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Cardiomyocyte apoptosis in the ischemic and failing heart
Vol. 2, No. 1 2005
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Cardiovascular diseases MECHANISMS
Cardiomyocyte apoptosis in the ischemic and failing heart Richard D. Patten*, Richard H. Karas Molecular Cardiology Research Institute and Division of Cardiology, Tufts-New England Medical Center, Box #80, 750 Washington Street, Boston, MA 02111, USA
Despite modern medical therapy, heart failure continues to be an important public health problem caus-
Section Editor: Cam Patterson – Farmal Biomedicines, LLC, USA
ing significant morbidity and mortality. There is now increasing evidence from both animal models and humans supporting that apoptosis contributes to cardiomyocyte cell loss during acute ischemic injury and chronic heart failure, and that cardiomyocyte apoptosis contributes to heart failure progression. Therapies directed at preventing apoptosis within the heart have, therefore, emerged as attractive avenues of investigation.
Cardiomyocyte apoptosis and acute ischemic injury
Introduction Heart failure is a growing public health problem accounting for nearly 1 million hospitalizations and 250,000 deaths annually, with an estimated prevalence in the U.S. of 5 million cases [1]. Despite modern therapies including antagonists of the renin-angiotensin aldosterone and sympathetic nervous systems, 5-year mortality rates following a diagnosis of heart failure remain as high as 40–50%, indicating that heart failure remains a progressive syndrome associated with significant mortality. Factors that contribute to acute ischemic myocardial injury and progressive left ventricular (LV) dysfunction in heart failure are numerous, but mounting evidence supports that programmed cell death (apoptosis) contributes to cardiomyocyte cell loss observed in acutely ischemic and failing hearts [2]. Apoptosis is a tightly regulated, ATP-dependent process in which intracellular proteo*Corresponding author: R.D. Patten ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
lytic enzymes, termed caspases, are activated causing nuclear chromatin condensation and organized disassembly of the cell (see reviews [3–8]). This review will explore current data supporting a role for cardiomyocyte apoptosis in heart failure progression in both human and experimental studies. A detailed discussion of apoptotic signaling cascades will be presented along with potential therapeutic strategies aimed at reducing apoptosis and slowing heart failure progression.
DOI: 10.1016/j.ddmec.2005.05.018
In animal models of myocardial injury, apoptosis has been increasingly recognized as an important contributor to cardiomyocyte cell loss. In permanent coronary artery occlusion models of myocardial infarction (MI), we and others have demonstrated the presence of apoptotic cardiomyocytes and activated caspase 3 (GenBank accession no. NM_004346) within the infarct and peri-infarct zones [2,9–11] (Fig. 1). Cardiomyocyte apoptosis has also been recognized as an important mode of cell death in ischemia-reperfusion models mimicking the clinical scenario of an ST-elevation MI treated with thrombolytic therapy or percutaneous coronary intervention [12,13]. These experimental studies support that apoptosis contributes importantly to myocyte cell loss within the infarct and peri-infarct zones in the setting of both myocardial infarction and ischemia-reperfusion. The clinical literature also supports this notion. Elegant autopsy studies have shown that cardiomyocytes in normal hearts undergo cell death at very low rates [14], but the rate of apoptosis increases sharply in acutely ischemic hearts [15]. www.drugdiscoverytoday.com
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Figure 1. Cardiomyocyte apoptosis in the peri-infarct zone. Examples of terminal deoxynucleotidal transferase uridine nucleotide end-labeling method (TUNEL) staining of the peri-infarct zone in myocardial sections from placebo and 17-beta-estradiol (E2)-treated mice 24 h following coronary ligation. Here, cardiomyocytes are stained with a specific antibody that appears red; nuclei are blue. TUNEL-positive nuclei are green. Arrows indicate examples of TUNEL-positive cardiomyocyte nuclei. Scale bars, 20 mm. In this model, E2 reduced myocardial infarct size in association with a reduction in peri-infarct zone cardiomyocyte apoptosis.
Hearts from patients who died of an acute MI demonstrate a substantial increase in apoptotic cardiomyocytes in the infarct- and peri-infarct zones [16]. Baldi et al. [17] reported that the rate of apoptotic cells (defined as positive for both terminal deoxynucleotidal transferase uridine nick end labeling (TUNEL) and caspase 3 immuno-staining) averaged 25% within infarct zones. Using technetium99-labeled Annexin V (GenBank accession no. NM_001154), an agent which binds phosphatidylserine in the outer layer of the plasma membrane in apoptotic cells, Hofstra et al. [18] showed that acute MI patients exhibit measurable uptake of this apoptosis marker within the infarct-zone by radionuclide imaging. Furthermore, Abbate et al. reported that the degree of cardiomyocyte apoptosis in patients with MI correlated with more adverse LV remodeling and heightened mortality [19], supporting further that cardiomyocyte apoptosis contributes to cell death in acute myocardial injury, and that the degree of apoptosis coincides directly with LV remodeling and heart failure.
Cardiomyocyte apoptosis in chronically failing hearts Within animal models of chronic and progressive LV dysfunction and heart failure, cardiomyocyte apoptosis occurs at an increased rate above that observed in sham or control animals. In a rat pressure overload model, Candorelli et al. [20] demonstrated evidence of increased cardiomyocyte apoptosis following the transition from compensated hypertrophy to LV dysfunction. In a mouse MI model, Sam et al. noted a gradual increase in cardiomyocyte apoptosis defined both by TUNEL staining and caspase 3 activity within the non-infarcted myocardium over a span of 6 months that was associated with progressive LV dilation and heart failure [21]. Both of these representative experimental studies support 48
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that progressive cardiomyocyte cell loss contributes to progressive LV dysfunction. Moreover, multiple transgenic models of dilated cardiomyopathy are characterized by increased rates of cardiomyocyte apoptosis. For example, cardiac-specific overexpression of the alpha subunit of the G protein, Gq (Gaq) (GenBank accession no. NM_002067), leads to the development of compensated LV hypertrophy followed by progressive LV dysfunction, associated with a significant increase in cardiomyocyte apoptosis [22]. Similarly, mice with a cardiac-targeted overexpression of the cytokine, tumor necrosis factor alpha (GenBank accession no. NM_000594), develop increased cardiomyocyte apoptosis, significant LV dysfunction, heart failure and premature death [23,24]. These transgenic models represent a cross-section of many studies linking cardiomyocyte apoptosis to dilated cardiomyopathy and progressive LV dysfunction. Wencker et al. [25] recently developed a transgenic mouse with cardiac-specific expression of an artificially activated procaspase 8 (GenBank accession no. NM_001228) construct. Activation of procaspase 8 in these mice dose-dependently resulted in accelerated cardiomyocyte apoptosis, leading to severe LV dysfunction and death. One of the transgenic lines demonstrated low-level caspase 8-activation coupled with low-level but ongoing cardiomyocyte apoptosis at a rate tenfold higher than wild type mice, and a comparable to that observed in failing human hearts. These animals developed progressive LV dysfunction and premature death supporting further that cardiomyocyte apoptosis contributes to heart failure progression. Several clinical studies have demonstrated evidence of cardiomyocyte apoptosis in chronically failing human hearts [26,27]. In cardiac biopsy samples, Saraste et al. observed increased rates of cardiomyocyte apoptosis in the myocardium of patients with dilated cardiomyopathy and heart failure. In this study, the percent of apoptotic cardiomyocytes correlated with the rapidity of heart failure progression [28]. Narula et al. also reported increased TUNEL-positive cardiomyocytes in failing human myocardium, and demonstrated increased levels of cytoplasmic cytochrome C (GenBank accession no. NM_018947) and activated caspase-3 (GenBank accession no. NM_004346) as compared to normal controls [29]. We recently found that mechanical unloading with ventricular assist device therapy attenuated cardiomyocyte apoptosis in failing human hearts [30]. These clinical data support further that cardiomyocyte apoptosis is present at significantly increased rates in chronically failing hearts and that loss of cardiac myocytes in this manner could contribute to heart failure progression.
Apoptosis signaling pathways Apoptosis occurs following the activation of two general pathways: (1) the extrinsic pathway initiated through agonist binding to ‘‘death receptors;’’ and (2) the intrinsic or mitochondrial
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Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Figure 2. Apoptosis signaling pathways. Two major pathways converge on caspase 3, triggering apoptosis. Activation of the extrinsic pathway occurs by activation of the death receptor, Fas, by Fas ligand (FasL) causes cleavage and activation of procaspase 8 to caspase 8 which cleaves the pro-apoptotic protein, BID, that contributes to mitochondrial disruption. Caspase 8 also activates caspase 3 which then cleaves target proteins and activates other downstream caspases. The intrinsic or mitochondrial pathway is activated by stimuli such as hypoxia, reactive oxygen species, or sudden increases in intracellular calcium causing disruption of the mitochondrial membrane via activation of pro-apoptotic family members such as Bax and Bid leading to the formation of the mitochondrial permeability transition pore. This results in cytoplasmic leakage of inter-membrane contents including the pro-apoptotic factor, cytochrome C (Cyt C) that binds and activates apoptosis protease activating factor-1 (apaf-1) causing sequential activation of caspases 9 and 3. Pro-survival Bcl-2 family members including Bcl-2 and Bcl-xL protect the cell from apoptosis by inhibiting mitochondrial pore formation.
pathway activated by reactive oxygen species, hypoxia and sudden increases in intracellular Ca2+ that cause a loss of integrity of the outer mitochondrial membrane leading to the release of pro-apoptotic initiating factors [4,31] (Fig. 2). Extrinsic pathway
Apoptosis can be initiated through activation of death receptors (e.g. Fas (GenBank accession no. NM_000043)) by ‘‘deathinducing’’ ligands (e.g. Fas ligand (GenBank accession no. NM_000639)) that lead to the recruitment of death domaincontaining adaptor proteins such as FADD (Fas-associated via Death Domain (GenBank accession no. NM_003824)) and the formation of a membrane-associated, death-inducing signaling complex (DISC). FADD then recruits procaspase 8 into the complex in the form of a dimer, leading to its cleavage and activation; caspase 8 then cleaves and activates procaspase 3 to its active form, caspase 3 [27] (Fig. 1). Caspase 8 also activates the pro-apoptotic Bcl-2 family member, Bid (GenBank accession no. NM_001196) (see below), after which the C-terminal
portion of Bid (tBid) translocates to the mitochondria and inserts into the outer mitochondrial membrane, facilitating the formation of the mitochondrial permeability transition pore (MPTP). This leads to the release of cytochrome C and other pro-apoptotic factors into the cytoplasm. In this manner, the extrinsic pathway is mechanistically linked to the intrinsic or mitochondrial pathway. Intrinsic pathway
The intrinsic pathway of programmed cell death [4] is triggered by the accumulation of reactive oxygen species, sudden increases in intracellular calcium, or hypoxic insults [32]. Following these stimuli, the outer mitochondrial membrane becomes disrupted via mechanisms that are not entirely understood, although mitochondrial localization of proapoptotic Bcl-2 family members leading ultimately to the formation of the MPTP appears crucial in initiating this process. Upon outer mitochondrial membrane disruption, pro-apoptotic factors are released from the inter-membrane www.drugdiscoverytoday.com
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space into the cytosol. The heme-containing electron transport protein, cytochrome C, is one pro-apoptotic factor released from the inter-membrane space. Once in the cytoplasm, cytochrome C binds to and induces the oligomerization of Apaf-1 (GenBank accession no. NM_001160) (apoptosis protease activating factor), which then recruits procaspase 9 (GenBank accession no. NM_001229) forming the complex known as the apoptosome. Upon recruitment to the apoptosome, procaspase 9 is cleaved and activated and then binds and cleaves procaspase 3, resulting in caspase 3 activation. Caspase 3 is an important terminal effecter of apoptosis, cleaving substrate proteins and activating other downstream caspases within the cell (Fig. 2). Bcl-2 protein family
The Bcl-2 family of apoptosis-related proteins is evolutionarily conserved, consisting of both pro-survival and pro-apoptotic factors [14,22,32]. The pro-survival members of the Bcl-2 family, Bcl-2 (GenBank accession no. NM_000633) and Bcl-xl (GenBank accession no. NM_001191), inhibit apoptosis, in part, by maintaining mitochondrial membrane integrity and preventing the release of pro-apoptotic factors from the intermembrane space (e.g. cytochrome C). The anti-apoptotic Bcl2 proteins have been shown to inhibit the pro-apoptotic members via direct protein–protein interactions. Pro-apoptotic proteins within the Bcl-2 family, such as Bad (GenBank
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accession no. NM_004322), comprise a Bcl-2 homology (BH3) domain that induces hetero-dimerization with Bcl-2 and Bclxl, thereby blocking their anti-apoptotic effects. Other BH3 containing Bcl-2-family members such as Bid and Bax (GenBank accession no. NM_004324) exert their pro-apoptotic effects by inducing the formation of the mitochondrial permeability transition pore. Although the precise mechanisms are not clear, activation of pro-apoptotic, Bid and Bax, might themselves form a protein-permeable pore in the outer mitochondrial membrane.
The case for modulating cardiomyocyte apoptosis in acute ischemic syndromes and heart failure Numerous experimental studies have demonstrated that inhibiting cardiomyocyte apoptosis leads to improved functional recovery of the myocardium in acute ischemic syndromes and in chronically failing hearts. Inhibition of apoptosis has been achieved using both pharmacologic and molecular approaches that either activate pro-survival pathways or inhibit pro-apoptotic pathways (also see Table 1).
Pharmacologic strategies Several groups have recently studied the benefit of pharmacological inhibition of cardiomyocyte apoptosis in animal models of heart failure and myocardial infarction. The broad caspase inhibitor IDN1965 has been reported to prevent
Table 1. Targets and related therapies Apoptosis target
Therapeutic agent a
Caspases
IDN-1965
Caspases
Z-Asp 2,6-DCBMkb c
p38 MAPK e
d
RWJ-67657 f
Lab(s) working on this
Refs
Kitsis Lab Dorn Lab
[25,33] [33]
Anand Lab
[34]
Krum Lab
[35]
JNK (MAP kinase family member)
AS601245
Ferrandi Lab
[36]
Fas/Fas ligand
Mice lacking functional Fas receptor or Fas ligand Use of soluble Fas receptor by adenoviral gene transfer
Kitsis Lab Fujiwara Lab
[38] [39]
Caspase 9
Dominant negative construct in transgenic mice
Kitsis Lab
[40]
BCl2
Cardiac overexpression in transgenic mice Adenoviral-mediated gene transfer induced overexpression
Kirshenbaum Lab Gardner Lab Capetanaki Lab
[41] [42] [43]
ARCg
ARC-TAT peptide construct. The TAT moiety facilitates protein diffusion across cell membranes
Gottlieb Lab
[44]
Akt (serine-threonine kinase)
Overexpression strategies of constitutively active construct Hormone manipulation/Akt activation
Walsh Lab Rosensweig Lab Patten Lab
[48] [49] [31]
Overexpression strategies of constitutively active construct
Molkentin Lab
[50]
ERKh 1/2 (MAP kinase family member) a
IDN-1965: broad caspase inhibitor (N-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; Idun Pharmaceuticals). Z-Asp 2,6-DCBMk: broad caspase inhibitor (Alexis pharmaceuticals). MAPK: mitogen-activated protein kinase. d RWJ-67657 (Johnson & Johnson Pharmaceutical Research and Development). e JNK: Jun-N-terminal kinase (also known as stress-activated protein kinases or SAPK). f AS601245: 1,3-benzothiazol-2-yl(2-{[2-(3-pyridinyl)ethyl]amino}-4-pyrimidinyl)acetonitrile: Serono Pharmaceutical Research Institute, Geneva, Switzerland. g ARC: apoptosis repressor with caspase recruitment domain. h ERK: extracellular signal-regulated kinases 1 and 2 (p42/44). b c
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apoptosis, cardiac remodeling and death in transgenic mice with cardiac-specific expression of activated caspase 8 [25], and in transgenic mice with cardiac-targeted overexpression of the alpha subunit of Gq [33]. In the rat ischemia/reperfusion model, several studies have demonstrated that pharmacologic inhibition of caspases with peptide inhibitors reduces infarct size [12,13]. In the chronic rat MI model, Chandrashekhar et al. [34] administered a cellpermeable, broad caspase inhibitor, Z-Asp 2,6-DCBMk, immediately following left coronary ligation. At 4 weeks, treatment with the caspase inhibitor markedly reduced TUNEL-positive cardiomyocytes in the surviving myocardium, associated with improved LV function, and reduced LV hypertrophy and dilation, although infarct size and survival were not affected. Pharmacologic inhibitors of both p38 MAPK and Jun-N-terminal kinases (JNKs) are currently under development and have been shown in animal models to limit myocardial injury in response to ischemia-reperfusion [35,36]. Because of the observed gender differences in both heart failure survival and cardiomyocyte apoptosis in normal and failing hearts, we explored the effects of the hormone, 17betaestradiol (the main circulating form of estrogen) on infarct size and remodeling in the mouse model of MI. Physiologic estrogen replacement resulted in a modest but significant reduction in infarct size compared to placebo-treated females and was associated with less cardiomyocyte apoptosis and caspase 3 activation in the peri-infarct zone [31,37], the mechanism of which appears, in part, due to estrogen receptor-mediated activation of the PI3 kinase (GenBank accession no. NM_006218)-Akt signaling pathway.
Molecular approaches Numerous molecular targets have been examined using dominant negative, knockout, or overexpression transgenic strategies to inhibit apoptosis. For example, a naturally occurring mouse strain that harbors a non-functional death receptor, Fas, exhibits more than 60% reduction in infarct size compared to wild type mice following ischemia-reperfusion injury [38]; coincident with this reduction in infarct size was a nearly equivalent relative reduction in the percentage of apoptotic cardiomyocytes in the area of the myocardium at risk [38]. This same mouse strain deficient in the Fas receptor also develops less heart failure and LV dilatation in response to permanent coronary occlusion [39]. Thus, inhibiting Fas ligand by the adenoviral-mediated delivery of a construct encoding a soluble Fas receptor may hold promise [39]. These data, therefore, support that activation of the death receptor, Fas, contributes significantly to cardiomyocyte apoptosis following ischemiareperfusion injury, and disruption of this pathway might hold therapeutic value in reducing infarct size. Moreover, mice that express a dominant negative caspase 9 construct within the heart develop smaller areas of myocardial injury and less cardiomyocyte apoptosis when subjected to ischemia-reperfu-
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
sion injury [40]. Cardiomyocyte-specific overexpression of the pro-survival protein, Bcl-2, diminishes hypoxia-induced apoptosis in cultured cardiomyocytes, and ischemia/reperfusion injury [41,42]. Moreover, BCl2 overexpressed within the myocardium using replication deficient adenoviral-mediated gene transfer can reduce cardiomyocyte apoptosis and LV dysfunction in the desmin (GenBank accession no. NM_001927) knockout model of dilated cardiomyopathy [43]. Levels of the anti-apoptotic protein, ARC (GenBank accession no. NM_003946) (apoptosis repressor with caspase recruitment domain), can be increased in the heart via intra-coronary infusion of an ARC-TAT peptide construct. The TAT-peptide moiety facilitates the transport of ARC across cell membranes [44]. Increasing intracellular ARC in this manner was recently shown to decrease myocardial injury in isolated perfused hearts [44]. Thus, targeting specific proteins within the apoptosis cascade using molecular strategies can reduce both cardiomyocyte apoptotic cell death and infarct size, leading to better preservation of LV function in these rodent models. Other investigators have focused on increasing the activity of pro-survival signaling kinases as a means of promoting cell survival during myocardial infarction or ischemia-reperfusion [45]. It is well established that the serine-threonine kinase, Akt, has many anti-apoptotic effects that include phosphorylating and inhibiting pro-apoptotic proteins such as BAD and caspase 9. Akt (GenBank accession no. NM_001014431) also inhibits the Forkhead transcription factor (GenBank accession no. NM_005429) that activates transcription of Fas ligand, thus decreasing the level of this pro-apoptotic secreted factor. Through phosphorylation of the CREB (GenBank accession no. NM_004379) transcription factor, Akt also increases the expression of the pro-survival protein, Bcl-2 [46,47]. Based on numerous anti-apoptotic and pro-survival downstream effects, it is not surprising that cardiac-targeted expression of a constitutively active form of Akt reduces infarct size following ischemia-reperfusion injury [48,49]. The mitogen-activated protein kinase (MAPK) family of intracellular, serine-threonine kinases includes three major groups: the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (GenBank accession nos. NM_002746 and NM_002745), p38 MAPK (GenBank accession no. NM_001315) and Jun-N-terminal kinases (GenBank accession no. NM_002750). The ERK subgroup of signaling kinases has been shown to exert predominantly pro-survival downstream effects, whereas the p38 and JNK cascades are primarily proapoptotic when activated [45]. Overexpression of the kinase upstream to ERK1/2 (denoted MEK1) (GenBank accession no. NM_002755) within the myocardium confers resistance to apoptotic stimuli [50]. Alternatively, disrupting pro-apoptotic signaling kinases has also been shown to reduce cardiomyocyte cell loss in animal models. For example, mice that harbor a cardiac-targeted, dominant negative p38 MAPK construct demonstrate improved LV function following ischemia-reperwww.drugdiscoverytoday.com
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fusion injury. Moreover, mice that harbor a disrupted Jun Nterminal kinase (JNK1) gene also show resistance to myocardial injury in ischemia-reperfusion models.
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Conclusions
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Despite modern medical therapy, heart failure continues to be an important public health problem causing significant morbidity and mortality. There is now increasing data supporting that apoptosis contributes to cardiomyocyte cell loss during acute ischemic injury and in chronic heart failure, and that cardiomyocyte apoptosis contributes importantly to heart failure progression. In fact, current data in vitro animal heart failure and ischemic models support that inhibition of apoptosis can reduce myocardial damage, cardiac dysfunction, and perhaps increase survival. Therapies directed at preventing apoptosis within the heart have, therefore, emerged as attractive avenues of investigation. Multiple approaches have been employed in these animal models to reduce cardiomyocyte apoptosis and improve LV function following acute or chronic myocardial injury. Molecular and pharmacologic approaches to enhance activation of pro-survival signaling cascades (such as Akt and ERK1/2) or those aimed at inhibiting the pro-apoptotic molecules (caspases) or signaling cascades (e.g. p38 MAPK or JNK inhibitors) certainly hold promise. The primary challenge of these therapies is one of cell type or tissue specificity. Indeed, promoting cell survival broadly could potentially increase the risk of uncontrolled cell growth/proliferation in non-cardiac tissues where it is not desired such as early, unidentified malignancies. In the case of acute myocardial injury, a short duration of anti-apoptotic therapy might pose minimal risk of facilitating the growth of malignant cells. However, the long-term treatment of patients with heart failure and LV dysfunction poses increased risks of undesired effects in non-target tissues. Successful therapies will require strategies to target specific populations of cells.
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Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Cardiovascular diseases MECHANISMS
Cardiomyocyte apoptosis in the ischemic and failing heart Richard D. Patten*, Richard H. Karas Molecular Cardiology Research Institute and Division of Cardiology, Tufts-New England Medical Center, Box #80, 750 Washington Street, Boston, MA 02111, USA
Despite modern medical therapy, heart failure continues to be an important public health problem caus-
Section Editor: Cam Patterson – Farmal Biomedicines, LLC, USA
ing significant morbidity and mortality. There is now increasing evidence from both animal models and humans supporting that apoptosis contributes to cardiomyocyte cell loss during acute ischemic injury and chronic heart failure, and that cardiomyocyte apoptosis contributes to heart failure progression. Therapies directed at preventing apoptosis within the heart have, therefore, emerged as attractive avenues of investigation.
Cardiomyocyte apoptosis and acute ischemic injury
Introduction Heart failure is a growing public health problem accounting for nearly 1 million hospitalizations and 250,000 deaths annually, with an estimated prevalence in the U.S. of 5 million cases [1]. Despite modern therapies including antagonists of the renin-angiotensin aldosterone and sympathetic nervous systems, 5-year mortality rates following a diagnosis of heart failure remain as high as 40–50%, indicating that heart failure remains a progressive syndrome associated with significant mortality. Factors that contribute to acute ischemic myocardial injury and progressive left ventricular (LV) dysfunction in heart failure are numerous, but mounting evidence supports that programmed cell death (apoptosis) contributes to cardiomyocyte cell loss observed in acutely ischemic and failing hearts [2]. Apoptosis is a tightly regulated, ATP-dependent process in which intracellular proteo*Corresponding author: R.D. Patten ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
lytic enzymes, termed caspases, are activated causing nuclear chromatin condensation and organized disassembly of the cell (see reviews [3–8]). This review will explore current data supporting a role for cardiomyocyte apoptosis in heart failure progression in both human and experimental studies. A detailed discussion of apoptotic signaling cascades will be presented along with potential therapeutic strategies aimed at reducing apoptosis and slowing heart failure progression.
DOI: 10.1016/j.ddmec.2005.05.018
In animal models of myocardial injury, apoptosis has been increasingly recognized as an important contributor to cardiomyocyte cell loss. In permanent coronary artery occlusion models of myocardial infarction (MI), we and others have demonstrated the presence of apoptotic cardiomyocytes and activated caspase 3 (GenBank accession no. NM_004346) within the infarct and peri-infarct zones [2,9–11] (Fig. 1). Cardiomyocyte apoptosis has also been recognized as an important mode of cell death in ischemia-reperfusion models mimicking the clinical scenario of an ST-elevation MI treated with thrombolytic therapy or percutaneous coronary intervention [12,13]. These experimental studies support that apoptosis contributes importantly to myocyte cell loss within the infarct and peri-infarct zones in the setting of both myocardial infarction and ischemia-reperfusion. The clinical literature also supports this notion. Elegant autopsy studies have shown that cardiomyocytes in normal hearts undergo cell death at very low rates [14], but the rate of apoptosis increases sharply in acutely ischemic hearts [15]. www.drugdiscoverytoday.com
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Figure 1. Cardiomyocyte apoptosis in the peri-infarct zone. Examples of terminal deoxynucleotidal transferase uridine nucleotide end-labeling method (TUNEL) staining of the peri-infarct zone in myocardial sections from placebo and 17-beta-estradiol (E2)-treated mice 24 h following coronary ligation. Here, cardiomyocytes are stained with a specific antibody that appears red; nuclei are blue. TUNEL-positive nuclei are green. Arrows indicate examples of TUNEL-positive cardiomyocyte nuclei. Scale bars, 20 mm. In this model, E2 reduced myocardial infarct size in association with a reduction in peri-infarct zone cardiomyocyte apoptosis.
Hearts from patients who died of an acute MI demonstrate a substantial increase in apoptotic cardiomyocytes in the infarct- and peri-infarct zones [16]. Baldi et al. [17] reported that the rate of apoptotic cells (defined as positive for both terminal deoxynucleotidal transferase uridine nick end labeling (TUNEL) and caspase 3 immuno-staining) averaged 25% within infarct zones. Using technetium99-labeled Annexin V (GenBank accession no. NM_001154), an agent which binds phosphatidylserine in the outer layer of the plasma membrane in apoptotic cells, Hofstra et al. [18] showed that acute MI patients exhibit measurable uptake of this apoptosis marker within the infarct-zone by radionuclide imaging. Furthermore, Abbate et al. reported that the degree of cardiomyocyte apoptosis in patients with MI correlated with more adverse LV remodeling and heightened mortality [19], supporting further that cardiomyocyte apoptosis contributes to cell death in acute myocardial injury, and that the degree of apoptosis coincides directly with LV remodeling and heart failure.
Cardiomyocyte apoptosis in chronically failing hearts Within animal models of chronic and progressive LV dysfunction and heart failure, cardiomyocyte apoptosis occurs at an increased rate above that observed in sham or control animals. In a rat pressure overload model, Candorelli et al. [20] demonstrated evidence of increased cardiomyocyte apoptosis following the transition from compensated hypertrophy to LV dysfunction. In a mouse MI model, Sam et al. noted a gradual increase in cardiomyocyte apoptosis defined both by TUNEL staining and caspase 3 activity within the non-infarcted myocardium over a span of 6 months that was associated with progressive LV dilation and heart failure [21]. Both of these representative experimental studies support 48
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that progressive cardiomyocyte cell loss contributes to progressive LV dysfunction. Moreover, multiple transgenic models of dilated cardiomyopathy are characterized by increased rates of cardiomyocyte apoptosis. For example, cardiac-specific overexpression of the alpha subunit of the G protein, Gq (Gaq) (GenBank accession no. NM_002067), leads to the development of compensated LV hypertrophy followed by progressive LV dysfunction, associated with a significant increase in cardiomyocyte apoptosis [22]. Similarly, mice with a cardiac-targeted overexpression of the cytokine, tumor necrosis factor alpha (GenBank accession no. NM_000594), develop increased cardiomyocyte apoptosis, significant LV dysfunction, heart failure and premature death [23,24]. These transgenic models represent a cross-section of many studies linking cardiomyocyte apoptosis to dilated cardiomyopathy and progressive LV dysfunction. Wencker et al. [25] recently developed a transgenic mouse with cardiac-specific expression of an artificially activated procaspase 8 (GenBank accession no. NM_001228) construct. Activation of procaspase 8 in these mice dose-dependently resulted in accelerated cardiomyocyte apoptosis, leading to severe LV dysfunction and death. One of the transgenic lines demonstrated low-level caspase 8-activation coupled with low-level but ongoing cardiomyocyte apoptosis at a rate tenfold higher than wild type mice, and a comparable to that observed in failing human hearts. These animals developed progressive LV dysfunction and premature death supporting further that cardiomyocyte apoptosis contributes to heart failure progression. Several clinical studies have demonstrated evidence of cardiomyocyte apoptosis in chronically failing human hearts [26,27]. In cardiac biopsy samples, Saraste et al. observed increased rates of cardiomyocyte apoptosis in the myocardium of patients with dilated cardiomyopathy and heart failure. In this study, the percent of apoptotic cardiomyocytes correlated with the rapidity of heart failure progression [28]. Narula et al. also reported increased TUNEL-positive cardiomyocytes in failing human myocardium, and demonstrated increased levels of cytoplasmic cytochrome C (GenBank accession no. NM_018947) and activated caspase-3 (GenBank accession no. NM_004346) as compared to normal controls [29]. We recently found that mechanical unloading with ventricular assist device therapy attenuated cardiomyocyte apoptosis in failing human hearts [30]. These clinical data support further that cardiomyocyte apoptosis is present at significantly increased rates in chronically failing hearts and that loss of cardiac myocytes in this manner could contribute to heart failure progression.
Apoptosis signaling pathways Apoptosis occurs following the activation of two general pathways: (1) the extrinsic pathway initiated through agonist binding to ‘‘death receptors;’’ and (2) the intrinsic or mitochondrial
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Figure 2. Apoptosis signaling pathways. Two major pathways converge on caspase 3, triggering apoptosis. Activation of the extrinsic pathway occurs by activation of the death receptor, Fas, by Fas ligand (FasL) causes cleavage and activation of procaspase 8 to caspase 8 which cleaves the pro-apoptotic protein, BID, that contributes to mitochondrial disruption. Caspase 8 also activates caspase 3 which then cleaves target proteins and activates other downstream caspases. The intrinsic or mitochondrial pathway is activated by stimuli such as hypoxia, reactive oxygen species, or sudden increases in intracellular calcium causing disruption of the mitochondrial membrane via activation of pro-apoptotic family members such as Bax and Bid leading to the formation of the mitochondrial permeability transition pore. This results in cytoplasmic leakage of inter-membrane contents including the pro-apoptotic factor, cytochrome C (Cyt C) that binds and activates apoptosis protease activating factor-1 (apaf-1) causing sequential activation of caspases 9 and 3. Pro-survival Bcl-2 family members including Bcl-2 and Bcl-xL protect the cell from apoptosis by inhibiting mitochondrial pore formation.
pathway activated by reactive oxygen species, hypoxia and sudden increases in intracellular Ca2+ that cause a loss of integrity of the outer mitochondrial membrane leading to the release of pro-apoptotic initiating factors [4,31] (Fig. 2). Extrinsic pathway
Apoptosis can be initiated through activation of death receptors (e.g. Fas (GenBank accession no. NM_000043)) by ‘‘deathinducing’’ ligands (e.g. Fas ligand (GenBank accession no. NM_000639)) that lead to the recruitment of death domaincontaining adaptor proteins such as FADD (Fas-associated via Death Domain (GenBank accession no. NM_003824)) and the formation of a membrane-associated, death-inducing signaling complex (DISC). FADD then recruits procaspase 8 into the complex in the form of a dimer, leading to its cleavage and activation; caspase 8 then cleaves and activates procaspase 3 to its active form, caspase 3 [27] (Fig. 1). Caspase 8 also activates the pro-apoptotic Bcl-2 family member, Bid (GenBank accession no. NM_001196) (see below), after which the C-terminal
portion of Bid (tBid) translocates to the mitochondria and inserts into the outer mitochondrial membrane, facilitating the formation of the mitochondrial permeability transition pore (MPTP). This leads to the release of cytochrome C and other pro-apoptotic factors into the cytoplasm. In this manner, the extrinsic pathway is mechanistically linked to the intrinsic or mitochondrial pathway. Intrinsic pathway
The intrinsic pathway of programmed cell death [4] is triggered by the accumulation of reactive oxygen species, sudden increases in intracellular calcium, or hypoxic insults [32]. Following these stimuli, the outer mitochondrial membrane becomes disrupted via mechanisms that are not entirely understood, although mitochondrial localization of proapoptotic Bcl-2 family members leading ultimately to the formation of the MPTP appears crucial in initiating this process. Upon outer mitochondrial membrane disruption, pro-apoptotic factors are released from the inter-membrane www.drugdiscoverytoday.com
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space into the cytosol. The heme-containing electron transport protein, cytochrome C, is one pro-apoptotic factor released from the inter-membrane space. Once in the cytoplasm, cytochrome C binds to and induces the oligomerization of Apaf-1 (GenBank accession no. NM_001160) (apoptosis protease activating factor), which then recruits procaspase 9 (GenBank accession no. NM_001229) forming the complex known as the apoptosome. Upon recruitment to the apoptosome, procaspase 9 is cleaved and activated and then binds and cleaves procaspase 3, resulting in caspase 3 activation. Caspase 3 is an important terminal effecter of apoptosis, cleaving substrate proteins and activating other downstream caspases within the cell (Fig. 2). Bcl-2 protein family
The Bcl-2 family of apoptosis-related proteins is evolutionarily conserved, consisting of both pro-survival and pro-apoptotic factors [14,22,32]. The pro-survival members of the Bcl-2 family, Bcl-2 (GenBank accession no. NM_000633) and Bcl-xl (GenBank accession no. NM_001191), inhibit apoptosis, in part, by maintaining mitochondrial membrane integrity and preventing the release of pro-apoptotic factors from the intermembrane space (e.g. cytochrome C). The anti-apoptotic Bcl2 proteins have been shown to inhibit the pro-apoptotic members via direct protein–protein interactions. Pro-apoptotic proteins within the Bcl-2 family, such as Bad (GenBank
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accession no. NM_004322), comprise a Bcl-2 homology (BH3) domain that induces hetero-dimerization with Bcl-2 and Bclxl, thereby blocking their anti-apoptotic effects. Other BH3 containing Bcl-2-family members such as Bid and Bax (GenBank accession no. NM_004324) exert their pro-apoptotic effects by inducing the formation of the mitochondrial permeability transition pore. Although the precise mechanisms are not clear, activation of pro-apoptotic, Bid and Bax, might themselves form a protein-permeable pore in the outer mitochondrial membrane.
The case for modulating cardiomyocyte apoptosis in acute ischemic syndromes and heart failure Numerous experimental studies have demonstrated that inhibiting cardiomyocyte apoptosis leads to improved functional recovery of the myocardium in acute ischemic syndromes and in chronically failing hearts. Inhibition of apoptosis has been achieved using both pharmacologic and molecular approaches that either activate pro-survival pathways or inhibit pro-apoptotic pathways (also see Table 1).
Pharmacologic strategies Several groups have recently studied the benefit of pharmacological inhibition of cardiomyocyte apoptosis in animal models of heart failure and myocardial infarction. The broad caspase inhibitor IDN1965 has been reported to prevent
Table 1. Targets and related therapies Apoptosis target
Therapeutic agent a
Caspases
IDN-1965
Caspases
Z-Asp 2,6-DCBMkb c
p38 MAPK e
d
RWJ-67657 f
Lab(s) working on this
Refs
Kitsis Lab Dorn Lab
[25,33] [33]
Anand Lab
[34]
Krum Lab
[35]
JNK (MAP kinase family member)
AS601245
Ferrandi Lab
[36]
Fas/Fas ligand
Mice lacking functional Fas receptor or Fas ligand Use of soluble Fas receptor by adenoviral gene transfer
Kitsis Lab Fujiwara Lab
[38] [39]
Caspase 9
Dominant negative construct in transgenic mice
Kitsis Lab
[40]
BCl2
Cardiac overexpression in transgenic mice Adenoviral-mediated gene transfer induced overexpression
Kirshenbaum Lab Gardner Lab Capetanaki Lab
[41] [42] [43]
ARCg
ARC-TAT peptide construct. The TAT moiety facilitates protein diffusion across cell membranes
Gottlieb Lab
[44]
Akt (serine-threonine kinase)
Overexpression strategies of constitutively active construct Hormone manipulation/Akt activation
Walsh Lab Rosensweig Lab Patten Lab
[48] [49] [31]
Overexpression strategies of constitutively active construct
Molkentin Lab
[50]
ERKh 1/2 (MAP kinase family member) a
IDN-1965: broad caspase inhibitor (N-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; Idun Pharmaceuticals). Z-Asp 2,6-DCBMk: broad caspase inhibitor (Alexis pharmaceuticals). MAPK: mitogen-activated protein kinase. d RWJ-67657 (Johnson & Johnson Pharmaceutical Research and Development). e JNK: Jun-N-terminal kinase (also known as stress-activated protein kinases or SAPK). f AS601245: 1,3-benzothiazol-2-yl(2-{[2-(3-pyridinyl)ethyl]amino}-4-pyrimidinyl)acetonitrile: Serono Pharmaceutical Research Institute, Geneva, Switzerland. g ARC: apoptosis repressor with caspase recruitment domain. h ERK: extracellular signal-regulated kinases 1 and 2 (p42/44). b c
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apoptosis, cardiac remodeling and death in transgenic mice with cardiac-specific expression of activated caspase 8 [25], and in transgenic mice with cardiac-targeted overexpression of the alpha subunit of Gq [33]. In the rat ischemia/reperfusion model, several studies have demonstrated that pharmacologic inhibition of caspases with peptide inhibitors reduces infarct size [12,13]. In the chronic rat MI model, Chandrashekhar et al. [34] administered a cellpermeable, broad caspase inhibitor, Z-Asp 2,6-DCBMk, immediately following left coronary ligation. At 4 weeks, treatment with the caspase inhibitor markedly reduced TUNEL-positive cardiomyocytes in the surviving myocardium, associated with improved LV function, and reduced LV hypertrophy and dilation, although infarct size and survival were not affected. Pharmacologic inhibitors of both p38 MAPK and Jun-N-terminal kinases (JNKs) are currently under development and have been shown in animal models to limit myocardial injury in response to ischemia-reperfusion [35,36]. Because of the observed gender differences in both heart failure survival and cardiomyocyte apoptosis in normal and failing hearts, we explored the effects of the hormone, 17betaestradiol (the main circulating form of estrogen) on infarct size and remodeling in the mouse model of MI. Physiologic estrogen replacement resulted in a modest but significant reduction in infarct size compared to placebo-treated females and was associated with less cardiomyocyte apoptosis and caspase 3 activation in the peri-infarct zone [31,37], the mechanism of which appears, in part, due to estrogen receptor-mediated activation of the PI3 kinase (GenBank accession no. NM_006218)-Akt signaling pathway.
Molecular approaches Numerous molecular targets have been examined using dominant negative, knockout, or overexpression transgenic strategies to inhibit apoptosis. For example, a naturally occurring mouse strain that harbors a non-functional death receptor, Fas, exhibits more than 60% reduction in infarct size compared to wild type mice following ischemia-reperfusion injury [38]; coincident with this reduction in infarct size was a nearly equivalent relative reduction in the percentage of apoptotic cardiomyocytes in the area of the myocardium at risk [38]. This same mouse strain deficient in the Fas receptor also develops less heart failure and LV dilatation in response to permanent coronary occlusion [39]. Thus, inhibiting Fas ligand by the adenoviral-mediated delivery of a construct encoding a soluble Fas receptor may hold promise [39]. These data, therefore, support that activation of the death receptor, Fas, contributes significantly to cardiomyocyte apoptosis following ischemiareperfusion injury, and disruption of this pathway might hold therapeutic value in reducing infarct size. Moreover, mice that express a dominant negative caspase 9 construct within the heart develop smaller areas of myocardial injury and less cardiomyocyte apoptosis when subjected to ischemia-reperfu-
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sion injury [40]. Cardiomyocyte-specific overexpression of the pro-survival protein, Bcl-2, diminishes hypoxia-induced apoptosis in cultured cardiomyocytes, and ischemia/reperfusion injury [41,42]. Moreover, BCl2 overexpressed within the myocardium using replication deficient adenoviral-mediated gene transfer can reduce cardiomyocyte apoptosis and LV dysfunction in the desmin (GenBank accession no. NM_001927) knockout model of dilated cardiomyopathy [43]. Levels of the anti-apoptotic protein, ARC (GenBank accession no. NM_003946) (apoptosis repressor with caspase recruitment domain), can be increased in the heart via intra-coronary infusion of an ARC-TAT peptide construct. The TAT-peptide moiety facilitates the transport of ARC across cell membranes [44]. Increasing intracellular ARC in this manner was recently shown to decrease myocardial injury in isolated perfused hearts [44]. Thus, targeting specific proteins within the apoptosis cascade using molecular strategies can reduce both cardiomyocyte apoptotic cell death and infarct size, leading to better preservation of LV function in these rodent models. Other investigators have focused on increasing the activity of pro-survival signaling kinases as a means of promoting cell survival during myocardial infarction or ischemia-reperfusion [45]. It is well established that the serine-threonine kinase, Akt, has many anti-apoptotic effects that include phosphorylating and inhibiting pro-apoptotic proteins such as BAD and caspase 9. Akt (GenBank accession no. NM_001014431) also inhibits the Forkhead transcription factor (GenBank accession no. NM_005429) that activates transcription of Fas ligand, thus decreasing the level of this pro-apoptotic secreted factor. Through phosphorylation of the CREB (GenBank accession no. NM_004379) transcription factor, Akt also increases the expression of the pro-survival protein, Bcl-2 [46,47]. Based on numerous anti-apoptotic and pro-survival downstream effects, it is not surprising that cardiac-targeted expression of a constitutively active form of Akt reduces infarct size following ischemia-reperfusion injury [48,49]. The mitogen-activated protein kinase (MAPK) family of intracellular, serine-threonine kinases includes three major groups: the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (GenBank accession nos. NM_002746 and NM_002745), p38 MAPK (GenBank accession no. NM_001315) and Jun-N-terminal kinases (GenBank accession no. NM_002750). The ERK subgroup of signaling kinases has been shown to exert predominantly pro-survival downstream effects, whereas the p38 and JNK cascades are primarily proapoptotic when activated [45]. Overexpression of the kinase upstream to ERK1/2 (denoted MEK1) (GenBank accession no. NM_002755) within the myocardium confers resistance to apoptotic stimuli [50]. Alternatively, disrupting pro-apoptotic signaling kinases has also been shown to reduce cardiomyocyte cell loss in animal models. For example, mice that harbor a cardiac-targeted, dominant negative p38 MAPK construct demonstrate improved LV function following ischemia-reperwww.drugdiscoverytoday.com
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fusion injury. Moreover, mice that harbor a disrupted Jun Nterminal kinase (JNK1) gene also show resistance to myocardial injury in ischemia-reperfusion models.
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Despite modern medical therapy, heart failure continues to be an important public health problem causing significant morbidity and mortality. There is now increasing data supporting that apoptosis contributes to cardiomyocyte cell loss during acute ischemic injury and in chronic heart failure, and that cardiomyocyte apoptosis contributes importantly to heart failure progression. In fact, current data in vitro animal heart failure and ischemic models support that inhibition of apoptosis can reduce myocardial damage, cardiac dysfunction, and perhaps increase survival. Therapies directed at preventing apoptosis within the heart have, therefore, emerged as attractive avenues of investigation. Multiple approaches have been employed in these animal models to reduce cardiomyocyte apoptosis and improve LV function following acute or chronic myocardial injury. Molecular and pharmacologic approaches to enhance activation of pro-survival signaling cascades (such as Akt and ERK1/2) or those aimed at inhibiting the pro-apoptotic molecules (caspases) or signaling cascades (e.g. p38 MAPK or JNK inhibitors) certainly hold promise. The primary challenge of these therapies is one of cell type or tissue specificity. Indeed, promoting cell survival broadly could potentially increase the risk of uncontrolled cell growth/proliferation in non-cardiac tissues where it is not desired such as early, unidentified malignancies. In the case of acute myocardial injury, a short duration of anti-apoptotic therapy might pose minimal risk of facilitating the growth of malignant cells. However, the long-term treatment of patients with heart failure and LV dysfunction poses increased risks of undesired effects in non-target tissues. Successful therapies will require strategies to target specific populations of cells.
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