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1
* Cell Stress and Apoptosis Research Group, Department of Biochemistry and
National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland; and
The Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois 60153, USA
1Correspondence: Department of Biochemistry, National University of Ireland, Galway, University Road, Galway, Ireland. E mail: afshin.samali@nuigalway.ie
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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Key Words: cell death • cardiomyopathy • heat shock proteins • ischemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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12 million deaths annually. To enable the development of viable treatment strategies, the complex pathogenesis of this disease must first be understood. Cardiovascular disease can be initiated by multiple factors; in recent years it has emerged that a major contributory factor to its initiation and progression is the loss of cardiomyocytes. Adult cardiomyocytes are terminally differentiated cells and, once destroyed, are rarely replaced. Thus, their loss can contribute to the functional decline of the myocardium leading to heart disease as we know it. Until recently, the mode of cell death involved was not clear and was attributed to necrosis. It is now believed, however, that necrosis is not the only form of cell death in cardiomyocytes and that apoptosis may play an important role.
Necrosis is a rapid and irreversible process that occurs when cells are severely damaged. Necrosis involves swelling of the cell and its organelles, disruption of mitochondria, membrane rupture, and cell lysis (1)
. It is a destructive process, as release of cellular content into the surrounding environment can cause further damage or death to neighboring cells. This contrasts with apoptosis, which plays a more ‘constructive’ role in organisms (2)
. Apoptosis is a highly organized, energy-dependent mechanism whereby a cell neatly commits suicide without causing damage to surrounding tissue and occurs normally during development, tissue turnover, and in the immune system. In the heart, for instance, apoptosis is involved in postnatal shaping of the right ventricle by eliminating unnecessary cells (3)
. Apoptosis was originally defined by its morphological characteristics, which include cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing, and formation of apoptotic bodies. More recent research on the underlying biochemical events leading to these distinct features of apoptosis has revealed the pivotal role of a family of proteases termed caspases (4)
. Caspases are present in the cell as inactive procaspases that are cleaved and activated in response to apoptotic stimuli. Initial activation of caspases may involve transduction of a signal from membrane receptors belonging to the tumor necrosis factor receptor (TNF) family, such as Fas or TNF receptor 1 (TNFR1) (5)
, or may be mediated by mitochondrial cytochrome c release (6)
. Stimulation of death receptors results in activation of caspase-8, which goes on to activate caspase-3, a key effector protein of the apoptotic machinery. Release of cytochrome c, on the other hand, leads to activation of caspase-9 through formation of a cytosolic complex known as the apoptosome and subsequent activation of caspase-3, involved in disassembly of the cell (Fig. 1
).
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Unlike necrosis, which is a passive, unregulated process, apoptosis is both energy dependent and highlyregulated. Apoptosis is controlled by the complex interaction of numerous prosurvival and prodeath signals. These include the Bcl-2 family of proteins, which may be antiapoptotic (Bcl-2, Bcl-xL) or proapoptotic (Bax, Bid), and exerts its effect primarily at the level of mitochondria (7)
. Other important regulators of apoptosis act at the level of caspases. Such proteins include cellular FADD-like inhibitory protein (cFLIP) and the inhibitor of apoptosis (IAP) family (8
, 9)
. There are proteins that counteract the effect of the caspase inhibitors themselves such as the recently characterized Smac/DIABLO, a mitochondrial protein that, when released, binds and neutralizes IAPs, promoting caspase activity (10)
. Several other proteins are also believed to be involved in the regulation of apoptosis including stress proteins, growth factors, calcium, and oxidants. The complex interaction of all of these molecules determines the ultimate fate of the cell: life or death.
Dysregulated apoptosis has been implicated in many diseases including cancer, autoimmune and degenerative disorders, and is now emerging as a likely suspect in cardiomyocyte death. Over the past few years, there have been several reports of the occurrence of cardiomyocyte apoptosis during such conditions as cardiomyopathy, myocardial infarction, and ischemia/reperfusion injury (11
12
13
14
15
16)
. Although the significance of cell death by apoptosis to cardiac pathology is yet to be established, there is now great interest in the field. This is because unlike necrosis, which is thought to be an essentially irreversible process, the step-by-step nature of apoptosis suggests it may be amenable to therapeutic intervention.
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APOPTOSIS IN THE HEART |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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, 18)
. Techniques such as TUNEL are prone to false positive results, as they may detect necrotic cells or living cells that are undergoing DNA repair (19)
. It has been shown that with careful standardization, however, the TUNEL assay provides reproducible results of the occurrence of apoptosis in samples (17)
. Detection of active components of apoptotic pathways such as caspases can help to confirm findings. It is now accepted that various techniques should be used in parallel to demonstrate the occurrence of apoptosis in the heart.
Another difficulty in studying apoptosis of heart cells lies in determining the ‘true’ rate of apoptosis in disease. Although in early studies the measured value varied widely and was in some cases very high (11
, 15)
, more recent reports have yielded values that appear at the outset to be very low, e.g., <1% (20)
. However, such measurements represent only the number of cells undergoing apoptosis at a single point in time. It is now widely postulated that given the relatively short time course of apoptosis, the gradual loss of such numbers of myocardial cells could have serious consequences over an extended period. Thus, when investigating the role of apoptosis in heart disease, it is essential to consider not only the techniques used to detect and quantify apoptosis, but also the potential implications of such cell death over time.
Cell death in ischemia/reperfusion injury
Myocardial ischemia is caused by an inadequate blood supply, and thereby an inadequate supply of oxygen and glucose to the heart. If blood flow is not rapidly restored, massive cardiomyocyte death may follow. Reperfusion of ischemic tissue is not without risk, however, as it is also associated with cardiomyocyte death. It remains controversial whether cell death during reperfusion is a completion of that which was initiated during ischemia or caused directly by reperfusion itself—for example, by oxidative stress.
Regardless of the cause of cardiomyocyte death—ischemia, reperfusion, or both—studies conducted with several animal models of ischemic-reperfused heart and on tissue from humans after myocardial infarction (a clinical counterpart of ischemia/reperfusion injury) suggest that both apoptosis and necrosis are responsible for cell death (16
, 21
22
23
24)
.
One of the first studies of apoptosis as a result of ischemia/reperfusion injury found that whereas ischemia alone resulted in ISEL-labeled DNA, reperfusion was required to induce internucleosomal DNA fragmentation (16)
. It was concluded that apoptosis initiated during ischemia could not proceed without reperfusion. Given that ATP is required for the completion of apoptosis, it follows that reoxygenation and restoration of glucose supply would be required for cell death to proceed. Since this study, there have been several investigations of the type of cell death in the heart, with most citing both apoptosis and necrosis as culprits. One such study found that after myocardial infarction, cell death was primarily due to apoptosis, but over time became entirely necrotic (21)
. In contrast, others have found that in hearts from cases of fatal myocardial infarction, apoptosis and necrosis occurred simultaneously in all instances (22)
.
One hypothesis that explains the coexistence of apoptosis and necrosis after ischemia/reperfusion is that damage produced by ischemia is capable of initiating apoptosis, but if ischemia is prolonged, necrosis ensues. If, on the other hand, critical metabolic processes such as energy production are restored, as happens during reperfusion, the apoptotic cascade as initiated by ischemia may proceed. This is supported by a recent study of a canine model of ischemia/reperfusion injury which found that prolonged ischemia without reperfusion induced necrosis but not apoptosis. In contrast, 1 h of ischemia followed by 6 h reperfusion induced apoptosis as defined by DNA fragmentation and TUNEL labeling, but induced significantly less necrosis than ischemia alone (23)
. Another report confirms that the apoptotic cascade is triggered by ischemia and that reperfusion is required for completion of apoptosis, but suggests that the low level of cell death occurring by apoptosis means that it is not a decisive factor in tissue damage (24)
. This may indeed be the case for an acute injury as incurred by the global ischemia used in this experiment (24)
. It has frequently been reported in cases of regional ischemia such as myocardial infarction that the central area of infarct, i.e., the area exposed to the most severe stress, is predominantly necrotic, whereas apoptosis is found in the adjacent border areas (15)
. In the global ischemia model, all layers of the heart are subjected to equally severe stress, which may explain why necrosis is predominant (24)
.
An alternative explanation for the presence of necrosis after ischemia/reperfusion is that the extent of apoptosis after such an injury overwhelms the system. The heart therefore is unable to efficiently remove the apoptotic cells and secondary necrosis ensues (3)
. Thus, whether apoptosis plays an important role in ischemia/reperfusion injury is not clear; even if it is not found to be a decisive factor in initial tissue damage, it is possible that apoptosis in the border zone may contribute to the ongoing deterioration that may follow myocardial infarction. This is supported by the fact that apoptosis is found in the heart days after the initial reperfusion therapy (15)
.
Evidence suggests that another mechanism of cell death is involved in the heart. For example, there is often a lack of morphological evidence of apoptosis such as formation of apoptotic bodies (15
, 16)
. Indeed, this phenomenon is not restricted to conditions such as ischemia/reperfusion and has been observed in association with heart failure (discussed later). Given the specialized function of cardiomyocytes, it is possible they have evolved a unique mechanism to eliminate cells. This novel category of cell death in myocardial cells may share common mechanisms with apoptosis in the early phase of death such as DNA fragmentation, but later manifest signs of necrosis such as disintegration of membranes and inflammation.
Cell death in heart failure
Several cardiac disorders culminate in heart failure. Examples include chronic overload such as that caused by hypertension or a defective heart valve, viral myocarditis, alcoholism, and ischemic heart disease. In response to these kinds of stress, the myocardium adapts itself in a process known as remodeling. This generally involves changes in gene expression and hypertrophy of cardiomyocytes whereby the cells increase in size. Although this structural change is an adaptive response to stress, it can eventually lead to heart failure. The mechanism behind the transition from hypertrophy to failure is poorly understood and, until recently, cell death was largely ignored as a potential pathogenic factor. This was due to lack of evidence of necrosis in the failing myocardium. With the discovery that apoptosis plays a pathogenic role in disease, however, it has been proposed that this mode of cell death may be partly responsible for the progression to heart failure.
Data obtained from numerous studies reinforce the possible role for apoptosis in heart failure. Examination of human heart tissue (usually obtained at transplantation) has shown apoptosis in association with conditions such as idiopathic dilated cardiomyopathy, ischemic cardiomyopathy, arrhythmogenic right ventricular dysplasia, and hypertrophic cardiomyopathy (11
12
13
14
, 20
, 25)
. The degree of apoptosis found in association with such conditions is often quite low, but it is thought that the gradual loss of cardiomyocytes over time may contribute to the eventual failure of the heart. Most of the tissue examined is obtained at the end stage of the disease, and it is proposed that apoptosis may not play a major role at this late stage. A recent study that measured the levels of proapoptotic Bax in relation to the severity of heart failure found levels of Bax to be significantly higher in association with mild heart failure compared with moderate or severe heart failure (26)
. Thus, cardiomyocytes are more prone to apoptosis in the earlier stages of the disease. This susceptibility to apoptosis showed close correlations with echocardiographic and hemodynamic parameters that indicate the severity of heart failure, and it was concluded that apoptosis may have a role in the transition from mild to end-stage heart failure.
Apoptosis is not the only form of cell death involved in heart failure, as necrosis has also been found in such conditions (13
, 20)
. It is also possible that (as has been proposed for ischemia/reperfusion injury) another mode of cell death is involved. This was suggested after an investigation of the role of apoptosis in end-stage heart failure. Explanted hearts from patients undergoing transplantation demonstrated significant cytosolic accumulation of cytochrome c (27)
. In several other cell types, mitochondrial release of cytochrome c has been shown to initiate apoptosis involving activation of caspase-3 and cleavage of its substrates protein kinase C
(PKC) and poly ADP-ribose polymerase (PARP). In this case, release of cytochrome c was associated with activation of caspase-3 and PKC. Cleavage of PARP did not occur, nor were the typical ultrastructural alterations of apoptosis observed. One potential explanation is that apoptosis or programmed cell death in cardiomyocytes follows a different course from that typically seen in undifferentiated cells. Thus, although apoptosis appears to have been initiated, nuclear alterations are not seen. The existence of an alternative mode of programmed cell death in association with heart failure is further supported by more recent findings. In cardiac tissue from patients with end-stage heart failure, TUNEL-positive cardiomyocytes failed to show caspase-3 activation. In addition, cardiomyocytes appeared to undergo caspase-independent ‘autophagic’ death rather than apoptosis (28)
. Autophagy is a mechanism that shares aspects of apoptosis and necrosis in that it is a programmed or regulated process but displays necrotic morphology (29)
. Thus, as for ischemia/reperfusion, there is likely to be more than one form of cell death involved.
There is little doubt that apoptosis is found in association with myocardial disease. Whether it is involved in the pathogenesis of such disease remains unclear, although the fact that other mechanisms of death may also be found alongside apoptosis does not infer that modulation of apoptosis would not be of value. In the case of myocardial infarction, for example, although necrosis appears to be responsible for much of the damage in the central area of infarct, it would be of interest to determine whether attenuation of apoptosis in the border zone would minimize overall damage. Indeed, the ‘necrosis’ typically observed under these circumstances may represent a novel form of cell death that, although having necrotic morphology, may also be amenable to therapeutic intervention. There is increasing evidence that apoptosis or at least a closely related mechanism is involved in heart failure. Further studies using endomyocardial biopsies rather than tissue obtained at transplantation or postmortem may provide better insight into its role in disease progression.
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PATHWAYS OF APOPTOSIS IN CARDIOMYOCYTES |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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Death receptor pathway of apoptosis
There have been reports of the expression of Fas and Fas ligand in the heart (21
, 30
, 31)
. Enhanced expression of Fas is found in association with increased apoptosis in experimental models of myocardial infarction (21)
, hypoxia (31)
, and overstretched myocardium (32)
. The cardiomyopathy associated with the anticancer drug Adriamycin involves apoptotic death executed through a Fas-mediated pathway (33)
and Fas is directly involved in cell death after myocardial ischemia (34)
. Simulated ischemia/reperfusion in a cell culture model increased the sensitivity of myocytes to Fas-mediated death. It was therefore suggested that ischemia/reperfusion might lead to down-regulation of inhibitors of the Fas pathway such as cFLIP. cFLIP is highly expressed in the heart under normal physiological conditions but is degraded after ischemia/reperfusion (8)
. Thus, loss of cFLIP may be important in enhancing sensitivity of cardiomyocytes to apoptosis after ischemia/reperfusion.
This is supported by recent finding that whereas cFLIP RNA and protein are abundantly expressed in cardiomyocytes, cFLIP protein was down-regulated in TUNEL-positive cardiomyocytes in grafted cardiac tissue (35)
. The same study found that in failing human hearts, cFLIP-positive cardiomyocytes rarely showed evidence of apoptosis. Activated monocytes, which release soluble Fas ligand after stimulation, induced death of neonatal rat cardiomyocytes via the Fas-mediated pathway (35)
. These results suggest that a Fas-mediated pathway of cell death exists in cardiomyocytes, but that under normal conditions is down-regulated by inhibitors such as cFLIP. After stress such as ischemia, however, cFLIP becomes inactivated, leaving the cells susceptible to death via the Fas pathway.
Although activation of Fas by its ligand is known to induce apoptosis, this is not always the case, with Fas ligation being associated with induction of transcription in some cell types (36)
. This phenomenon has been demonstrated in cardiomyocytes where up-regulation of Fas expression did not result in apoptosis in a rat model of volume overload hypertrophy (37)
. Recombinant Fas ligand did not promote cell death in cultured cardiomyocytes but induced AP-1 DNA binding. Induction of transcription factors such as AP-1 has been shown to be associated with a hypertrophic response (38)
. Thus, induction of death is not the only role of the Fas pathway in the heart. Depending on the stimulus, it may be involved in adaptive responses such as hypertrophy or cell death by apoptosis.
Signaling through the TNF receptor also plays a dual role in the heart. Like Fas, binding of the TNFR1 by its ligand (TNF-
) results in the recruitment of adaptor proteins that serve to transduce signals downstream (5)
. The TNFR1 pathway is a mediator of diverse physiological events including inflammation, cell growth, differentiation, and apoptosis. This is reflected in the different adaptor proteins and downstream signaling events associated with receptor activation.
In recent years, it has emerged that TNF plays a role in the progression of myocardial disease. Increased TNF- and TNFR1 expression are found in association with heart failure (39)
. Agents that suppress TNF-, such as pentoxifylline, have shown positive therapeutic effects in trials, emphasizing the negative role of elevated TNF- (40)
. As TNF- can induce apoptosis of cardiomyocytes (41)
, it is thought that at least part of its pathogenic effect in the heart is due to the induction of cell death. In contrast, there is also evidence to support a prosurvival role of TNF in the heart whereby it is involved in regulation of adaptive responses to biomechanical stress (42)
. Examples of such adaptive responses include the induction of cellular hypertrophy in response to pressure overload and modulation of contractile function after ischemia. A recent study found that TNF protects against ischemia-induced apoptosis (43)
. Thus, whether the cell chooses a survival pathway or a death pathway in response to TNFR activation depends on the interaction of various signaling pathways and regulators of these pathways. For example, the presence of endogenous inhibitors such as cFLIP may promote activation of transcription rather than apoptosis.
Mitochondrial pathway of apoptosis
Mitochondria play a key role in apoptosis. Not only do they provide the energy essential for the completion of apoptosis, they also release important proapoptotic factors into the cytosol. Mitochondria are a primary site of action of the apoptosis regulatory proteins of the Bcl-2 family and a major source of reactive oxygen species (ROS), which have been implicated in cellular damage and death (6)
.
Like skeletal muscle cells, cardiomyocytes have a very large energy requirement and so contain the highest density of mitochondria of all mammalian cells. It has been proposed that the strain on cardiomyocyte mitochondria as a result of their huge workload could lead to leakage of proapoptotic factors into the cytosol, promoting ‘accidental’ apoptosis. Thus, mitochondrial-dependent stimulation of apoptosis may be down-regulated in these cells, possibly by increased levels of endogenous inhibitors or by a lack of an essential component of the apoptotic pathway. The latter is the case in skeletal myocytes, which lack Apaf-1 and are thereby resistant to cytochrome c-mediated caspase activation (44)
.
Recent studies suggest that cytochrome c-mediated apoptosis is important in cardiomyocytes. Serum and glucose deprivation induce cytochrome c release in vitro, resulting in activation of caspases-9 and -3 and apoptosis as determined by nuclear fragmentation, internucleosomal DNA cleavage, and processing of caspase substrates (45)
. The role of cytochrome c as a mediator of cell death is further emphasized by the fact that inhibition of caspase processing by the broad range caspase inhibitor zVAD-fmk did not affect cytochrome c release, indicating that this event occurs upstream of caspase activation. It is therefore a cause rather than a consequence of this event. As serum and glucose deprivation are components of ischemia in vivo, these results suggest that this pathway may be involved in cell death in relation to heart disease. ROS have also been implicated in ischemia/reperfusion-induced damage, and it has recently been reported that mitochondrial cytochrome c release, activation of caspase-3, and PARP cleavage are involved in H2O2-induced cardiomyocyte apoptosis (46)
.
These in vitro studies demonstrate that cardiomyocyte apoptosis can occur via a cytochrome c-mediated pathway, but it remains to be seen whether this pathway plays a significant role in the pathogenesis of cardiac disease. To our knowledge, there has been only one report of cytochrome c release in relation to apoptosis in human cardiomyocytes. Narula and colleagues (27)
examined explanted hearts from cardiomyopathy patients undergoing transplantation and found that cytochrome c release was associated with caspase-3 activation and PKC cleavage, but the typical ultrastructural alterations of apoptosis were not observed. This may be due to cytochrome c-mediated death following a different pathway from the one typically observed in other cell types, which is consistent with the view that there is a novel mechanism of cell death in the myocardium that is neither entirely apoptotic nor necrotic.
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Regulators of apoptosis with therapeutic potential |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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. This is of particular importance in fully differentiated cardiomyocytes in order to avoid unnecessary death of salvageable cells and to promote apoptosis in response to irreversible cellular damage, as opposed to necrosis, which could further harm the myocardium. Although most of our knowledge of regulation of apoptosis applies to nonmyocytes, emerging evidence suggests that similar controls are found in the heart.
Caspase inhibitors
Apoptosis may be initiated by a variety of different signals, but the ultimate result is the same in most cells. Thus, it appears that the final steps of apoptotic death are highly conserved and likely to be mediated by a similar set of caspases. As described earlier, various regulatory mechanisms exist within cells that target caspases. These include cFLIP and the IAP family, inhibitors present in a variety of cell types that may also play an important role in the heart. Recently an inhibitor of apoptosis that is expressed almost exclusively in skeletal muscle and heart has been characterized. ARC (apoptosis repressor with caspase recruitment domain) was first shown to interact with caspases-8 and -2 and to attenuate apoptosis induced by stimulation of death receptors (47)
. More recently it was demonstrated that ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis (48)
, suggesting that ARC can exert its effect at different levels in the apoptotic pathway and may be a key regulator of apoptosis in the heart. As overexpression of ARC had a protective effect compared with cells expressing only endogenous levels of ARC, up-regulation of this protein in vivo could provide a means of attenuating cell death in relation to disease.
The use of synthetic inhibitors of apoptosis presents another potential therapeutic avenue. The broad range caspase inhibitor zVAD-fmk was effective in reducing myocardial reperfusion injury in rats, which was attributed in part to the attenuation of cardiomyocyte apoptosis (49)
. The same inhibitor is reported to attenuate apoptosis in rabbit cardiomyocytes (50)
. Unfortunately, any potentially beneficial effects this inhibitor may have in vivo could be far outweighed by its side effects, whereby caspase inhibition may increase ROS production resulting in secondary toxicity as observed in other cell types (51)
.
Others have investigated the effects of specific inhibitors of caspases. One study found that cardiomyocyte DNA fragmentation and caspase activation were prevented by inhibitors of caspase-1 and -3 without reduction of the infarct size in ischemia/reperfused rat hearts (52)
. This contrasts with another study that found that, in addition to zVAD-fmk, inhibitors of caspase-8, -9, and -3 all limited infarct size as a result of reperfusion injury (53)
. The different outcomes of these studies may reflect the times the inhibitors were administered. In the former study, inhibitors were administered before ischemia; in the latter, they were administered during early reperfusion. This underscores the importance of determining the optimum time for therapeutic intervention. Like zVAD-fmk, specific caspase inhibitors might not be of major therapeutic value. They do, however, provide a starting point for the development of more sophisticated inhibitors for use in treating cardiac disease.
Bcl-2 family
Cardiomyocytes are known to express Bcl-2 family proteins, but the extent to which they influence apoptosis in the heart is unknown. It has been proposed that expression of these proteins is very low relative to the numbers of mitochondria in heart cells and therefore they may not play an important role. Several reports, however, suggest otherwise. A dramatic increase in Bax expression and decreased expression of Bcl-2 have been reported during left ventricular adaptations to chronic pressure overload in the rat (54)
, and it is thought that such changes are likely to influence cells to undergo apoptosis. In a model of reactive oxygen species-induced apoptosis, H2O2 increased expression of proapoptotic Bad and elicited translocation of Bax and Bad to the mitochondria, resulting in cytochrome c release, activation of caspase-3, and cleavage of PARP (46)
. An up-regulation of Bcl-2 family protein has also been reported in human end-stage heart failure where increases in proapoptotic Bak and Bax and antiapoptotic Bcl-2 and Bcl-xL were observed in association with apoptosis (55)
. Expression of Bax was significantly higher than that of the antiapoptotic proteins, which suggests that in the failing human heart, the balance between prosurvival and prodeath signals is tipped to favor the latter, resulting in apoptosis.
As with caspase inhibition, manipulation of the Bcl-2 system may also provide new treatments to forestall cardiomyocyte apoptosis. The potential strategies could involve up-regulating the antiapoptotic Bcl-2 pathway or inhibiting proapoptotic pathways. As the mechanisms involved are not fully understood, there have been few reports regarding the manipulation of these pathways in cardiomyocytes. It has been shown that ischemic preconditioning with reduced apoptosis is associated with up-regulation of Bcl-2 in rats (56)
and that overexpression of Bcl-2 in ventricular myocytes prevents apoptosis (57)
, but further investigation is required to determine whether such a strategy could be used in a clinical setting.
IGF-1
It has recently become evident that growth factors such as IGF-1 are involved in the regulation of apoptosis. IGF-1 is an important growth and survival factor in cardiomyocytes, and has been shown to inhibit ischemia/reperfusion and serum withdrawal/doxorubicin-induced apoptosis (58
, 59)
and to prevent activation of cell death after infarction, limiting ventricular dilation, myocardial loading, and cardiac hypertrophy (60)
. Although the mechanism has not been fully elucidated, it is known to involve signaling by phosphatidylinositol (PI) 3-kinase and Akt (protein kinase B) (61)
and attenuation of Bax induction and caspase-3 activation (59)
. A recent report indicates that Akt also exerts its cardioprotective effect by improving the function of surviving cardiomyocytes after transient ischemia (62)
.
Several reports support a therapeutic application for IGF-1 in the treatment of cardiac disease. As discussed, IGF-1 has been shown to inhibit apoptosis induced by a variety of stimuli, including ischemia/reperfusion and serum withdrawal (58
, 59)
. More recently, IGF-1 suppressed apoptosis of cardiomyocytes in a canine model of dilated cardiomyopathy (63)
. It has been shown that adenoviral gene transfer of the downstream signaling molecules of IGF-1, PI-3 kinase, and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro (64)
. Similarly, expression of a constitutively active form of Akt greatly attenuated apoptosis and infarct size in a rat model of ischemia/reperfusion injury. Taken together, these results indicate that manipulation of the IGF-1-mediated survival pathway may be of therapeutic value.
Heat shock proteins
It is well established that heat shock proteins (hsps) are synthesized in response to a variety of stressful stimuli and that their expression coincides with increased resistance to subsequent cellular damage (65)
. This resistance is due at least in part to interference with apoptosis, and there are several examples of the involvement of hsps in the suppression of cell death (66
67
68)
.
The hsps are recognized as important regulators of cardiomyocyte apoptosis. Different families of hsps are expressed in the heart, including the hsp90, -70, -60, -27, and -10 families; induction of these proteins by a variety of agents including cardiotrophin-1 (67)
, herbamycin A (69)
, heat shock, and ethanol (70)
protects cardiomyocytes against stress such as ischemia. Inducible hsps70 and -27 are the two best studied of the myocardial hsps; overexpression of these proteins can protect cells against ceramide, serum withdrawal, and lethal hypoxia (68)
, all of which are known to be inducers of apoptosis. Thus, hsps are likely to be key regulators of apoptosis in the heart during stressful conditions such as ischemia, and a failure to up-regulate these proteins in response to stress may be a pathogenic factor in the progression of disease. This helps to explain the finding that increased expression of hsp27 and -60 but no increase in hsp72, hsc70, or hsp90 are found in association with heart failure (71)
.
Although hsps play a key role in the regulation of apoptosis, the mechanisms by which they do so are poorly understood. Recent reports have provided insight into the mechanism of action of hsp70 and hsp27, however. Hsp70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation (72)
; this event is mediated through direct association with Apaf-1 and prevention of apoptosome formation (73)
. Our group and others have shown that hsp27 can prevent cytochrome c release at the level of the mitochondria (74)
or can interact with cytochrome c or procaspase-3, preventing apoptosome formation and caspase-3 activation, respectively (75
76
77)
. Another recent study has demonstrated that overexpression of hsp60 and hsp10, in combination or individually, protects cardiomyocytes from ischemia/reperfusion-induced apoptosis and that this protection is due at least in part to maintenance of mitochondrial integrity and ATP generation (78)
.
Given the cardioprotective effect of heat shock protein induction in in vitro and animal studies, these proteins represent a potentially valuable treatment for cardiac disease. As application of a thermal or other hsp-inducing stress may cause more harm than good in the clinical situation; delivery of hsps by gene therapy is the more promising option. Gene therapy offers many potential benefits such as the ability to specifically target heart cells without inducing the additional metabolic and structural alterations associated with stress-induced hsp-induction. Transfection results in higher levels of hsp expression than heat stress (79)
. Hsps have been successfully delivered to cardiac cells in vitro using a variety of vectors (68
, 78)
, resulting in a cardioprotective effect. Efficient transfer of genes to the heart of animal models has been demonstrated using herpes simplex virus and adenoviral vectors (80
, 81)
.
Calcium
Calcium (Ca2+) plays an important signaling role in the cell. It is well established that changes in intracellular Ca2+ are associated with apoptosis, and numerous Ca2+-regulated effectors have been identified. These include the Ca2+/calmodulin-regulated phosphatase calcineurin, which promotes apoptosis in several cell types (82)
. One mechanism it uses is thought to be dephosphorylation of Bad, which then translocates to the mitochondria, influencing cytochrome c release (83)
. Similarly, the Ca2+-dependent cysteine protease calpains required for apoptosis in several models of cell death can cleave Bax, promoting its proapoptotic activity (84)
. Ca2+ is also believed to modulate cytochrome c release directly by regulating the mitochondrial permeability transition pore, a proposed mechanism for cytochrome c release during apoptosis (85)
. Another proposed mechanism by which Ca2+ regulates apoptosis is via Ca2+-dependent endonucleases involved in DNA fragmentation. One such enzyme is DNase I, which is found in the ER and is released in response to depletion of ER calcium, i.e., ER stress (86)
. ER stress also leads to activation of caspase-12, providing a novel death receptor/mitochondria-independent pathway of cell death (87)
.
Under normal physiological conditions, Ca2+ has several important functions in the heart, including driving the contraction of heart muscle. Its role in heart disease and cell death is less well understood although excessive Ca2+ influx has long been linked to damage incurred by ischemia. As in other cell types, changes in levels of intracellular calcium may trigger pathways that lead to apoptosis. Given that a selective calpain inhibitor was effective in reducing infarct size and DNA fragmentation in ischemic-reperfused rat heart, a Ca2+-regulated apoptotic pathway may be important under these conditions (88)
. A high molecular weight calmodulin binding protein (HMWCaMBP), a homologue of the calpain inhibitor calpastatin, had reduced expression, and activity after ischemia/reperfusion. This was correlated with increased calpain expression, Bax expression and apoptosis (89)
. This decrease in HMWCaMBP activity is consistent with an earlier finding that calpastatin activity is decreased after ischemia/reperfusion (90)
. These inhibitors may play an important role in sequestering calpain from its substrates in normal myocardium, but are proteolyzed during ischemia/reperfusion, leaving calpain free to act on substrates leading to apoptosis or other forms of myocardial injury. One such substrate may be Bid, as calpain can cleave Bid to an active fragment capable of mediating cytochrome c release (91)
. Calpain has been linked to the activation of caspase-12, providing another mechanism by which Ca2+ may trigger the apoptotic effector machinery (92)
.
Calcineurin has also been implicated in proapoptotic pathways in the heart. Stimulation of ß-adrenergic receptors induces apoptosis in a Ca2+/calcineurin-dependent manner in association with dephosphorylation of Bad and cytochrome c release (93)
. Others contradict this finding, however, as it has been reported that calcineurin is a mediator of myocardial hypertrophy rather than apoptosis and that it can, in fact, protect against apoptosis (94
, 95)
. As for previously described regulators of apoptosis, this discrepancy is explained by the fact that the calcineurin pathway may act in either a pro- or antiapoptotic manner depending on the stimulus. This was recently demonstrated in a mouse leukemic cell line where the Ca2+ ionophore A23187 and the ER Ca2+-ATPase inhibitor thapsigargin activated opposing prosurvival and death pathways via calcineurin (96)
.
Although our understanding of the relationship between changes in intracellular Ca2+ and myocardial apoptosis is still limited, the association between Ca2+ and ischemia/reperfusion and ischemia/reperfusion and apoptosis would suggest a link between Ca2+ and apoptosis in the heart. As such, modulation of Ca2+ may serve as a potential therapeutic target. Given the multiple roles of Ca2+ in the cell, it is essential we first clarify its involvement in apoptosis so that we may target specific molecules such as proapoptotic calpain or calcineurin pathways. Some of the treatments already in use for heart disease may exert some of their effect by modulation of calcium-dependent apoptosis. For example, the calcium channel blocker nifedipine was shown to reduce the extent of Ca2+-mediated cardiomyocyte apoptosis (97)
. ß-Blockers, which antagonize ß-adrenergic receptors, may exert part of their effect by modulation of Ca2+-mediated apoptosis. It would also be of value to determine whether caspase-12 plays an important role in Ca2+-mediated cell death since caspase-12 is highly expressed in heart tissue (92)
.
Antioxidants
Cellular antioxidants represent one of the most potent mechanisms of combating damage and cell death. They act by removing free radicals from the cell and thereby minimize oxidative stress resulting from a variety of insults, including ischemia/reperfusion injury. It has been proposed that apoptosis is modulated by oxidative stress and antioxidants play a part in its prevention (98)
. Several endogenous antioxidants are found in cardiomyocytes including glutathione (GSH), glutathione peroxidase (GSHPx), superoxide dismutase (SOD) catalase, and vitamins E and C (99)
. In relation to apoptosis, GSH has been extensively studied in a variety of cell types; it is thought that during apoptosis, a drop in intracellular levels of GSH is associated with increased oxidative stress. This leads to damage of cellular macromolecules within the cell and subsequent cell death (98)
.
In the heart, GSHPx has a role in regulating ischemia/reperfusion-induced apoptosis. Experiments with GSHPx knockout mice demonstrated increased levels of apoptosis in response to ischemia/reperfusion whereas mice overexpressing GSHPx were more resistant to such damage (100)
. Overexpression of manganese-SOD (MnSOD) protects against ischemia/reperfusion injury in transgenic mice, an effect likely to be mediated in part by modulation of apoptosis (101)
.
Given the ability of antioxidants to protect against apoptosis, particularly in ischemia/reperfusion injury, the use of antioxidants in vivo may attenuate the effects of such stress. A recent study showed that long-term treatment with the antioxidants probucol and pyrrolidine dithiocarbamate in rats after myocardial infarction reduced oxidative stress, blocked the increased expression of p53, Bax, and caspase-3, and inhibited caspase-3 activation (102)
. Attenuation of cardiac dysfunction using antioxidant treatment has been demonstrated in other models of cardiac disease such as adriamycin-associated cardiomyopathy (103)
. In the clinical setting, agents with antioxidant properties such as carvedilol or those that promote antioxidant activity, such as propranolol, are already used to treat heart disease (104)
. With the knowledge that these antioxidants function in part by modulation of apoptosis, the development of more potent therapeutic agents may be possible.
Whereas regulation of apoptosis provides appealing targets for therapeutic intervention, current data are limited and a much wider investigation is required to determine whether modulation of apoptotic pathways will provide useful therapeutic strategies. Several important questions regarding the effect of apoptosis prevention in the heart remain unanswered. Injury such as myocardial infarction causes apoptosis not only of cardiomyocytes, but also of nonmyoctes such as coronary endothelial cells and interstitial macrophages. The importance of apoptotic elimination of these cells after myocardial injury is not fully characterized. It may be critical for the rapid healing of tissue by removing damaged cells; prevention of apoptosis in these cells could aggravate tissue damage. Even if therapeutic agents such as caspase inhibitors could be targeted specifically to cardiomyocytes, it is not yet clear whether inhibition of apoptosis would be ultimately beneficial or would result in a switch to another, more destructive mode of death, such as necrosis. Despite these uncertainties, modulation of several of the aforementioned therapeutic targets has been shown to have a positive effect both in vitro and in vivo. This area of study is in its infancy, however, and it will be some time before we can fully evaluate the potential benefits of such therapies.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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. Similarly, NO donors used in the treatment of myocardial infarction have both proapoptotic and antiapoptotic properties, although it has yet to be shown that this is part of the therapeutic effect of these agents (105)
. It may turn out that other conventionally used pharmaceuticals exert their effect via apoptotic pathways. It must be noted that apoptosis is not the only mode of cell death in the heart. There is still controversy as to whether necrosis or apoptosis is the main form of cardiomyocyte death involved in the pathogenesis of cardiac disease or whether there is a third, as yet uncharacterized, form of cell death that combines aspects of both apoptotic and necrotic death. Further research should provide answers to these questions and determine the therapeutic value of antiapoptotic intervention in the treatment of cardiovascular disease.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONAPOPTOSIS IN THE HEARTPATHWAYS OF APOPTOSIS IN...Regulators of apoptosis with...CONCLUSIONSREFERENCES |
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