosis to a greater extent than either p53 or STAT-1 alone.60 Interestingly, the STAT-1/p53 association can occur with the C-terminal region of STAT-1 lacking a DNA-inducing domain, paralleling the ability of this domain to enhance apoptosis in cardiac myocytes.60 In contrast, it has been reported that p53 is able to inhibit STAT-3 activation.61 Thus, these studies indicate that STAT-1, but not STAT-3, is able to mediate its effects on gene expression, at least in part, by acting as a coactivator and modulator of the functional activity of p53. Previous studies demonstrated that I/R-induced apoptosis required Curr Probl Cardiol, March 2006

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serine 727 of STAT-1, but not tyrosine 701, in cardiac myocytes. Therefore, the activity of the C-terminal TD of STAT-1 (via phosphorylation on serine 727) mediated the effects of cell death in cardiac myocytes exposed to I/R. This is supported by the observation of enhanced cell death in a model using a STAT-1 construct encoding only the C-terminal TD and lacking the DNA-binding domain in cardiac myocytes exposed to I/R.62 Moreover, the exchange of serine 727 to a nonphosphorylatable alanine reduced the ability of the isolated Cterminal STAT-1 construct in promoting cell death in cardiac myocytes exposed to simulated I/R. Likewise, cardiac myocytes isolated from mice lacking the N-terminal domain of STAT-1 (amino acids 1 to 134), but expressing the C-terminal domain, were more sensitive to I/R-induced cell death.62 The isolated intact hearts from these mice exposed to I/R injury had larger infarct sizes and a greater number of TUNEL-positive myocytes than control hearts.62 Interestingly, it has been shown that STAT-1 can be cleaved by caspases such as caspase-3 at position 694.63 As previously mentioned, different groups have demonstrated that caspases play an active role in apoptotic cell death in cardiac myocytes exposed to I/R.54,55 Cleavage of STAT-1 by caspase-3, at position 694, will ultimately release the C-terminal STAT-1 TAD. The N-terminal fragment containing the DNA-binding domain may function as a dominant negative against intact STAT-1 protein, while the caspase-mediated generation of the proapoptotic C-terminal TAD fragment may be involved in amplifying and perpetuating the apoptotic loop in hearts exposed to I/R injury.

STAT-1 and STAT-3: Therapeutic Targets Against the Damaged Myocardium Following I/R Injury The aforementioned studies suggest that modulation of STATsignaling may be an attractive therapy against the damaged myocardium. For example, identification of pharmacological and specific inhibitors of STAT-1 activation may be a feasible way to reduce the apoptotic effects of STAT-1. Recently, it has been reported that the polyphenolic agent epigallocatechin-3-gallate (EGCG), a major constituent of green tea, is a potent inhibitor of STAT-1 phosphorylation and activation.64 Recently, the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart have been assessed. EGCG reduced STAT-1 phosphorylation and protected cardiac myocytes against I/R-induced apoptotic cell death. EGCG also reduced the expression of a known STAT-1 200

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pro-apoptotic target gene, Fas receptor.65 More interestingly, oral administration of GTE, as well as EGCG infusion, limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction in cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart.65 This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Since GTEmediated cardioprotection is achieved, at least in part, through inhibition of STAT-1 activity, one may postulate that a similar action can be implemented in the clinical setting, to minimize STAT-1 activation levels in patients with acute coronary artery disease (CAD). In contrast to STAT-1, STAT-3 activation would need to be enhanced to have any beneficial effects in protecting the damaged myocardium following an ischemic insult. One feasible way to enhance STAT-3 activation is via a cytokine that is known to primarily induce STAT-3 signaling in the heart, such as CT-1.53,54 CT-1 has previously been shown to protect both neonatal and adult cardiac myocytes against I/R-induced apoptosis.53,54 Another feasible, but yet untested, method is a gene therapy approach in which the STAT-3 viral vector expresses a constitutively active form of STAT-3 that is only expressed in the heart and inducible when required. Thus, it is clear that STAT-1 plays a critical role in inducing pro-apoptotic genes and apoptosis in cardiac myocytes exposed to I/R, while STAT-3 is able to protect against apoptosis in the heart. Therefore, the relative levels of activated STAT-1 or STAT-3 may determine the balance between death and survival of cardiac myocytes following I/R-induced myocardial damage. Moreover, STAT-1 is known to enhance the functional activity of the p53 pro-apoptotic transcription factor.60 p53 is also known to inhibit the activation of STAT-3; consequently, the level of p53 will shift the relative balance towards cell death rather than survival. Thus, the STAT-1 or the STAT-3 activated pathways are potential therapeutic targets in the prevention of ischemic heart disease. Agents that are likely to inhibit STAT-1, but not STAT-3, activity, and vice versa, may guide the development of therapeutic strategies, which may subsequently prevent the progression to heart failure. Another group of proteins that may affect cell survival is the Bag-1 family. A detailed discussion of this protein family is provided in the following section. Curr Probl Cardiol, March 2006

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The Role of Bag-1 as a “Cardioprotective” Survival Molecule The Identification and Structure of Bag-1. The Bag-1 family of proteins was identified some 10 years ago by two separate research laboratories, whose goal was to search for novel partners for the previously identified anti-apoptotic molecule, Bcl-2 protein, and the activated nuclear hormone glucocorticoid receptor, respectively.66,67 The protein was named by virtue of its binding to Bcl-2 and its pro-survival properties, hence Bcl-2-associated athanoGene-1 (Greek: athanos, antideath). Over the last decade, Bag-1 has been a particularly intensive focus of research, especially in cancer cell biology, where Bag-1 has been shown to exist as multiple isoforms, and to interact with a wide range of cellular targets.66-71 Although originally identified as a Bcl-2 binding protein,66 it is now clear that Bag-1 isoforms interact with a wide range of cellular targets including the 70-kDa heat shock chaperone proteins, Hsc70 and Hsp70, nuclear hormone receptors, signaling molecules (eg, Raf-1), and components of the protein ubiquitylation/degradation machinery (eg, the E3 ligases Siah and CHIP and the proteasome), as well as DNA itself.66-72 In-depth biochemical studies have suggested that Bag-1 is thought to function by coupling the activity of the Hsc70/Hsp70 chaperones to specific protein targets, therefore, potentially acting as a co-chaperone. Therefore, through its multiple partners, Bag-1 can regulate cellular proliferation and survival activities, including transcription and apoptosis, important for both normal and diseased cells (Fig 10). The pleiotropic nature and multifaceted “pro-survival” behavior of Bag-1 in the modulation of these assorted pathways raised intense curiosity in examining and deciphering both the precise function(s) and the expression of Bag-1 in normal and pathological cardiac physiology, as a route for prospective molecular prophylaxis therapy. Although originally appreciated as a protective regulator against thermal stress, the so-called heat shock response, Bag-1, is likely to be activated by a wide range of other critical cardiac-related stresses, both physiological and pathological, including cardiac development, aging, osmotic changes, and ischemia. Bag-1 exists as multiple protein isoforms through alternate translation initiation of a single mRNA.73 The gene for human Bag-1 resides at chromosome 9 band 12 and is composed of seven exons (Fig 11). The most abundant protein (short) isoform, Bag-1S (36 kDa), is translated from the AUG codon (position ⫹348) and has a predominantly cytoplasmic localization. The largest (long) isoform, Bag-1L, 50 kDa, is translated 202

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apoptosis stress responses proliferation

BAG-1 signalling

transcription

FIG 10. BAG-1 pleiotropic function. (Color version of figure is available online).

from the CUG codon (position ⫹1), contains a nuclear localization sequence within its N-terminal extension, and is localized within the nucleus. There is also an intermediate (middle) isoform, Bag-1M, of 46 kDa, which initiates from the first in-frame AUG at position ⫹216. Within each Bag-1 isoform, an assortment of protein domains have been recognized.68,72,74 The Bag domain (BD) is a carboxy-terminal domain of ⬃70-amino-acid residues present in all isoforms. The core of the Bag domain is involved in mediating the interaction with the heat-shock chaperone molecules, where Bag-1:chaperone binding complexes play a critical role in many of Bag-1 functions. Similarly, all Bag-1 isoforms contain an ubiquitin-like domain (ULD).75,76 Ubiquitin is a ubiquitous 76-amino-acid residue protein that is covalently attached to protein substrates by a series of substrate recognition, activation, and conjugation reactions. A key function of ubiquitin is in targeting proteins for degradation by the proteasome, the major nonlysosomal proteolytic complex in cells.77 The ULD is essential for some activities of Bag-1 and appears to be important for Bag-1:proteasome binding.78 In addition, two potential nuclear localization signals (NLS) have been identified within Bag-1 proteins; one is within the unique amino-terminal domain of Bag-1L (therefore directing its nuclear localization), and the second NLS Curr Probl Cardiol, March 2006

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FIG 11. Molecular organization of BAG-1

lies within Bag-1S. Finally, there are multiple copies (up to nine) of a 6-amino-acid repeat within all of the human Bag-1 isoforms at their amino-termini. The precise function of these acidic acid repeats remains unknown, yet this part of the molecule is thought to be important for some of Bag-1 functions, including DNA binding.79 The Role of Bag-1 in the Heart. Isoform-specific expression of Bag-1 in mouse development has been demonstrated previously.80 In situ hybridization and immunohistochemistry established that Bag-1 expression is stage- and site-specific, with Bag-1L being ubiquitously expressed early in development and progressively downregulated during later stages. Notably, Bag-1S was only detected in the myocardium during early developmental stages before being present in other organs during later development. More recent data established a vital role for Bag-1 in the heart. Bag-1 was shown to be highly expressed in cardiac tissues and assist in cytoprotection in injured heart cells or, indeed, the whole heart.81 More specifically, using model systems of primary isolated neonatal and adult cardiac myocytes or the intact rat heart, it was documented that only the S and L isoforms of Bag-1 are expressed in cardiac cells, which is in keeping with the lack of the internal AUG for the Bag-1M isoform, within the rodent sequence. Most noticeably, not only were specific Bag-1 protein isoforms induced following injury, but their subcellular localization was altered following the reintroduction of oxygen. In primary 204

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Elevated cytoplasmic BAG-1 expression – independent of the ULD

I/R expression levels

BAG-1S HSC70

complex formation

BAG-1:HSC70

CM Apoptosis

BAG-1:RAF-1

FIG 12. The mechanism of BAG-1-mediated cardioprotection is independent of the interaction between Bag-1 and ULD. For further explanation, refer to the text. (Color version of figure is available online).

cultures of cardiac myocytes exposed to simulated ischemia and reperfusion injury, Bag-1 relocalized to the nucleus from the cytoplasm following ischemia and, once there, offered substantial levels of cardioprotection,81 as documented by a dramatic reduction in the magnitude of myocyte apoptosis. Molecular analyses using specifically constructed overexpression DNA vectors also demonstrated that the short isoform of Bag-1, Bag-1S, which is predominantly cytoplasmic, was the only isoform conferring cardioprotection. In addition, contrary to most previous descriptions presented for Bag-1 in transformed cells, the expression of gross domain and point mutant expression constructs revealed that cardioprotection was solely dependent upon chaperone binding, not on the cell survival regulator Raf-1, and did not require the N-terminal ubiquitin-like domain81 (Fig 12). A series of coimmunoprecipitation experiments, carried out in primary cultures of rat cardiac myocytes, showed that the interaction of Bag-1 with Hsc70 and Raf-1, which was clearly documented in control conditions, dramatically diminished following simulated ischemia/reperfusion, to the benefit of Bag-1:Hsc70 complexes, suggesting that Bag-1-mediated cardioprotection does not involve interaction of Bag-1 with components of the ubiquitylation/proteasome machinery (Fig 11). Taken together, these data exemplify that Bag-1 proteins act unexpectedly in cardiac cells, being consistent with the model that Bag-1 directs chaperones to specific cellular targets to mediate cytoprotection. HowCurr Probl Cardiol, March 2006

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ever, the growth inhibitory or pro-apoptotic molecules that could also control stress responses and are targeted by the Bag-1/chaperone complex remain to be identified. Having discussed basic mechanisms of cell death, and how death/ survival may be modulated by factors such as STAT-1, STAT-3, and Bag-1, we now turn to the evidence for apoptosis as a distinct form of cell death in various cardiac pathologies, beginning with ischemia/reperfusion injury, and demonstrating the range of techniques in common use for the identification of apoptosis in the heart. Francis Blankenberg: Other Mechanisms of Endogenous Cardioprotection. As mention in the previous section “Mitoptosis,” apoptosis of the mitochondria as distinct from cellular apoptosis, is observed during periods of myocardial stress/ischemia. The role of mitoptosis in cellular apoptosis, however, remains far from certain. The induction of mitochondrial permeability transition pores and cytochrome-c released in the absence of caspase activation is an insufficient stimulus for apoptosis in some experimental systems.xxi Paradoxically, the release of NAD⫹ from injured mitochondrion, which cluster around nuclei during apoptosis, can have salutary effects on cell survival by providing an essential substrate for certain nuclear DNA repair enzymes.xxii The release of mitochondrial NAD⫹ to the cytoplasm requires an active oxidation of the mitochondrion’s own NADH/NADPH stores, which are necessary for its function and survival. This act of self-sacrifice does not go unreciprocated as the cell’s nuclear DNA is repaired, an essential requirement not only for cell survival but also for the generation of new mitochondria. Mitochondrial DNA fragments during periods of ischemic or drug-induced stress (ie, bleomycin DNA strand breaks). Contrary to the situation in the cell nucleus, mitochondrial DNA fragmentation may indicate self-repair and protection against oxidation.xxiii,xxiv Fragments of mitochondrial DNA not essential for function or replication (as opposed the crucial circular 16-kB mitochondrial DNA) are preferentially oxidized. These fragments act as “mini-sumps” for DNA damaging free radicals agents within the mitochondrion. Oxidation is accompanied by a significantly increased rate of DNA fragment production via mitochondrial DNA transcription (as opposed to destruction). Increased rates of synthesis produces all sizes of DNA fragments that are needed to mop up more free radicals from the mitochondrial matrix and protect circular mitochondrial DNA. Annexin V as a Stress-Related Protective Protein?. Annexin V (MW ⫽ 35.7 kDa) is a widely distributed intracellular protein with many proposed functions based on its nanomolar affinity for PS. Annexin V may play significant roles in cell physiology including controlling membrane permeability to calcium and inhibition of pro-apoptotic signals from protein kinase C and phospholipase A2. Also of interest, annexin V in some cell lines inhibits apoptosis based on its ability to increase intracellular calcium concentration.xxv Circulating levels of annexin V are virtually nil (1.7 ng/mL); however, they can rise several hundred-fold with myocardial infarction, suggesting that this protein may behave as an acute phase reactant.xxvi Annexin V is ubiquitously distributed in cardiomyocytes and, to a greater extent, endothelial cells and fibroblast.xxvii Annexin V is normally found in the 206

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sarcolemma, T-tubules, and intercalated disks of myocytes and in the cytoplasm of fibroblasts and endothelial cells. In heart failure annexin V is upregulated, with increased amounts of protein translocated to the interstitial tissues, suggesting a role in interstitial fibrosis and myocardial remodeling. Externalized PS, now known to be a powerful signal for cell removal by phagocytes, may be masked by annexin V, thereby inhibiting the local inflammatory response.

Apoptosis in Ischemia/Reperfusion Injury Introduction It is now generally accepted that extensive cardiac cell death occurs not only during cardiac ischemia but also during reperfusion following the ischemic episode. Considerable controversy exists, however, concerning the nature of the cell death that occurs during either ischemia or reperfusion. In particular, there is much debate as to the relative contributions of necrotic and apoptotic cell death during both ischemia and reperfusion. This debate is of some importance since apoptosis is a highly regulated energy-consuming process, whereas this is not the case for necrosis. Hence, in principle, apoptosis should be more amenable to inhibition by specific agents to produce a therapeutic benefit. However, the debate has been confused by attempts to observe, in the ischemic or postischemic heart, individual markers of apoptosis that have been defined on the basis of studies in noncardiac cells. Depending on whether or not one or other of these markers have been identified, various authors have concluded that apoptosis either does or does not make a significant contribution to cell death in the ischemic and postischemic heart. Evidently, however, the process of apoptosis may not be identical in cardiac cells to that previously observed in noncardiac cells. What is required, therefore, is an analysis of the various processes that are involved in cardiac cell death during ischemia and reperfusion so that the mechanism of such death can be defined, thereby paving the way for its therapeutic inhibition. This section will therefore consider the various features of the apoptotic process that have been defined in noncardiac cells and will analyze their occurrence in cardiac cells during ischemia and reperfusion. In this manner, it will be possible to analyze the overall nature of the cell death processes that occur in the heart in this situation and relate these to the classical apoptotic program.

DNA Fragmentation One of the defining features of apoptosis in noncardiac cells is the specific fragmentation of DNA within its normal chromatin structure. Curr Probl Cardiol, March 2006

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FIG 13. DNA fragmentation is a typical hallmark of apoptotic cell death. (Color version of figure is available online).

DNA, in chromatin, is organized so that approximately 200 base-pairs of DNA are associated with histone proteins to form a nucleosome (Fig 13). Hence, specific digestion of the DNA between individual nucleosomes results in DNA fragments of 200 base-pairs or multiples thereof (ie, 400, 600, 800 base-pairs, etc.). One popular method of detecting such DNA fragmentation is to use terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, the terminal transferase enzyme is used to achieve the labeling of free 3=-ends of fragmented DNA with labeled dUTP. Although this method has been widely used, it has been criticized on the grounds that it will also detect the random degradation of DNA that occurs during cardiac cell necrosis (82, for review see 83). Hence, a number of investigators have also carried out DNA laddering experiments in which DNA is isolated from the appropriate portion of the heart and subjected to gel electrophoresis. In this method, the ordered fragmentation of DNA characteristic of apoptosis will produce a ladder of DNA bands of 200, 400, 600, etc., base-pairs, whereas the random degradation characteristic of necrosis will produce a smear. Studies using both these methods have provided evidence that ischemia/ reperfusion induces ordered DNA fragmentation in the heart but have 208

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differed as to its time-course. Thus, for example, Gottlieb and coworkers84 did not identify TUNEL-positive cells or DNA laddering in the rabbit heart exposed to ischemia alone but did detect DNA fragmentation by both these assays during reperfusion following ischemia. In contrast, Kajstura and coworkers85 did observe DNA fragmentation by both these assays in rat hearts exposed to prolonged ischemia without reperfusion. In a more recent study,86 very few TUNEL-positive cells were detected in the dog heart exposed to ischemia alone and no DNA fragmentation was observed upon gel-electrophoresis. In contrast, a very large number of TUNEL-positive cells were observed in the peri-necrotic area after 6 hours of reperfusion and extensive DNA laddering was also observed at this time point. These studies suggest that the ordered DNA fragmentation characteristic of apoptosis does indeed occur in cardiac cells. Moreover, although there may be differences between different species and different experimental systems, it is likely that the vast majority of DNA fragmentation is confined to the postischemic period rather than occurring during ischemia itself. This conclusion is reinforced by the work of Scarabelli and coworkers,87 who took advantage of the fact that, unlike DNA laddering procedures, TUNEL staining is able to label individual cells (Fig 14A and B). They were therefore able to differentiate DNA fragmentation in endothelial cells from that occurring in the cardiac myocytes. In neither case was TUNEL positivity observed in the rat heart exposed to ischemia alone. However, TUNEL positivity was detected in endothelial cells after as little as 5 minutes of reperfusion and peaked at 60 minutes of reperfusion, decreasing at 2 hours of reperfusion. In contrast, the proportion of TUNEL-positive cardiac myocytes slowly increased over 2 hours of reperfusion. As expected, DNA laddering (which cannot differentiate endothelial from myocyte apoptosis) detected DNA fragmentation in samples prepared after reperfusion but not in samples exposed to ischemia alone. These studies, therefore, indicate that DNA fragmentation does occur in the heart, particularly during reperfusion, and has a different time-course in endothelial cells and cardiac myocytes. The importance of such DNA fragmentation is confirmed by a recent study in which TUNEL-positive cells were detected at postmortem in human hearts of patients with severely unfavorable cardiac remodeling, after left-ventricular myocardial infarction.88 Similarly, TUNEL-positive cells were also observed in biopsy samples from patients undergoing cardiopulmonary bypass, warm Curr Probl Cardiol, March 2006

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FIG 14. A: TUNEL-positive endothelial cells labeled by an anti-von Willebrand antibody. B: TUNEL-positive myocyte labeled by an anti-desmin antibody, which produces a red banding running perpendicularly to the long axis of the cell. (Color version of figure is available online). (Color version of figure is available online).

blood cardioplegia, and subsequent reperfusion, but not in similar biopsies taken before the onset of these procedures.89 The importance of DNA fragmentation as a key step in cardiac damage-induced apoptosis is supported by studies in which such DNA fragmentation was inhibited by treatment with aurintricarboxylic acid (ATA), an inhibitor of DNA endonucleases.90 In these experiments, such addition of ATA at the onset of reperfusion following cardiac ischemia resulted in reduced infarct size and enhanced regional contractile function. Hence, inhibition of DNA fragmentation inhibits reperfusioninduced cell death.

Translocation of Phosphatidylserine A widely used assay of apoptosis relies on the translocation of phosphatidylserine to the outside of the cell membrane that occurs during 210

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FIG 15. Translocation of phosphatidylserine residues from the inner to the outer side of the cell membrane during the early stages of the apoptotic process.

apoptosis. Hence, apoptotic cells exhibit surface staining with labeled Annexin V, which binds to phosphatidylserine (Fig 15). In experiments in an in vivo mouse model of ischemia/reperfusion, surface staining with Annexin V was demonstrated in the intact heart only during reperfusion and not during the ischemic episode.91,92 Hence, as with DNA fragmentation, it appears that this feature of apoptosis occurs predominantly during reperfusion. As with TUNEL labeling, surface staining with Annexin V has also been demonstrated in human patients. Thus, six of seven patients with acute myocardial infarction who were treated with primary coronary angioplasty showed increased uptake of labeled Annexin V in the infarct area, indicating that apoptosis was occurring.93 This use of labeled Annexin V to examine apoptosis in living patients offers an important diagnostic tool as well as a means of potentially examining the effects of specific therapies on apoptosis in the heart (for review see 94). Francis Blankenberg: Reversible Expression of PS and Myocardial Ischemic Stress. Although the localization of exogenously administered annexin V appears to depend on ongoing apoptosis, recent experimental data suggest that the physiologic stresses such as that induced by ischemia may lead to Curr Probl Cardiol, March 2006

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FIG 16. Colocalization of TUNEL and Caspase-3-positive staining used as marker of apoptotic cell death. (Color version of figure is available online). (Color version of figure is available online).

transient and reversible PS expression, which, if unchecked, will lead to cell death by apoptosis.xxviii Thimister’s clinical study found that annexin V localization partially resolved by day 3 to 4 and completely by day 8 in regions of ischemic injury following acute myocardial infarction. These results suggest that either the injured cells that concentrated tracer were removed from the ischemic zone or these cells recovered in terms of both function and viability with loss of PS positivity. The reduction of perfusion abnormalities with restoration of regional wall motion 1 week following infarction suggests the latter explanation. If true, then annexin V imaging may be vastly more sensitive to cellular stress than previously thought and may be a true marker of tissues at risk that have the potential for salvage with prompt therapeutic intervention.xxix,xxx

Caspase Activation Among the effector caspases, caspase-3 has been shown to have an important role in the heart. Thus, for example, activation of caspase-3 has been observed in the hearts of several different species exposed to ischemia-reperfusion (95,96, for review see 97). Interestingly, in the experiments of Scarabelli and coworkers,87 active caspase-3 was observed in rat hearts exposed to ischemia alone, whereas, 212

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as noted above, TUNEL-positivity was observed only during reperfusion. However, during reperfusion, staining for active caspase-3 colocalized with TUNEL staining (Fig 16). This suggests that cleavage of caspase-3 may represent a relatively early event in apoptosis that occurs during cardiac ischemia, with subsequent DNA laddering occurring only as a later event during reperfusion. The importance of caspase activation in the cell death caused by ischemia/reperfusion is supported by studies in which either a generalized caspase inhibitor or a specific inhibitor of caspase-3 can reduce infarct size.98,99 Moreover, when given at reperfusion, such inhibitors are not only able to reduce infarct size but can also protect left ventricular function and attenuate remodeling.100 Hence, these experiments establish an important role for caspases in cell death in the heart exposed to ischemia/reperfusion and indicate a particularly critical role for caspase-3 as an important effector caspase in the heart. Moreover, the role of caspase-3, which has been established by inhibitor experiments, is also supported by findings in which overexpression of caspase-3 targeted to the heart of mice resulted in increased infarct size and reduced cardiac function.101 Moreover, the importance of caspase-3 in the cardiac response to ischemia/reperfusion is also supported by studies in human patients where activation of caspase-3 has been observed during postinfarction left-ventricular remodeling102 and in patients undergoing coronary bypass surgery.103 Although these studies establish the importance of caspases and, in particular, of the effector caspase-3 in cell death in the heart exposed to ischemia-reperfusion, it is also necessary to determine which initiator caspases activate the effector caspases such as caspase-3 in the heart. Evidence is now available that both initiator caspase-8 and initiator caspase-9 play important but distinct roles in cardiac cell death in response to ischemia/reperfusion. Thus, an initial study99 demonstrated that specific inhibitors of either caspase-9 or caspase-8 given at reperfusion were able to reduce infarct size in the isolated rat heart. More detailed studies in cultured cardiac cells have indicated that both chemical and gene-based inhibitors of caspase-9 can reduce apoptotic cell death in cardiac myocytes exposed to simulated ischemia alone, whereas inhibition of caspase-8 has no effect. In contrast, inhibition of either caspase-8 or caspase-9 was able to reduce apoptotic cell death in response to ischemia/reperfusion.104 These studies in cultured cardiac cells were supported by further studies in the intact isolated heart exposed to ischemia/reperfusion that demonstrated activation of caspase-9 during ischemia alone with further activation during reperfusion, whereas caspase-8 was only activated by reperCurr Probl Cardiol, March 2006

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FIG 17. Caspase-9 activation (solid arrow) was mainly observed in endothelial cells, while caspase-8 activation (broken arrow) was only detected in cardiac myocytes. For further explanation, see text. (Color version of figure is available online).

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fusion following ischemia.105 Moreover, as well as this difference in the activation of the two caspases during ischemia and reperfusion, it was also observed that their activation differs in endothelial cells and cardiac myocytes. Thus, activation of caspase-9 was observed primarily in endothelial cells and only to a much lesser extent in cardiac myocytes, whereas activation of caspase-8 was only observed in cardiac myocytes (Fig 17). In agreement with this, a specific caspase-9 inhibitor prevented endothelial apoptosis in this system, whereas a specific caspase-8 inhibitor affected only cardiac myocyte apoptosis.105 When taken together with earlier results described above on the time-course of apoptosis in the different cell types,87 these findings suggest a model in which activation of caspase-9 during ischemia results in endothelial cell apoptosis, which continues during reperfusion, whereas activation of caspase-8 specifically during reperfusion is responsible for cardiac myocyte apoptosis. In turn, such activation of caspase-8 and caspase-9 will activate effector caspases such as caspase-3, which is observed in both endothelial cells and cardiac myocytes87 and in turn leads to cleavage of cell survival proteins and DNA fragmentation with TUNEL-staining and DNA-laddering. This key role for caspase-8 and caspase-9 is further supported by the detection of activated forms of both caspases in patients undergoing coronary artery bypass grafting when biopsies are taken following a period of cardioplegic arrest and subsequent reperfusion.89 The key role of caspase-8 and caspase-9 in the activation of effector caspases and the setting off of the apoptotic cascade focuses attention on the upstream signals that induce activation of caspase-9 and caspase-8 in the heart exposed to ischemia/reperfusion. These signals are discussed in the next sections.

The Mitochondrial Pathway Although originally defined in noncardiac cells, cytochrome-c release occurs also in a number of different situations in cardiac cells. Thus, release of cytochrome-c has been observed in the intact heart exposed to ischemia/reperfusion with the movement of cytochrome-c from the mitochondria to the cytosol becoming maximal during the reperfusion phase.105-107 Such release of cytochrome-c has also been observed in human cardiac cells in patients with cardiomyopathy107 and in failing human myocardium where it was associated with caspase-9 activation.108 The key role for cytochrome-c release that is suggested by its detection in animal models and failing human hearts is further supported by studies Curr Probl Cardiol, March 2006

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in which inhibition of cytochrome-c release was found to block apoptosis,109 whereas its addition to heart cytosol was shown to be sufficient to induce apoptosis.110

Death Receptors In terms of death receptors, there is evidence indicating that Fas and Fas ligand are involved in cell death in response to ischemia/reperfusion in the heart. Thus, both Fas itself85 and Fas ligand111 show increased expression during experimental cardiac ischemia/reperfusion with significant amounts of Fas ligand being released into the coronary effluent from postischemic hearts during reperfusion. Moreover, increased expression of Fas ligand112,113 and of Fas itself114 has also been observed in human cardiac patients. More direct evidence for the role of the Fas/Fas ligand system in cell death during cardiac ischemia/reperfusion has been obtained from lpr mice which lack functional Fas. Exposure of these mice to ischemia/ reperfusion leads to reduced cell death and infarct size directly indicating a role for Fas in these processes.115 Similarly, overexpression of Fas ligand in the heart is sufficient to induce cell death in some but not all situations (for review see 97). Taken together, therefore, these findings suggest that the Fas/Fas ligand system plays an important role in cardiac ischemia/reperfusion and in the observed activation of caspase-8, which occurs during reperfusion. It is possible, however, that other changes that occur during cardiac ischemia/ reperfusion may be necessary to sensitize the cardiac cells to the elevated levels of Fas ligand that are observed during this process and therefore to induce cell death via the Fas receptor.

Other Proteins Regulating Apoptosis As described above, several other protein families, such as Bcl-2 and p53, can influence the outcome of an apoptotic signal, such as ischemia/ reperfusion injury. In a detailed study in the intact heart exposed to ischemia/reperfusion (for review see 86), upregulation of the pro-apoptotic Bax and p53 proteins was observed during reperfusion with decreased expression of the anti-apoptotic Bcl-2 protein, whereas none of these proteins showed altered expression during ischemia alone. Hence, changes in these proteins may play a role in the cell death, which occurs during the reperfusion phase following ischemia. In agreement with the potential role of Bcl-2 in cell death in cardiac cells, overexpression of Bcl-2 in the heart, either in transgenic animals or by virally mediated gene delivery, reduces both infarct size and apoptosis 216

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in hearts exposed to ischemia/reperfusion.116,117 Similarly, such overexpression of Bcl-2 in cultured cardiac cells exposed to hypoxia not only reduces apoptosis but decreases cytochrome-c release from the mitochondria, suggesting that Bcl-2 achieves its anti-apoptotic effect in cardiac cells at the level of the mitochondrion.118 Again, as described above, a further connection between the Bcl-2 family and the mitochondrial pathway of apoptosis is provided by the Bid protein, a member of the Bcl-2 family. Thus, during reperfusion following ischemia, Bid is cleaved by caspase-8, with cleavage not occurring in the presence of a caspase-8 inhibitor.105 However, the cleaved Bid then induces release of cytochrome-c from the mitochondria, supplementing the cytochrome-c release that occurred during ischemia and early in reperfusion and resulting in further activation of caspase-9. Hence, in the presence of a caspase-8 inhibitor, an early phase of cytochrome-c release occurs, but this is not maintained as reperfusion goes on due to the lack of Bid cleavage.105 Thus, the findings discussed in this review indicate conclusively that a wide variety of features considered as characteristic of apoptosis do occur in the heart exposed to ischemia/reperfusion. These include DNA fragmentation, caspase activation, release of cytochrome-c, and altered expression of proteins such as Fas, Fas ligand, members of the Bcl-2 family, and p53. It appears, however, that the great majority of these changes are only observed during reperfusion following ischemia rather than during the ischemic phase itself. Although there is evidence that the apoptotic pathway can be initiated during ischemia, it appears that it is only fully executed during reperfusion. As such, apoptosis offers an attractive therapeutic target to modulate cell death and remodeling that occurs during reperfusion following an ischemic episode. One way of minimizing the cell death that accompanies ischemia/reperfusion injury is, of course, preconditioning.

Apoptosis and Preconditioning Introduction Ischemic preconditioning has long been recognized to be a potent cardioprotective intervention, resulting in a reduction in infarct size of up to 90%.119 Ischemic reconditioning has been shown to reduce apoptosis by fivefold in a model of 30 minutes of ischemia followed by 3 hours of reperfusion.120 Pharmacologic preconditioning has also been shown to reduce apoptosis.121 Since preconditioning can reduce infarct size by as much as 90%, it is reasonable to conclude that a large percentage of cell Curr Probl Cardiol, March 2006

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death associated with I/R is preventable. Whether this cell death is apoptosis or some other form of cell death becomes a matter of less concern, the focus moves towards identifying the targets of preconditioning that may impact cell homeostasis and survival. Many studies have been published examining delayed preconditioning, and it is generally accepted that gene transcription plays an essential role.122-126 However, this is not necessarily the case in immediate preconditioning, which depends more critically on posttranslational modifications such as phosphorylation. In this review, we will focus primarily on the signal transduction events of early preconditioning as they relate to cell survival.

Metabolic Features of Preconditioning Protection The earliest studies of ischemic preconditioning noted that intracellular pH in preconditioned hearts did not drop as low (as control hearts) during ischemia.127 Similarly, calcium (Ca2⫹) did not rise as high, and ATP levels were better preserved during the ischemic phase. These observations directed interest to the ischemic phase and supported the concept that injury developed during ischemia and that tissue preservation would depend on ameliorating injury during ischemia. Attempts to preserve ion homeostasis, such as inhibition of the sodium/hydrogen (Na⫹/H⫹) exchanger, were only successful as pretreatment, reinforcing this notion.128,129

ATP Necrotic cell death increases with longer ischemia, largely as a consequence of loss of adequate ATP to maintain cell integrity. The Na⫹/K⫹ ATPase is the least sensitive to ATP reductions and therefore is able to function long after protein synthesis and other cellular functions are lost.130 However, a more recent study has contested this, providing evidence that protein synthesis and sodium (Na⫹) pumping are suppressed to a similar degree when respiration is inhibited.131 Nevertheless, when ATP levels drop below a critical threshold, ion homeostasis will fail, resulting in cell swelling and rupture. Thus, with prolonged ischemia, necrotic cell death will predominate, while shorter periods of ischemia followed by reperfusion will be accompanied by regulated cell death.

Calcium Steenbergen and coworkers127 showed that Ca2⫹ rises during ischemia; however, subsequent studies132 have demonstrated little change in Ca2⫹ during ischemia compared to the massive elevation in systolic and 218

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diastolic intracellular Ca2⫹ during reperfusion. The smaller rise in intracellular Ca2⫹ in preconditioned hearts is beneficial through multiple mechanisms. Ca2⫹ can lead to excessive activation of phospholipases (both PLC and PLA2), which will destabilize membranes and liberate fatty acids, including sphingosine (which can be converted to ceramide or to sphingosine-1-phosphate, each with distinct biological effects) and arachidonic acid. While arachidonic acid may itself be pro-apoptotic in part through effects on the mitochondrial permeability transition pore (MPTP),133,134 it can be further metabolized by lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases. Arachidonic acid is converted to 20-HETE by -hydroxylase,135 which in turn can activate the action of a cytochrome P450 p38 mitogen-activated protein kinase (p38MAPK)136; 20-HETE can also trigger vasoconstriction, thus contributing to the “no-reflow” phenomenon.137 Ca2⫹ can activate calpain, which will cleave a variety of intracellular targets including the proapoptotic protein, Bid,138 and the membrane skeletal protein fodrin, which is important in maintaining calcium homeostasis.139 Elevated Ca2⫹ will also lower the threshold for opening of the MPTP, whose opening triggers mitochondrial swelling and release of pro-apoptotic factors. These events will be discussed in greater detail below, but it is noteworthy that overexpression of Bcl-2, which preserves mitochondrial integrity, enhances mitochondrial tolerance to Ca2⫹ loading and is also reported to limit endoplasmic reticulum Ca2⫹ release.140,141 Inhibition of the Ca2⫹dependent protease, calpain, reduces infarct size and contractility in part through preserving mitochondrial integrity and fodrin function.142,143

Intracellular pH The third metabolic parameter of interest is intracellular pH, which drops as low as 6.3 during ischemia. However, in preconditioned hearts, acidosis is attenuated, with the pH remaining above 6.5.127,144 This has been attributed to decreased glycolysis145 as well as limited Na⫹/H⫹ exchange.129 Acidosis has been shown to activate proapoptotic Bnip3, a BH3-only member of the Bcl-2 family.146 Bnip3 binds tightly to mitochondria at low pH, and this coincides with opening of the MPTP and is followed by caspase-independent cell death.147 Overexpression of Bcl-2 in murine hearts attenuates cytosolic acidification and consumption of ATP during ischemia,148 possibly through limitation of ATP hydrolysis by the F0F1-ATPase. This effect may be indirect, as it has been suggested that Bcl-2 may regulate VDAC to control ATP flux through the mitochondrial outer membrane.149 It should also be noted that hexokinase reversibly associates with the mitochondrial outer membrane, and this Curr Probl Cardiol, March 2006

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interaction is pH dependent.150 Hexokinase interacts with VDAC and opposes the release of cytochrome-c triggered by Bid or Bax.151,152 Although a low matrix pH opposes the opening of the MPTP,153 acidosis is reported to trigger release of mitochondrial matrix Ca2⫹.154

Reactive Oxygen Species A fourth consideration is the production of reactive oxygen species (ROS), which plays a dual role. Preconditioning triggers a modest burst of ROS that activates a signal transduction pathway that confers protection against the subsequent ischemic insult.155-157 However, preconditioning suppresses the large and sustained production of ROS following ischemia and reperfusion. Reactive oxygen causes lipid peroxidation of mitochondrial and plasma membranes, triggers mitochondrial MPTP opening, activates phospholipases, inhibits SERCA function, and activates a host of signal transduction pathways, some of which are pro-apoptotic.158-162 Interventions that limit ROS production or detoxify ROS are protective.163,164 Cellular detoxification requires glutathione (GSH) and glutathione peroxidase, as well as mechanisms to regenerate GSH. A recent study demonstrates the importance of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate shunt, in regeneration of GSH and amelioration of ischemia/reperfusion injury.165 Heme oxygenase, another enzyme involved in the response to oxidative stress, has also been shown to reduce infarct size and apoptosis.166,167

Signal Transduction Pathways in Preconditioning at the Intersection with Cell Death Despite the evidence that events during ischemia contributed to cell death, subsequent findings raised the possibility that events during reperfusion were equally important to tissue salvage, if not more so. The observations that apoptosis occurred in connection with reperfusion,84 and that preconditioning prevented apoptosis,168-170 led investigators to focus attention on reperfusion injury. Efforts to inhibit apoptosis have shown promise in a posttreatment setting.95,171 More recently, the identification of several interventions that are protective when given after ischemia, at the onset of reperfusion, support the concept that cell death is not established until some time during reperfusion.172-174 While ischemic preconditioning and pharmacologic preconditioning differ in some aspects, they share in common the activation of protein kinase C (usually the epsilon isoform), a requirement for the opening of the mitochondrial KATP channel (mitoKATP), and an early burst of ROS 220

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production.157,175-177 Additional studies have implicated ERK, PI3K, Akt/PKB, and p70S6K.178,179 Nitric oxide (NO) is considered to be important in both immediate and delayed preconditioning,180-182 and a growing number of studies suggest that exogenous NO activates guanylyl cyclase, leading to activation of cGMP-dependent kinase and subsequent effects on mitoKATP.183,184

Protein Kinase C The importance of protein kinase C has been demonstrated through use of inhibitors such as chelerythrine,185,186 small peptide agonists and antagonists,172,187 and through genetic manipulation.188,189 Most evidence points to protein kinase C, although some studies have implicated the delta isoform.190,191 Ping189 showed that preconditioning triggered translocation of PKC⑀ to mitochondria, while PKC␦ translocated out of cytosol to an unspecified compartment, presumably the Triton X-100insoluble fraction. Phosphorylation of cytoskeletal constituents by PKC could alter contractility, Ca2⫹ sensitivity, and ATP utilization, with potentially favorable effects on survival190,192-196; therefore, a beneficial role for PKC␦ cannot be excluded. However, a peptide antagonist of PKC␦ has been shown to reduce infarct size in transgenic mice.188 The downstream targets of PKC␦ are unknown, although the BH3-only Bcl-2 family member, Bad, has been implicated.197 Other studies identify mitoKATP as the ultimate target, although additional protein kinases may be involved.189

Nitric Oxide In most studies, NO has been shown to play a beneficial role, and many studies have demonstrated a pathway involving guanylyl cyclase, PKG, and the mitoKATP. NO may not be entirely benign, however, since it can combine with superoxide to generate the highly reactive peroxynitrite radical, which can interact with the mitochondrial electron transfer complexes to permanently inhibit respiration and ATP production, while NO can reversibly suppress respiration.198-200 This may explain why some studies have shown a beneficial effect from overexpression of NO synthase (NOS) or superoxide dismutase.201-203 The dynamic balance between NO and superoxide is important to regulation of vascular tone204 and may modulate responses within the cardiomyocyte as well.

Receptor-Dependent Signaling of Apoptosis Fas and FasL are downregulated by ischemic preconditioning.170,205 Although a recent study failed to detect changes in Fas, FADD, and Curr Probl Cardiol, March 2006

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caspase-8 activity, previous work remonstrated caspase-8 processing in endothelial cells.105 Low doses of TNF␣ can trigger preconditioning through a pathway involving ROS production.206 However, like ischemia/reperfusion and Fas ligation, TNF␣ can also trigger production of ceramide,207-209 which is well known to impair mitochondrial respiration210 and trigger apoptosis through opening of the MPTP.211,212 The cardioprotective effects of TNF␣ may be mediated through a ceramideindependent pathway, since ischemic preconditioning reduced ceramide production, and the administration of an inhibitor of sphingomyelinase reduced ceramide production and infarct size.213 Preconditioning also triggers the release of diacylglycerol, which activates protein kinase C isoforms and inhibits ceramide production.214

Mitochondria Preservation of mitochondrial integrity is widely viewed as crucial to cardioprotection.215,216 Caspase activation is attenuated in preconditioned hearts after I/R,217 but this is more likely to be a consequence of better preservation of mitochondrial integrity, since similar studies demonstrated reduced cytochrome-c release, suppression of MPTP opening, and decreased the ratio of Bax to Bcl-2.218,219 Preconditioning also triggers phosphorylation of Bad, thus preventing its association with mitochondria,220 an effect mediated by Akt and/or PKC⑀.189,220 Akt may also be protective by triggering the association of hexokinase to mitochondria,152 where it prevents Bax binding to VDAC.151 The clinical regimen of glucose, insulin, and potassium (GIK) may be protective in part through effects on Akt and hexokinase.221,222 However, it remains unclear whether the cardioprotective effect is due to enhanced intracellular glucose utilization or to inhibition of apoptotic pathways. Overexpression of Bcl-2 or Bcl-xL is cardioprotective,148,223 and administration of a short peptide corresponding to the BH4 domain of Bcl-xL has been shown to reduce infarct size.140,224 Thus, regulation of Bcl-2 family members is a critical determinant of cell survival after I/R. ARC (apoptosis repressor with CARD domain) has been shown to play an important cardioprotective role at the mitochondria, although it is unknown whether preconditioning modulates its activity.225

The MitoKATP Channel and the MPTP In nearly every case where it has been examined, the mitoKATP channel has been shown to be required for cardioprotection. However, no clear connection to classical apoptotic pathways has been established, and mitoKATP openers do not protect Jurkat cells against Fas 222

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ligation or UV-induced apoptosis (Gottlieb, unpublished data). Most studies have shown a role for the mitoKATP in oxidative stress or I/R, settings where activation of the MPTP seems to be required for apoptosis. Preconditioning or activation of the mitoKATP prevents opening of the MPTP by limiting matrix Ca2⫹ loading.226-230 Inhibition of MPTP opening by cyclosporin A or sanglifehrin A is cardioprotective,231-233 and MPTP opening has been demonstrated using radioactive tracers to measure matrix volume of rapidly isolated mitochondria from ischemic and reperfused hearts.234 It has been suggested that transient opening of the MPTP occurs during preconditioning and may represent a protective mechanism.235 The MPTP has been proposed to comprise VDAC in the outer mitochondrial membrane, ANT in the inner membrane, and cyclophilin D.236-238 However, studies by Fontaine and Bernardi have also implicated Complex I.239 MPTP opening results in ROS production240 and release of mitochondrial NADH.232 MPTP opening also recruits Bax and triggers cytochrome-c release, initiating apoptosis.241 The recruitment of Bax may be important, since at least one study indicates that MPTP opening may not be sufficient to induce apoptosis.242

Caspases, Calpains, and Other Proteases While a number of early studies indicated a role for caspases in postischemic cell death,95,168,217 it is not clear that they are essential.142 Calpains also appear to play an important role and are readily activated during reperfusion.138,243 Lysosomal proteases (cathepsins) have been implicated in some forms of cell death, and inhibitors of the cathepsins have been shown to reduce infarct size.244-246 Finally, the ubiquitinproteasome system plays a role in intracellular signaling after ischemia, notably regulation of NF␬B.247,248 Recent work on metalloproteinase inhibitors suggests that this class of proteases may play an important role in remodeling.249 In conclusion, I/R is a complex injury leading to cell death by a variety of mechanisms. Preconditioning, whether ischemic or pharmacologic, is able to salvage 50 to 90% of the tissue that would otherwise die. Critical determinants rest on cellular homeostasis: maintenance of Ca2⫹, pH, ATP, and redox. Although cell death pathways may be initiated during ischemia, they are not put into effect until reperfusion. With the demonstration that some therapies are effective when administered at reperfusion, the focus shifts from the question of apoptosis versus necrosis to whether cell death is avoidable. Studies of preconditioning Curr Probl Cardiol, March 2006

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yield valuable insights into the pathways that regulate cell death and suggest potential therapeutic approaches that may be effective at reperfusion. Francis Blankenberg: Dr. Gottleib’s comments on the need to refine the goal of therapy as the avoidance of cell death as opposed to inhibition of any particular mode of cell death are well taken. As apoptosis is an energy requiring process any massive loss of ATP such as seen with hypoxia or excessive activation of PARP would essentially stall cell death prior to its completion (ie, the self-packaging of a cell into apoptotic bodies and/or DNA ladder formation). Once stalled, a cell will simply swell at a given point in the apoptotic cascade and swell and disintegrate, becoming histologically and even biochemically indistinguishable from a necrotic cell. This may occur despite the fact that the cell’s death was initiated by an apoptotic signal/ pathway. If one simply designs therapy based on what is seen histopathologically, that is, at the end stage of a series of complex events in a cells death, novel therapeutic interventions may be overlooked.

Myocyte Apoptosis in the Hibernating Myocardium Unlike myocardial infarction, in which cellular necrosis along with irreversible loss of contractile function occurs following a permanent coronary occlusion, the myocardium exposed to chronic or repetitive sublethal ischemic insults undergoes an adaptive phenomenon, known as hibernation, in which cardiac metabolism and function are concomitantly downregulated to match the limited energy supply (“perfusion-contraction” matching).250 Since the viability of the hibernating myocardium is preserved, despite the functional downregulation of the affected segments, inotropic stimulation triggers a temporary recuperation of cardiac contractility, which is however associated with decompensation of the metabolic balance achieved by the hibernating segments.251 Other evidence documenting the viability of the hibernating heart includes the almost immediate contractile recovery of dysfunctional segments following revascularization procedures,252 as well as the enhanced glucose uptake within the hibernating myocardium revealed by positron emission tomography (PET).253 Nonetheless, the assumption of anatomical integrity of the hibernating myocardium based on the functional and metabolic recovery of the affected segments has been sharply questioned by subsequent findings. For instance, myocytes undergoing a dedifferentiation process, showing structural and biochemical features of fetal cardiomyocytes, have been described in myocardial biopsies obtained from the hibernating regions of patients with delayed recovery in cardiac function following surgical revascularization.254 The observation of additional 224

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morphological alterations in the same biopsy specimens, including an increased interstitial space in the absence of atrophic involution of cardiac myocytes, raised the question of whether cardiac cell loss could concomitantly occur. Myocyte apoptosis has been documented in the hibernating myocardium of both animals and man. In a porcine model of hibernating myocardium, created by prolonged and subocclusive stenosis of the left anterior descending coronary artery, apoptosis was detected by the in situ end-labeling method and deoxyribonucleic acid laddering on agarose-gel electrophoresis. Apoptotic myocytes, assuming a patchy distribution pattern, were observed in the hibernating regions of all instrumented pigs, though not in nonischemic remote areas and in sham-operated animals. Apoptotic nuclei were noted in association with patchy necrosis and/or around areas of focal fibrosis. A significant correlation was also observed between the magnitude of myocyte apoptosis and regional coronary blood flow reduction.255 The occurrence of myocyte apoptosis was also detected, independently of necrosis, though in association with compensatory hypertrophy and mild replacement fibrosis, in a similar porcine model of hibernating myocardium, subjected to 3 months of severe, though not complete, stenosis of the left anterior descending coronary artery.256 In a clinical setting, cardiac biopsies from 38 patients with hibernating myocardium exhibited structural degeneration, reparative fibrosis, as well as myocyte apoptosis (detected by the TUNEL method and electron microscopy), while dedifferentiation was not observed, suggesting that cellular degeneration, rather than adaptation, occurs in the hibernating heart. A significant correlation between the severity of morphological alterations and the extent of functional recovery was also observed, supporting the idea that delays in reperfusion may reduce the likelihood of complete structural and functional recovery after restoration of coronary flow.257 A more recent study from the same group, carried out in 14 patients with cardiac hibernation, reported the occurrence of myocyte cell loss via both ubiquitin-related autophagic cell death (ascertained by ultrastructural changes including autophagic vacuoles, cellular degeneration, and nuclear disassembly) and apoptosis (detected by TUNEL and electron microscopy).258 Conversely, complement-9, used as a marker of myocyte necrosis, was only found in 1 of the 14 biopsies.258 In contrast with the previous findings, in an analogous study carried out in 28 human subjects with myocardial hibernation, no apoptotic nuclei were detected by TUNEL, or by electron microscopy, in normal or Curr Probl Cardiol, March 2006

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dedifferentiated cardiomyocytes, hinting that cardiomyocyte dedifferentiation, and not degeneration via apoptosis, occurs in the chronic hibernating myocardium.259 The morphological features of the hibernating heart are still the object of controversy. Indeed, there is debate over the relative contribution of cellular degeneration versus cellular dedifferentiation to the overall structural damage taking place in the hibernating segments. Apoptotic cell death affecting cardiac myocytes was detected by different techniques in the majority, though not all, of the studies carried out so far. Although lacking a quantitative connotation, these studies have important clinical implications. If a slow, though enduring, myocyte apoptosis occurs during cardiac hibernation, namely a chronic condition which can persist silently for a long time before eventually becoming symptomatic, the progressive cardiac cell loss which ensues, in the long run, can very well become one important factor contributing to the deficient recovery in cardiac function of the hibernating myocardial segments following surgical revascularization. Consistent with the above hypothesis, in patients with myocardial hibernation undergoing coronary bypass surgery, the postoperative recovery in cardiac function was shown to be inversely proportional to the severity of the morphological changes and the duration of hibernation.260 If these findings are confirmed by subsequent studies focusing on structural and functional repercussions of delayed reperfusion of the hibernating heart, early revascularization should be recommended in all patients to minimize the extension of tissue degeneration in the hibernating myocardium, thereby improving functional recovery, as well as postoperative outcome. Reperfusion may be achieved by various means: pharmacological (using thrombolytic therapy), mechanical (by percutaneous coronary intervention, such as angioplasty with or without coronary stents), or surgical (ie, coronary artery bypass graft surgery [CABG]). Surgical revascularization, via CABG, may involve the use of the cardiopulmonary bypass machine and cardioplegia. In the next section, the occurrence of apoptosis in this clinical setting is described and reviewed. Francis Blankenberg: One of the key features of chronically ischemic (hibernating) myocardium is the loss of mitochondria via the process of mitoptosis as mentioned previously. Targeted mitochondria ultimately fragment into lipid-containing multilamellar vesicles along with the loss of mitochondrial integrity and extrusion of mitochondrial contents into the cytoplasm, including cytochrome-c, which are additional promoters of apoptosis. Hibernating myocytes therefore face not only direct metabolic damage to their cellular contents from chronic ischemic stress but also from 226

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degenerating mitochondria. The degenerating mitochondria not only add proapoptotic proteins but with their loss there is also loss of contractility and increased mechanical stress on a myocyte, thereby setting a viscous positive feedback loop that can realistically only be broken by prompt early revascularization therapy as championed by Dr. Chen-Scarabelli.

Myocyte Apoptosis in the Iatrogenic Ischemia/Reperfusion Injury The controlled forms of cardiac arrest, which are intentionally given to the heart during on-pump cardiac surgery, to facilitate the surgical manipulation of the diseased heart, are likely to represent, together with the temporary, though complete, coronary occlusion induced by balloon inflation during percutaneous transluminal coronary angioplasty (PTCA), the most common expression of iatrogenic ischemia/reperfusion injury. Several cardioplegic techniques, such as crystalloid, cold, and warm blood cardioplegia, have been developed in the last several decades, in the attempt to prevent or, at least, to minimize this inevitable surgically related ischemic insult.261 Route and modality administration of cardioplegia (continuous versus intermittent; antegrade versus retrograde) have also been extensively modified and diversified to maximize the final degree of cardioprotection during cardiac surgery. Nonetheless, the protection afforded by the different techniques employed so far was shown to be oftentimes inadequate, above all when case-related technical difficulties considerably prolong the overall time during which the heart is maintained on cardiopulmonary bypass. Functional and ultrastructural alterations affecting both cardiac cells and the coronary circulation have been documented in patients undergoing cardiac surgery, despite the protection provided by crystalloid262 and cold blood cardioplegia.263,264 Apoptotic cell death has also been implicated in the pathogenesis of the iatrogenic ischemia/reperfusion injury associated with on-pump cardiac surgery. DNA fragmentation has been detected by TUNEL staining in atrial biopsies from patients protected by three different cold crystalloid cardioplegic solutions (St. Thomas II, Bretschneider, or Hamburg) given by the antegrade route.265 Qualitative occurrence of apoptotic cell death was documented in subendocardial myocytes and endothelial cells from human hearts exposed to cardioplegic arrest followed by reperfusion, though not in bioptic specimens harvested before aortic cross-clamping.265 The occurrence of apoptosis and the relative contribution of its signaling pathways in human myocytes from patients exposed to cardiopulmonary bypass, warm blood cardioplegia, and subsequent reperfusion Curr Probl Cardiol, March 2006

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FIG 18. Endogenous urocortin is overexpressed only in human cardiac cells which do not show TUNEL positivity. (Color version of figure is available online).

have been recently evaluated and quantified. Warm blood cardioplegia, which nowadays many consider to be the most effective way to protect the heart from the iatrogenic ischemia/reperfusion insult concurrent with on-pump cardiac surgery,261 was indeed associated with myocyte apoptosis, identified as colocalization between TUNEL and caspase-3positive staining.89 In the human heart exposed to 40 to 55 minutes of cardioplegic arrest followed by 10 minutes of reperfusion, over 3% of cardiac cells showed colocalization of TUNEL and cleaved caspase-3. The proportion of apoptotic myocytes was almost doubled in patients exposed to roughly twice the duration of cardioplegic arrest followed by the same reperfusion time, suggesting that the overall degree of cardiac cell loss correlates with the extent of the ischemic insult. With respect to the relative contribution of the two major apoptotic signaling pathways, mitochondrial damage, leading to caspase-9 activation, was shown to be the main initiator of apoptosis affecting cardiac myocytes.89 In contrast, death receptor ligation, which results in proteolytic activation of caspase-8, appeared to be a relatively minor contributor to myocyte apoptosis, although the magnitude of myocyte apoptosis mediated by 228

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caspase-8 activation might increase if its assessment was performed after a longer reperfusion phase, after the release of the aortic cross-clamp. In the same study, cardioplegic arrest was also associated with increased expression of urocortin at a protein level, and myocytes overexpressing urocortin never displayed TUNEL-positive staining (Fig 18), providing evidence that endogenous urocortin effectively protects those myocytes in which it is produced.89 In addition, urocortin-positive, TUNEL-negative myocytes were surrounded by TUNEL- and urocortin-negative myocytes, showing enhanced expression of the Kir 6.1 cardiac potassium channel subunit. Since it was previously showed that exogenous urocortin enhanced the expression of Kir 6.1 and potassium channel blockers abolished urocortininduced cardioprotection both in cultures of myocytes and in the intact heart,266 the overexpression of Kir 6.1 in myocytes unlabeled by TUNEL and urocortin antibody suggests that endogenous urocortin can protect not only the myocytes from which it is produced in an autocrine fashion but also, upon release in the extracellular matrix, those in the surroundings, by means of a paracrine pathway. As previously described, the apoptotic process is mediated by specialized proteases, called caspases, whose sequential activation is accountable for the cleavage of major cytosolic and nuclear cell components.267 Activation of caspase-3 and -7, the two principal effector caspases, was detected by immunohistochemistry and Western blot analysis in left ventricular cardiac myocytes from coronary artery bypass graft patients.268 In the same study, preoperative administration of N-acetylcysteine, a reactive oxygen-derived species scavenger, significantly reduced the degree of caspase-7 and -3 activation, although no improvement in clinical outcome was observed. Caspase activation independent from DNA fragmentation has also been associated with early myofibrillar protein cleavage, resulting in decreased ATPase activity and contractile dysfunction.269,270 Consistent with these experimental findings, myofibrillary loss, associated with massive myocyte activation of caspase-9 and caspase-3, largely independent of DNA fragmentation, was also documented in the human heart, in a case of sudden death temporally related to ephedra intake.271 These experimental and human data seem to suggest that activated caspases, inducing breakdown of myofilaments with subsequent contractile impairment, may be per se a sufficient and autonomous cause of postoperative cardiac dysfunction, acting before the completion of the apoptotic process, and independently from necrotic cell death. The above postulation seems to find confirmation in a recent experimental study, showing that prevention Curr Probl Cardiol, March 2006

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of caspase activation with z-VAD, a broad caspase inhibitor, attenuated contractile dysfunction, independently from myocyte cell loss, in primary cultures of isolated porcine left ventricular myocytes exposed to simulated hyperkalemic cardioplegic arrest.272 Although captivating, this hypothesis, suggesting a causative role of caspase activation in the induction of cardiac dysfunction, nonetheless still needs to be validated in the human heart exposed to cardioplegic arrest. In conclusion, the aforementioned studies consensually confirm that, in patients undergoing on-pump cardiac surgery, cardioplegia may ameliorate, though not totally prevent, myocyte apoptosis, or caspase activation, whose rates significantly correlate with the length of time on bypass. Since the loss and functional impairment of myocytes following cardioplegic arrest are known to directly reduce cardiac contractility, resulting in greater mortality and morbidity associated with on-pump cardiac surgery,3,4 interventions aimed at reducing the extent of apoptotic cell death postoperatively (such as supplementation of cardioplegic solutions with exogenous urocortin) as well the magnitude of caspase activation (such as clinical use of caspase inhibitors with proven safety) may possibly reduce the risk of postsurgical cardiac dysfunction in patients exposed to the inescapable ischemia/reperfusion injury associated with cardiopulmonary bypass surgery.

Francis Blankenberg: Recently, it has been found that zinc chloride (Zn) blocks caspase-3-dependent apoptosis occurring in a rat heterotopic heart transplant model of acute rejection.xxxi Caspase-3 is the terminal point in the cascade of proteolytic reactions that commit a cell towards apoptotic cell death. The dose of Zn used in this study would have translated to 70 mg for the average 70-kg adult. This is just over four times the 15 mg/day recommended daily allowance. While Zn toxicities such as gastrointestinal disorders and certain hematologic derangements do exist, their occurrence is exceedingly rare. The dose used in this study could be provided to patients in tablet form and would in all likelihood be well tolerated given Zn’s wide therapeutic range. Zn added to cardioplegia solutions would also be expected to benefit patients undergoing cardiopulmonary bypass surgery.

Novel Cardioprotective Agents In the battle for cardiomyocyte cell survival, various agents exerting cardioprotective effects have been identified. Two such agents are minocycline, a tetracycline antibiotic, and urocortin, an endogenous protein. A description of each agent is provided, along with its potential utilization in the arsenal of weapons against cell death. 230

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Minocycline: An Antibiotic with Pleiotropic Effects Minocycline, a second-generation tetracycline, is a broad spectrum antibiotic, which has been commonly used to treat pneumonia and acne, as well as infections of the skin, genital, and urinary systems. The antimicrobial mechanism of action is bacteriostatic and achieved through inhibition of bacterial protein synthesis.273 This antibiotic, which has an excellent bioavailability (90 to 100% absorption of an oral dose from the gastrointestinal tract in fasting adults),274 is primarily excreted in the urine by glomerular filtration, and partially, in bile.273 In addition to its established antimicrobial action, minocycline exerts various effects, which resulted in renewed interest by physicians and scientists. It has been shown, for instance, that minocycline protects the brain in rodent models of global and focal cerebral ischemia.275,276 Minocycline exhibited a wide therapeutic window, since the beneficial effect was obtained not only when the drug was administered before the onset of ischemia, but also a few hours after the delivery of the ischemic insult. The significant neuroprotection was attributed to decreased expression of caspase-1 and cyclooxygenase 2 (COX-2),275,276 as well as inhibition of the inducible form of nitric oxide synthase (iNOS).275 These effects would account for a reduction of the secondary inflammation that occurs during an ischemic stroke and consistently contributes to the extent of neuronal cell death.277 Remarkable neuroprotection was also observed in other experimental models of neurodegeneration. In a transgenic mouse model of Huntington’s disease, for instance, minocycline delayed disease progression and prolonged survival both inhibiting caspase-1 and caspase-3 mRNA upregulation and decreasing the activity of iNOS.278 In a following study, carried out in a similar model of Huntington’s disease, minocycline was also reported to inhibit the recruitment of both mitochondrial caspaseindependent (apoptosis-inducing factor) and caspase-dependent (Smac/ Diablo and cytochrome-c) apoptotic signaling pathways, with subsequent reduction of cell death/disease progression.279 In a mouse model of Parkinson’s disease, minocycline-induced prevention of neurodegeneration was associated not only with decreased iNOS and caspase-1 expression but also with inhibited phosphorylation of p38 MAPK.280 Minocycline was also shown to inhibit mitochondrial leakage of cytochrome-c and delay progression of amyotrophic lateral sclerosis in a transgenic mouse model of the disease.281 In a similar model of ALS, minocycline was reported to delay disease onset and extend dosedependent survival, with protection from loss of motor neurons and from vacuolization at 120 days.282 Curr Probl Cardiol, March 2006

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FIG 19. Minocycline induces downregulation of upstream and downstream caspases both at the mRNA (A) and protein levels (B).

Through modulation of cytokine expression, and attenuation of cell death and lesion size, minocycline also improved functional recovery in a rat model of spinal cord injury (SCI).283 In addition to the extensively reported neuroprotection, minocyclinemediated protection was also documented in other organs, including kidneys and testes. Minocycline reduced apoptotic cell death in hypoxic kidney epithelial cells, with a protection mechanism centered on mito232

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FIG 19. Continued

chondria and involving suppression of Bax accumulation, prevention of outer membrane damage, and reduction in cytochrome-c release.284 Pretreatment with minocycline also suppressed both in vitro and in vivo the mitochondrial release of cytochrome-c, and consensually, the magnitude of TUNEL-positive cells, in spermatogenic cells exposed to heat stress.285 Curr Probl Cardiol, March 2006

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More recently, minocycline was shown to effectively protect cardiac myocytes against I/R injury, inducing a marked reduction of both necrotic and apoptotic cell death. The cardioprotective effect has been validated at three levels: in vitro, using primary cultures of neonatal and adult cardiomyocytes; ex vivo, infusing minocycline to the isolated rat heart; and in vivo, injecting the animals with minocycline over a period of 3 days. Importantly, the reduction of infarct size and apoptotic cell death observed following in vivo treatment with minocycline was associated with a remarkable postischemic recovery of cardiac function.106 With respect to its antiapoptotic mechanism of action, minocycline was shown to induce profound inhibition of the activity level of several initiator and effector caspases, through the synergetic action of multiple mechanisms. Besides the well-documented downregulation of caspase-1 and -3, minocycline reduced the cardiac expression of caspase-7, -8, -9, and -12 in basal condition and prevented the postischemic upregulation of all the above caspases. In addition, minocycline effectively interfered with upstream and downstream mechanisms leading to secondary caspase activation and reactivation, inducing reduced decompartmentalization of cytochrome-c and Smac/DIABLO, together with increased ratio of XIAP to Smac/DIABLO (Fig 19).106 These combined actions concur to modulate the functional activity of caspases at three different levels: reducing the mitochondria-mediated activation of caspase-9; promoting the inhibition of activated caspases; and preventing the reactivation of dormant caspases. Therefore, the effects achieved with in vivo administration of minocycline effectively cooperate to keep in check the level of caspase activity in the heart, raising the point of commitment in ischemic/reperfused cardiac myocytes. Since this comprehensive action of caspase modulation is not dependent on a direct inhibition of caspase activity,278 clinical use of minocycline is not limited by the potential toxic effects of other conventional caspase inhibitors due to abrogation of normal homeostatic apoptosis in the human adult. Owing to this, minocycline could be valuable in acute but also in chronic clinical settings, where it may provide important synergism with conventional cardioprotective agents in counteracting the occurrence and the progression of myocyte cell loss.

Francis Blankenberg and H. William Strauss: In a recent article we also considered whether 99mTc-annexin V imaging using SPECT (single photon emission computed tomography) could be used to monitor a potential treatment response to minocycline in experimental stroke. Forty CB6F1 adult male mice underwent unilateral distal middle cerebral artery occlusion 234

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(dMCAO) and were imaged and sacrificed on 1, 3, 7, or 30 days after injury (personal communication). Animals were given 22.5 mg/kg minocycline (or vehicle) i.p. 30 minutes and 12 hours after dMCAO and then 22.5 mg/kg twice daily for up to 7 days. In each group, mice were injected with 5 to 10 mCi of 99mTc annexin V 2 hours before undergoing SPECT on days 1, 3 and 7, and 30. After imaging, brains were collected for histology and assessed for apoptosis using TUNEL stain and activated microglia using isolectin B4 (IB4). There was marked focal uptake of 99mTc-labeled annexin V in the left (ischemic) cerebral hemisphere as confirmed by SPECT that was significantly decreased 4- to 5-fold by minocycline. This was correlated to reduced infarct size (P ⬍ 0.01), numbers of TUNEL (P ⬍ 0.05), and IB4 (P ⬍ 0.01) positive cells among treated mice. In another experimental model we chose a highly specific form of antiinflammatory treatment based on the selective inhibition of Fas Ligand with anti-FasL monoclonal antibody (personal communication). Following brain ischemia there is a rapid onset of increased expression of FasL and Fas receptor within the neurons of the ischemic penumbra. The mechanisms for reduced injury in these models appear to be twofold: the first a direct blockade (or absence) of FasL on ischemically injured neurons and the second the inhibition of mediated Fas cell death of neutrophils and later on the macrophages/microglial cell recruited to regions of ischemia as part of the brain’s and the body’s postischemic inflammatory response. Adult Sprague-Dawley male rats (280 to 300 g) underwent 2 hours of unilateral occlusion with an intraluminal beaded thread followed by reperfusion. Immediately after recovery, rats received 400 ␮g of MFL4 anti-FasL antibody (BD Biosciences; Pharmingen) i.p. and again on day 3. On day 6 rats underwent microSPECT imaging 1 hour after tail vein injection of 3 to 5 mCi of 99mTc-HYNIC-annexin V (Theseus Imaging Corp.). On day 7 rats were sacrificed for histology. Results of ROI analyses on reconstructed masked axial images were expressed as the ratio of brain uptake to contralateral background uptake. Treated rats demonstrated significantly (0.05) less uptake of tracer in the ischemic hemisphere (n ⫽ 5, 52.4 ⫾ 18.9 cts/voxel) as compared with control (n ⫽ 6, 243.3 ⫾ 169.2 cts/voxel; P ⫽ 0.03). H&E stained sections also revealed a significantly smaller stroke volume in antibody treated rats (7.3 ⫾ 5.8%, cross sectional area) as compared with control (26.5 ⫾11.9%), as well as fewer TUNEL-positive cells (6.4 ⫾ 8.8 versus 83.5 ⫾ 62.6, P ⫽ 0.024). Interestingly, caspase-8 activity within neurons also was significantly decreased 24 hours after injury (6.0 ⫾ 1.0 versus 14.2 ⫾ 5.2, P ⫽ 0.022). Radiolabeled annexin V SPECT imaging may therefore also be a novel way to monitor the progression of myocardial infarction and its response to novel anti-inflammatory therapies.

The Cardioprotective Mechanism of Action of Urocortin and Its Homologues Introduction Coping with stressful stimuli is key to survival of the species. In mammals, this is achieved by a group of molecules related to corticotroCurr Probl Cardiol, March 2006

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phin-releasing hormone (CRH), which is the central mediator of the hypothalamic-pituitary axis and the stress response.286,287 First isolated from bovine hypothalamus almost 25 years ago288 and originally cloned from brain tissue, CRH is present in the heart and widespread throughout the periphery.289 Although the stress response is finely tuned and exquisitely complex, molecules involved in its pathway can be traced back through evolutionary time. There are fish and amphibian homologues of CRH, urotensin 1, and sauvagine, respectively.290,291 Urocortin is a novel member of the CRH family and was cloned from the rat midbrain.6 It was so called because of its high sequence homology to both urotensin 1 and CRH, where it shares 65 and 45% at the amino acid level, respectively. This family is presently expanding further, with two new urocortins having been identified: urocortin II and urocortin III have been isolated from mouse genomic libraries.292-294 Human versions of these novel rodent sequences have also been isolated: stresscopinrelated peptide (SRP), which is equivalent to urocortin II, and stresscopin (SCP), which is equivalent to mouse urocortin III.292-294 Therefore, there are currently four related molecules in mammals. All of these small, active peptide members of the CRH family are processed into active forms by cleavage from a much larger inactive propeptide.291,295

Receptors for the Urocortins At present, two classes of mammalian receptor for CRH family members are known. Termed CRHR1 and CRHR2, they represent two different gene products.296 Further variety in receptor structure is achieved by extensive alternative splicing of these basic structures.297 The CRHR1 gene is expressed as subtypes 1a-h,298 whereas the CRHR2 gene has only three isoforms, ␣, ␤, and ␥.299 All CRH receptor isoforms, to date, conform to the classic G-protein-coupled, seven hydrophobic transmembrane spanning domain structure.300 This suggests that once the ligand has bound to its receptor, downstream signaling should follow a common pathway. However, some interesting studies have found that different G-protein species are coupled to different receptor subtype, presumably eliciting different downstream responses and subsequently increasing the variety and complexity of the responses and signaling produced by the different CRH family members.301,302 The N-terminal region of both the CRHR1 and the CRHR2 receptor has been demonstrated to be responsible for ligand binding.303 CRH and the urocortins bind to both CRHR1 and CRHR2; however, the CRHR2 receptor has between 10- and 50-fold higher affinity for the urocortins.291 236

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Urocortins and Cardioprotection Originally CRH, urocortins were isolated from mammalian brain samples. However, more recently, they have been found in several other peripheral tissue types including heart.304 Early pieces of circumstantial evidence implicating urocortin as being involved in cardiac physiology and pathology was the discovery that, during simulated I/R in primary cardiomyocytes, the levels of their mRNA increased dramatically, based on a sensitive 5=RACE assay.305 Furthermore, this increase was also seen at the protein level as demonstrated by Western blot studies. Indeed, conditioned media derived from cardiomyocytes exposed to I/R were able to protect naïve cells from the damaging effects of I/R.305 This led to the suggestion that endogenous urocortin could be upregulated during I/R and released into the local environment where it can bind back onto the cardiac sarcolemmal CRH R2 receptor in an autocrine/paracrine manner. Studies expanded from these initial observations with exogenously applied urocortin demonstrated unequivocally that urocortin protects primary cardiomyocytes from apoptotic cell death, measured using both Annexin V surface staining and TUNEL positivity.306,307 Furthermore, these cardioprotective peptides were also able to protect the whole heart ex vivo by reducing infarct size in the Langendorff perfusion model and in vivo.308-310These findings have recently been extended to demonstrate that Ucn II and Ucn III were also potent cardioprotective agents, in vitro and ex vivo.311,312 The ability of urocortin and its homologues to protect the heart from I/R injury is now overwhelmingly recognized. However, the precise mechanism of action of these cardioprotective agents is less well understood. The vast majority of mechanistic studies of cardioprotection has been performed on urocortin. In these studies, it became apparent early on that urocortin’s cardioprotective mechanism of action was complex, requiring activation of several diverse kinases for the acute effects of urocortin, and necessitating altered gene expression for the later effects of urocortin, since some of the cardioprotection induced by urocortin was lost in the presence of cyclohexamide.305

Early Cardioprotective Effects of Urocortin Several major kinase pathways are affected by urocortin treatment. A number of early studies using primary cardiomyocyte preparations implicated MAPK as being involved in one cardioprotective pathway employed by urocortin.307 A subfamily of MAPK, the p42/p44 MAPK, is phosphorylated and activated by the MAPK kinase (MEK1/2). Interestingly, specific pharmacological inhibition of MEK1/2 by PD 98059 Curr Probl Cardiol, March 2006

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abolished cardioprotection produced by urocortin when assayed by trypan blue exclusion, Annexin V, and TUNEL positivity.308 This abolition of urocortin’s cardioprotective effect was seen when PD 98059 was given during ischemia, but also when given during reperfusion. Although studies using primary cardiomyocyte preparations are important, it is crucial to extend studies to the whole heart. Again, we see that the inhibitor of the MEK1/2 pathway PD 98059 removes the ability of urocortin to reduce infarct size during I/R in an ex vivo heart model employing the Langendorff perfusion apparatus.312 These findings were also seen for the two urocortin homologues, SRP and SCP, in both in vitro studies and studies using the Langendorff perfused ex vivo heart model,311,312 suggesting that all three of the urocortins, at least in part, have a similar mechanism of action, via the activation of the MEK1/2 pathway. Additional to the MEK1/2 and p42/44MAPK pathway, activation of the phosphatidyl inositol 3-OH kinase (PI3K) and the serine threonine Akt, its downstream effector, has also been demonstrated to preserve cardiac function and to be involved in cardioprotection produced by urocortin during I/R.309 The use of chemical inhibitors of the PI3K pathway, such as Wortmannin and LY 294002, has been shown to remove urocortin’s cardioprotection in both neonatal and adult cardiomyocytes. Thus, both urocortin homologues seem to work also through the PI3K pathway.312 A third kinase, PKC, has for some time been implicated in cardioprotection during I/R injury. However, its involvement is complicated by the revelation that, to date, there are 12 different isoforms of PKC, contained within three different families: classical, atypical, and novel PKCs, with each phosphorylating diverse effectors and having a wide range of tissue and subcellular distribution.313,314 Until recently, it has been impossible to dissect the importance of individual isoforms in terms of a physiological function. Recently, however, small peptides of 6 to 8 amino acids have been used to inhibit specific isozymes of PKC from binding to their specific receptor for activated C kinase (RACK).315,316 These assays take the form of inhibition of a specific isozyme of PCK translocating from a cytosolic to a membrane fraction. Pseudo-RACK peptides have also been used to enhance the function of specific PKCs.317 These data, along with studies using knock-out mice and mice overexpressing PKC isozymes in cardiac cells and the whole heart, have strongly implicated the PKC⑀ isozyme as the major PKC involved in cardio238

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protection during ischemia and reperfusion injury, and in producing the phenomenon of ischemic preconditioning.318-324 Very recently,325 it has been demonstrated that a short 10-minute exposure of primary cardiomyocytes to urocortin caused a specific translocation/activation of PKC⑀ in vitro and in the Langendorff perfused ex vivo heart. Furthermore, a PKC⑀-specific inhibitor peptide, when introduced into cardiomyocytes, prior to simulated ischemia, resulted in the loss of urocortin’s cardioprotective effects.325 This loss of cardioprotection by Ucn was also seen in whole heart ex vivo from PKC⑀ knockout mice. These findings indicate that the cardioprotective effect of urocortin is also dependent upon PKC⑀ activation. In addition to its effects on diverse kinase pathways, urocortin has recently been shown to modulate L-type calcium channels.326 Using whole-cell patch-clamp recording on isolated adult rat cardiomyocytes, urocortin produced a concentration-dependent decrease in the inward calcium current after 10 minutes, which correlated with increased cell survival.326 Unfortunately, it is unclear whether urocortin had a direct effect on the channel moiety or whether its modulation involved activation of the cardiac CRHR2 receptor, as the effects of urocortin receptor antagonists were not investigated in this study.

A Diversity of Genes Whose Expression Is Modulated by Urocortin Based on candidate gene studies, there is some evidence for the induced expression of some heat shock protein species (hsp) by urocortin.327 Expression of the cardioprotective hsp 90 has been shown to be induced by urocortin, with this effect blocked by PD 98059.327 Thus, the induction of cardioprotective hsps may play a role in the cardioprotective effects of urocortin. Another cardioprotective agent, cardiotrophin-1 (CT-1), is also under intense investigation. Unlike urocortin, CT-1 is a member of the interleukin-6 family of cytokines328 and has a totally different cardioprotective pathway to urocortin. However, recently it was discovered that CT-1 message and protein levels were induced by urocortin and acted via the p42/p44 MAPK pathway.329 The obvious limitation to a candidate gene approach to unraveling genes affected by urocortin is the number that can be studied at a given time. Nevertheless, the use of Affymetrix gene chip technology has been used to great effect in unraveling the gene expression profile component of urocortin’s cardioprotective effect.330 In the only study of its kind, Curr Probl Cardiol, March 2006

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several genes of interest were altered by urocortin. They included genes that were found to be both upregulated and attenuated by the peptide.330 Three gene products, very diverse and seemingly unrelated in terms of functional protein product, were altered by urocortin and, upon further investigation, were found to be intimately involved in cardioprotection produced by urocortin. The first protein studied was an ATP-sensitive potassium channel (Katp channel) that is dependent upon the cellular concentration of ATP for activation. When the concentration of ATP falls, the KATP channels open, but remain closed under normal physiological concentrations of ATP. Thus, they are sensors of the metabolic state of a cell. These channels, when open, during stressful stimuli including I/R, are thought to be cardioprotective.331-335 There are two known subtypes of this channel, each a product of alternative RNA splicing: kir 6.1 and kir 6.2. These are two small transmembrane-spanning domain proteins that represent the pore of the KATP channel. However, to make this type of channel functional, they need to combine with another subunit derived from a totally different set of genes, the sulphonylurea receptors (SUR), so called because of their binding site for the sulphonylurea class of drugs used in the treatment of diabetes. These receptors are 12 large transmembrane-spanning domain proteins and are members of the ABC binding cassette superfamily. These subunits are responsible for sensing and binding ATP/ADP and ultimately gating the channel pore.336-339 Urocortin specifically enhanced expression of the Kir 6.1 potassium channel subunit only. No differences were seen in the expression of Kir 6.2 or the three isoforms of SUR (m). Through utilization of antagonists to the mitochondrial KATP channel, 5-hydroxydecanoate (5-HD), and dominant-negative constructs to Kir 6.1, it was possible to demonstrate the loss of the cardioprotective effect of urocortin, as assessed by TUNEL positivity assays,330 whereas openers of KATP channels, such as cromakalim, were cardioprotective during simulated I/R in vitro. Urocortin was also seen to induce kir 6.1 after a 1-hour exposure in the whole heart.89 A second gene modulated by urocortin is calcium-independent phospholipase A2 (iPLA2). This gene product belongs to a superfamily of phospholipases represented by three classes: cytosolic PLA2 (cPLA2); secretory PLA2 (sPLA2); and calcium-independent PLA2 (iPLA2). They are characterized on the basis of cellular localization, substrate specificity, Ca2⫹ dependency, and type of lipid modulator. PLA2 catalyses the breakdown of membrane phospholipids into arachidonic acid, which is a 240

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precursor of prostaglandins and leukotrienes and a minor metabolite lysophosphatidylcholine (LPC).340-342 It has been documented that the activity of the cardiac iPLA2 class of PLA2 only is increased during I/R, with a concomitant increase in the generation of LPC, which has been shown to be highly cardiotoxic.341 Very interestingly, urocortin was found to lower the expression levels of this isozyme only, by over twofold, and also lowered the generation of LPC in normoxic cardiomyocytes and in those exposed to I/R, as did a specific pharmacological inhibitor of iPLA2, bromoenol lactone (BEL), resulting in cardioprotection.343 The third gene product found to be altered by urocortin from the gene chip study was PKC⑀. Urocortin, as well as activating this kinase, also caused an increase in the mRNA and protein levels by over threefold.325 Hence, to date, three seemingly unrelated gene products have been demonstrated to be altered by urocortin. Is there any way these diverse genes can interact to produce a cardioprotective pathway activated by urocortin? For this answer, we need to study the cardiomyocyte mitochondrion.

A Unifying Theory of the Mechanism of Action of Urocortin Involves Cardiomyocyte Mitochondria It has been demonstrated that early during I/R cardiomyocyte mitochondrial function is compromised. There is a loss of membrane potential resulting in a decrease in oxidative phosphorylation, as well as increases in reactive oxygen species and the release of pro-apoptotic molecules including cytochrome-c.344-346 Nonetheless, why study cardiomyocyte mitochondria in relation to the cardioprotective effect of urocortin? Very recently, a link has been found between the genes regulated by urocortin and cardiomyocyte mitochondria. The three recently discovered proteins found to be involved in urocortin’s cardioprotective mechanism of action appear to be localized to the cardiomyocyte mitochondria, based on a combination of pharmacology, Western blotting, and immunocytochemistry.325,330,343,347 Thus, a clue as to how urocortin protects cardiomyocytes from I/R damage may lie at the level of the cardiomyocyte mitochondria. By using the mitochondrial selective dyes [mitotracker green and tetramethyl rhodamine methyl ester (TMRM)] to measure damage to the mitochondrial transmembrane potential, it was found that urocortin indeed protects cardiomyocyte mitochondria from damage produced by I/R.348 This protective effect from I/R injury was also observed in the Curr Probl Cardiol, March 2006

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presence of the KATP channel opener cromakalim and the iPLA2 inhibitor BEL, suggesting that both KATP channel opening and inhibition of LPC formation are crucial for the protection of cardiac myocyte mitochondria during I/R injury.348 When the mitochondrial KATP channel is blocked using 5-HD, exogenous LPC applied to primary cardiomyocytes, or PKC⑀ activation blocked by selective inhibitor peptides, mitochondrial damage is enhanced, compared with I/R alone, and crucially, the protective effect of Ucn under these conditions is lost.348 Interestingly, the KATP channel opener cromakalim also protects cardiomyocyte mitochondria from LPC-induced damage, suggesting a possible interaction between mitochondrial KATP channels and the iPLA2 metabolite LPC. As some studies suggest, this metabolite interacts with ion channels and may even be an antagonist of potassium channels.349-351 Therefore, some protection afforded by cromakalim may be due to pharmacological competition for the same binding site as LPC. However, when 5-HD is present with LPC, mitochondrial damage is enhanced, compared to cardiomyocytes treated with either agent alone. Therefore, damage to mitochondria by LPC may also be via mechanisms other than KATP channels. Thus, three end effector molecules modulated by Ucn are localized to cardiomyocyte mitochondria and are involved in I/R injury and cardioprotection. Furthermore, there is accumulating evidence that these three molecules can interact. For example, there is now evidence that LPC can modulate both KATP channels and PKC⑀ and that PKC⑀ can interact with KATP channels and iPLA2.351-354 Significantly, PKC⑀ has been shown to translocate to mitochondrial membranes and interact with mitochondrial proteins, including the mitochondrial permeability transition pore.355,356 Although further studies are necessary to define fully the mechanism of cardioprotection produced by urocortin, especially in relation to the other kinases which are important for its effect, namely P42/p44 MAP kinases and PI3 kinase also, it is clear that protection against I/R injury involves both early effects on specific kinases and more long-term gene changes and that protection at the subcellular level may occur at the level of the cardiomyocyte mitochondria. Much less work has been performed on the newer homologues of urocortin, SRP, and SCP, in relation to their cardioprotective mechanism of action. It will be interesting in the future to determine whether their cardioprotective pathways resemble that of urocortin, or whether they diverge, giving some novel twist to the cardioprotective story of the urocortins. 242

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H. William Strauss: This comprehensive review highlights the complexity of the cardiac myocyte response to ischemic injury. The Yin and Yang of protein interactions drives the cell to either complete its death cycle or recover. A major question to raise, as we consider the use of anti-apoptotic agents in patients with ischemia and reperfusion, is whether the drugs will postpone or halt the apoptotic process. The next question is how will the apoptosis delayed or arrested cells behave? Experiments will have to be done to determine if these therapies allow substantially damaged cells to survive without completing the repair process. If this is the case, there may be an enhanced potential for arrhythmogenesis or incomplete scar formation— possibly leading to cardiac rupture.

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