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The basic biology of apoptosis and its implications for cardiac function and viability
The Basic Biology of Apoptosis and Its Implications for Cardiac Function and Viability Guro Valen, MD, PhD Crafoord Laboratory of Experimental Surgery, Karolinska Hospital, Stockholm, Sweden
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Apoptosis or programed cell death is a continuous process of destruction of nonfunctional cells. It is a physiologic process whereby the body disposes of unwanted cells by self-destruction and is our utmost defense against damaged cells. There are several pathways leading to programed cell death. Apoptosis is seen in failing, infarcted, and hibernating human hearts, and during
open heart surgery. Apoptosis appears to be induced by myocardial ischemia-reperfusion injury and this is reduced by ischemic preconditioning. Antiapoptotic interventions may be a future target for myocardial protection. (Ann Thorac Surg 2003;75:S656 – 60) © 2003 by The Society of Thoracic Surgeons
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pathways may lead to apoptosis as demonstrated in Figure 1. It may be triggered by ligand binding to specific death receptors on the cell membrane, which will recruit adaptor proteins, leading to activation of the cystein protease (caspase) cascades, ending with cell death. In more detail, the tumor necrosis factor (TNF) family of death receptors comprises TNF receptor 1 (TNF-R1), Fas, death receptors DR 3, 4 (TRAIL R1), and DR5 (TRAIL R2). Ligand binding to receptors recruits adaptor proteins (FADD to Fas, TRAD to TNF-R1, RIP to both), which then converts caspase 8 from a proenzyme to an active form [2, 5]. This triggers a caspase cascade, ending with the activation of effector caspases 3, 6, 7. Effector caspases will induce proteins necessary for cell cleavage, such as nuclear laminins. The induction of apoptosis through death receptors is rapid and occurs within a few hours of ligand binding, does not require RNA or protein synthesis, and may be independent of mitochondria [2, 5]. Another main pathway of apoptosis in myocytes is by stress-induced activation of specific intracellular proteins, where especially the Bcl-2 family of proteins plays an important role. Stress signaling may be through G proteins and protein kinases, where mitogen activated protein kinases are important [2, 3, 6]. The Bcl-2 family consists of proapoptotic members such as Bax, Bad, and Bik, and antiapoptotic members Bcl-2 and Bcl-XL. The antiapoptotic Bcl-2 family members stabilize the mitochondrial membrane, while the proapoptotic members permeabilize it and induce release of cytochrome C. Released cytochrome C in the presence of dATP forms an activation complex with apoptotic protein activating factor-1 (Apaf-1) and caspase 9, activating downstream cascades such as caspase 3 [2, 3, 6]. A third mitochondriadependent but caspase-independent pathway of apoptosis has recently been discovered [4]. Apoptosis-inducing factor (AIF) is a mitochondria located protein colocalized with heat shock protein 60. On induction of apoptosis by namely c-myc or staurosporin, AIF translocates to the nucleus where it will cause chromatin condensation and
poptosis (Greek, meaning leaves or flowers falling off trees) or programed cell death is the physiologic process whereby the body disposes of unwanted cells by self-destruction. The biological importance of apoptosis is easy to grasp in embryology, where it is an important feature of remodeling and tissue and organ formation. In mature organisms apoptosis occurs in several renewing cell types such as intestinal epithelium and leukocytes where it maintains a balance between cell replication and cell death. Apoptosis is our utmost defense against damaged cells by a continuous process of destruction of nonfunctional cells. Thus failure of apoptosis would result in cancer or autoimmunity, whereas increased apoptosis may lead to degenerative processes [1–9]. As a general rule, necrosis and apoptosis are distinct mechanisms of cell death, whereby necrosis is “dirty death” and apoptosis is “clean death.” Ischemic necrosis is characterized by adenosine triphosphate (ATP) depletion, cell swelling, and loss of cell membrane integrity, leading to loss of cell content to adjacent cells, thereby initiating an inflammatory process. Apoptosis on the other hand usually requires energy and is associated with cell shrinkage and phagocytocis without loss of membrane integrity, sparing the adjacent tissue from inflammation. However, this dogma has recently been challenged. Several proinflammatory genes are activated during the apoptotic process. In complex tissue such as in atherosclerotic plaques, phagocytocis of apoptotic cells may be inefficient. Moreover, apoptotic cells may undergo a secondary necrosis under some circumstances [1– 8]. A hallmark of apoptosis is fragmentation of DNA into units of about 200 base pairs. Three distinct cellular
Presented at the 3rd International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, June 2– 6, 2002. Address reprint requests to Dr Valen, Crafoord Laboratory L6:00, Karolinska Hospital, 17176 Stockholm, Sweden; e-mail: guro.valen@ cmm.ki.se.
© 2003 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
0003-4975/03/$30.00 PII S0003-4975(02)04687-8
Fig 1. A schematic presentation of the intracellular pathways of apoptosis. Apoptosis can be initiated by stimulation of the membranebound tumor necrosis factor (TNF) family of death receptors and by stress-induced intracellular activation of the Bcl-2 family causing release of cytochrome C from the mitochondria, both leading to activation of caspase cascades. A third pathway, which is caspase independent, is stress-induced release of apoptosis-inducing factor from the mitochondia. See text for details.
large-scale DNA fragmentation. AIF translocation is inhibited by Bcl-2 [4]. In general apoptosis proceeds in two separate phases, the decision phase and the execution phase. In the decision phase integration of proapoptotic and antiapoptotic signals from the cell surface and within the cell takes place. If the balance favors apoptosis, the execution phase is triggered by the activation of the caspase cascades (or nuclear translocation of AIF).
Apoptosis Induction With Special Reference to Cardiac Surgery The signals promoting and inhibiting apoptosis may vary between cell type, and the same signal may have opposing effects [1–3]. For instance nitric oxide may stimulate apoptosis through several routes including upregulation of the proapoptotic transcription factor p53 and downregulation of Bcl-2, while it may inhibit apoptosis through upregulation of nuclear factor kappa B, which induces transcription of the inducible antiapoptotic protein-1, or by inhibiting cleavage of caspase 8 [2, 9]. Based on experimental studies in vitro, in nonhuman (or nonadult) cardiomyocytes, and in extremely controlled experimental situations, stimuli inducing cardiac apoptosis that may be relevant to open heart surgery are thought to be oxidative stress (oxygen free radicals), TNF␣, nitric oxide specially through inducible nitric oxide synthase with peroxynitrite formation, neurohormonal factors such as angiotensin II, and mechanical stress [3, 6, 8, 9]. Cardiopulmonary bypass (CPB) induces a “whole body inflammation” with generation of oxygen free radicals, cytokine release, and altered nitric oxide release due to
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the bloods contact with foreign surfaces [10 –12]. Possibly CPB per se may induce apoptosis, as patients on CPB have a serum increase of Fas and the the Fas ligand [13, 14] whereas serum from patients on CPB induces apoptosis in cultured endothelial cells [15]. This induction is aggravated by deep hypothermic arrest [16]. However, the role of temperature for apoptosis may be dual; a recent gene-array based study suggests that moderate hypothermia may reduce myocardial apoptosis as response to ischemia through modification of several genes in the signaling pathways [17]. CPB-induced apoptosis may be due to a tissue- or cell-specific source; for instance, in pigs on CPB the duodenum appeared more suspeptible to apoptosis than other tissues [16] whereas CPB appears to actually reduce the occurrence of apoptosis in neutrophil granulocytes [18]. The cardioplegicreperfused heart is also a possible source of circulating apoptosis markers, where a specific nuclear factor kappa B-driven inflammatory response partially triggered by ischemia-reperfusion (oxidative stress), mechanical stress, or the hypothermia of the cardioplegic solution is initiated [10 –12, 19].
Detection of Apoptosis A complete review of all methods of apoptosis detection is not included in the present review but rather the most usually employed methods and some limitations of their use. A summary of the apoptosis events is that signals will lead to changes of the mitochondrial membrane permeability, inducing release of soluble mitochondrial proteins. These may directly or indirectly translocate to the nuclei, inducing gene programs leading to DNA fragmentation, chromatin condensation, and ultimately cell death [2– 8]. Changes in the Bcl-2 family of proteins may be the first indicators that apoptosis is induced and are usually detected by immunostaining or immunoblotting. It should be kept in mind that it is not neccessarily an increased amount of protein that is of interest but rather whether there is a subcellular translocation of protein localization, ie, from the cytosol to the mitochondria. Thus extraction of subcellular protein fractions may be preferable to total protein extraction as usually employed for immunoblotting. Other possible targets in this context is the tumor supressor gene p53 but its role for apoptosis in ischemia-reperfusion injury of the heart is uncertain. Permeabilization of the outer mitochondrial membrane can be detected by measuring the mitochondrial membrane potential delta psi [20]. The mitochondrial KATP channel may be involved in apoptosis but whether this is directly or indirectly due to its role in apoptosis-limiting preconditioning is uncertain [20]. Proteins leaked out from the permeabilized mitochondrial membrane include cytochrome C, where again detection of translocated protein is more important than the total cellular protein content. In the cytosol activated as well as nonactivated components in of the caspase cascades, as well as cleavage products of their proteolytic activity such as poly (ADP) ribose, are good targets indicating an ongoing process of apoptosis. The other mitochondrial
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leakage enzyme, AIF, is potentially of great interest for cardiac apoptosis but so far we do not know whether this is an important pathway and there is currently no commercially available kit or antibody. Nuclear translocation of early immediate genes may be a part of an apoptotic signal transduction pathway through mitogen activated protein kinases but may also be unspecific for other intracellular events. Finally, the consequences of apoptosis (chromatin condensation, cell shinkeage, DNA fragmentation) may be employed as indicators. The gold standard of apoptosis is electron microscopy by which morphologic changes can be visualized. However, electron microscopy is a time-consuming technique not available to all laboratories. DNA fragmentation may be a good end-point and can be measured by electrophoresis where fragmented DNA shows up as “ladders.” Another alternative is in situ terminal deoxynucleotidyl transferase-labeled dUTP nick end labeling or TUNEL assay, which will stain fragmented DNA strands in situ and is a quantifiable method. A disadvantage with the TUNEL assay is that is overestimates apoptotic nuclei, as it labels not only fragmented DNA but also RNA or DNA in the process of repair as well as some cells undergoing necrosis [21]. Usually several end-points in different stages of the apoptotic process should be addressed to demonstrate that apoptosis indeed is taking place [1– 8, 20, 21].
Cardiac Apoptosis and Functional Implications
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In the human heart increased apoptosis is seen in clinical conditions such as myocardial infarction, heart failure, and hibernation [3, 22, 23]. Evidence suggests that apoptosis is induced in patients during open heart surgery; in the human cardioplegic/reperfused heart, early immediate genes leading to apoptosis are turned on, cytochrome C is released, DNA fragmentation is present, and chromatin condensation can be seen in the electron microscope [24, 25]. Furthermore soluble Fas and the Fas ligand are increased in patients on cardiopulmonary bypass [13, 14] and apoptosis may contribute to postbypass renal insufficiency [26] as well as to cerebral dysfunction [27]. In cardiomyocytes it appears that apoptosis is induced by reperfusion rather than by ischemia [28, 29]. In animal experiments apoptosis is induced by ischemia-reperfusion injury in parallel with necrosis in all species investigated, including in experimental models mimicking open heart surgery, regionally ischemic hearts in vivo, and isolated regionally or globally ischemic hearts [29 –34]. Apoptosis is a feature of the infarcted myocardium, in which fragmented DNA is frequently found in the area at risk in the infarct border zones. Apoptosis may function to terminate the tissue repair process and may contribute to increasing the bulk of necrotic cells, although this issue is still controversial [22]. Mice with cardiospecific caspase 3 overexpression have increased apoptosis, increased infarctions, and deteriorated heart function when subjected to induced ischemia [35]. Transgenic mice with cardiospecific Bcl-2 overexpression similarily have reduced infarctions as
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well as improved cardiac function when ischemia is induced [36]. Inhibition of apoptosis may protect against ischemiareperfusion injury also in models of stunning and may influence the contractile apparatus per se. In support of this are recent findings where unspecific caspase inhibition (but not caspase 3 or 9 inhibition) [34] or inhibition of caspase 3 improved the contractile recovery of the stunned myocardium [37]. In humans on CPB there appears to be a correlation between degree of apoptosis and loss of cardiac function [25], while in transplanation models apoptosis inhibition in the absence of necrosis is associated with loss of function [38]. However, blockage of apoptosis with an angiotensin receptor II inhibitor in the working rat heart with global ischemia did not influence functional performance during reperfusion [32]. Thus more studies are required to address whether inhibition of apoptosis has direct effects on myocardial contractility.
Apoptosis and Ischemic Preconditioning Brief episodes of ischemia and reperfusion adapt the heart to tolerate a sustained ischemic event. This phenomenon, which is termed ischemic preconditioning, was described first by Murry and associates [39] as an infarct-limiting intervention. Myocardial protection through preconditioning appears to apply to all species investigated including man. Preconditioning may also protect against myocardial stunning although this is a controversial issue as functional protection by preconditioning may be secondary reduced to myocardial necrosis. It has become apparent that preconditioning can be achieved by a range of different stimuli varying from local ischemia and reperfusion in the heart, systemic stimuli such as hypoxia, hyperoxia, or LPS, or ischemia directed toward other organs such as intestine or brain [40 – 42]. The different models may offer immediate or delayed (24 to 72 hours later) protection and probably act through separate signal transduction pathways. The last few years has supplied evidence that reduction of apoptosis is an important feature of the preconditioning response. In experimental models of stunning, heart function improvement as well as apoptosis reduction can be achieved by various preconditioning models [43]. The mechanisms underlying this are not yet fully clarifed but in classic or other immediate models of cardiac preconditioning upregulation of antiapoptotic proteins such as Bcl-2 and survivin and reduction of proapoptotic proteins such as Bax and p53 are involved [29, 44, 45]. Possibly the preconditioning model is important for the signaling pathway of apoptosis. In our laboratory a model of delayed, remote preconditioning targeting the hindlimbs reduced cardiac apoptosis through a caspase 3 dependent pathway without influencing Bcl-2. In rat myocyte culture studies, antiapoptotic signaling by hypoxia involves the opoid delta receptor, the KATP channel, and protein kinase C delta [46]. Overexpression of heat shock proteins of 10 and 60 kDA families, which may be tissue mediators of preconditioning, reduced DNA fragmenta-
tion, cytochrome C release, and caspase 3 activation induced by simulated ischemia and reoxygenation [47].
Summary and Future Directions of Research Apoptosis is induced in the human heart in conditions such as heart failure, myocardial infarction, and hibernation and is induced during open heart surgery where it may contribute to cardiac ischemia-reperfusion injury as well as to postbypass renal failure and cerebral insufficiency. In experimental studies apoptosis is induced during ischemia reperfusion injury in the border zones of myocardial infarction in all species investigated. Apoptosis may potentially be beneficial or detrimental; good in that it is a clean death without causing inflammation to adjacent tissue, bad in that it leads to loss of cells. In experimental studies inhibition of apoptosis contributes to reducing infarct size. Ischemic preconditioning not only protects against cell necrosis but also an antiapoptotic component of protection has become apparent the last years and this may be an important feature of endogenous protection. The functional consequences of apoptosis are currently not fully determined. Furthermore in cardiac surgery with cardiopulmonary bypass or organ preservation for transplantation, hypothermia is a stimulus that may both inhibit and enhance apoptosis. From a clinical point of view, a thorough definition of the temperature limits at which apoptosis is promoted or inhibited should be defined. Cardiopulmonary bypass is accompanied by systemic indices of apoptosis. It is not fully clarified whether activated blood components induces apoptosis in particularly susceptible organs, and if so which, or if a systemic apoptosis of the vasculature and circulating blood cells is the main source. Apoptosis reduction appears to be a goal to reduce the cardiac response to ischemia reperfusion and the systemic inflammatory response to cardiopulmonary bypass. Future therapeutic targets remain to be defined.
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18. 19. 20. 21. 22. 23.
Doctor Valen is supported by Grants from the Swedish Medical Research Council (12665), the Swedish Heart-Lung Foundation, the Fredrik O Ingrid Thuring Foundation, and the King Gustav V’s and Queen Victoria’s Foundation.
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39. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124 –36. 40. Ta¨ hepoˆ ld P, Valen G, Starkopf J, Karaine C, Zilmer M, Vaage J. Pretreating rats with hyperoxia attenuates ischaemiareperfusion injury of the heart. Life Sci 2001;68:1629 –40. 41. Gho BC, Schoemaker RG, Van den Doel MA, Duncker DJ, Verdouw PD. Myocardial protection by ischemia in noncardiac tissue. Circulation 1996;94:2193–200. 42. Tokuno S, Hinokiyama K, Tokuno K, Lo¨ wbeer C, Valen G. Spontaneous ischemic events in the heart and brain preconditions the hearts of severely atherosclerotic mice. Atheroscleros Thrombos Vasc Biol 2002;22:995–1001. 43. Stadler B, Phillips J, Toyoda Y, Federman M, Levitsky S, McCully JD. Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia-reperfusion. Ann Thorac Surg 2001;72:555–63. 44. Maulik N, Goswami S, Galang N, Das DK. Differential regulation of Bcl-2, AP-1, and NFkB on cardiomyocyte apoptosis during myocardial ischemic stress adaption. FEBS Lett 1999;443:331–6. 45. Zhu L, Fukuda S, Cordis G, Das DK, Maulik N. Antiapoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hepoxic preconditioning. FEBS Lett 2001;508:369. 46. Liu H, Zhang HY, McPherson BC, et al. Role of opoid delta receptors, mitochondrial K(ATP) channels, and protein kinase C during cardiocyte apoptosis. J Mol Cell Cardiol 2001;33:2007–14. 47. Lin KM, Lin B, Lian IY, Mestril R, Scheffer IE, Dillmann WH. Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation. Circulation 2001;103:1787–92.
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Apoptosis or programed cell death is a continuous process of destruction of nonfunctional cells. It is a physiologic process whereby the body disposes of unwanted cells by self-destruction and is our utmost defense against damaged cells. There are several pathways leading to programed cell death. Apoptosis is seen in failing, infarcted, and hibernating human hearts, and during
open heart surgery. Apoptosis appears to be induced by myocardial ischemia-reperfusion injury and this is reduced by ischemic preconditioning. Antiapoptotic interventions may be a future target for myocardial protection. (Ann Thorac Surg 2003;75:S656 – 60) © 2003 by The Society of Thoracic Surgeons
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pathways may lead to apoptosis as demonstrated in Figure 1. It may be triggered by ligand binding to specific death receptors on the cell membrane, which will recruit adaptor proteins, leading to activation of the cystein protease (caspase) cascades, ending with cell death. In more detail, the tumor necrosis factor (TNF) family of death receptors comprises TNF receptor 1 (TNF-R1), Fas, death receptors DR 3, 4 (TRAIL R1), and DR5 (TRAIL R2). Ligand binding to receptors recruits adaptor proteins (FADD to Fas, TRAD to TNF-R1, RIP to both), which then converts caspase 8 from a proenzyme to an active form [2, 5]. This triggers a caspase cascade, ending with the activation of effector caspases 3, 6, 7. Effector caspases will induce proteins necessary for cell cleavage, such as nuclear laminins. The induction of apoptosis through death receptors is rapid and occurs within a few hours of ligand binding, does not require RNA or protein synthesis, and may be independent of mitochondria [2, 5]. Another main pathway of apoptosis in myocytes is by stress-induced activation of specific intracellular proteins, where especially the Bcl-2 family of proteins plays an important role. Stress signaling may be through G proteins and protein kinases, where mitogen activated protein kinases are important [2, 3, 6]. The Bcl-2 family consists of proapoptotic members such as Bax, Bad, and Bik, and antiapoptotic members Bcl-2 and Bcl-XL. The antiapoptotic Bcl-2 family members stabilize the mitochondrial membrane, while the proapoptotic members permeabilize it and induce release of cytochrome C. Released cytochrome C in the presence of dATP forms an activation complex with apoptotic protein activating factor-1 (Apaf-1) and caspase 9, activating downstream cascades such as caspase 3 [2, 3, 6]. A third mitochondriadependent but caspase-independent pathway of apoptosis has recently been discovered [4]. Apoptosis-inducing factor (AIF) is a mitochondria located protein colocalized with heat shock protein 60. On induction of apoptosis by namely c-myc or staurosporin, AIF translocates to the nucleus where it will cause chromatin condensation and
poptosis (Greek, meaning leaves or flowers falling off trees) or programed cell death is the physiologic process whereby the body disposes of unwanted cells by self-destruction. The biological importance of apoptosis is easy to grasp in embryology, where it is an important feature of remodeling and tissue and organ formation. In mature organisms apoptosis occurs in several renewing cell types such as intestinal epithelium and leukocytes where it maintains a balance between cell replication and cell death. Apoptosis is our utmost defense against damaged cells by a continuous process of destruction of nonfunctional cells. Thus failure of apoptosis would result in cancer or autoimmunity, whereas increased apoptosis may lead to degenerative processes [1–9]. As a general rule, necrosis and apoptosis are distinct mechanisms of cell death, whereby necrosis is “dirty death” and apoptosis is “clean death.” Ischemic necrosis is characterized by adenosine triphosphate (ATP) depletion, cell swelling, and loss of cell membrane integrity, leading to loss of cell content to adjacent cells, thereby initiating an inflammatory process. Apoptosis on the other hand usually requires energy and is associated with cell shrinkage and phagocytocis without loss of membrane integrity, sparing the adjacent tissue from inflammation. However, this dogma has recently been challenged. Several proinflammatory genes are activated during the apoptotic process. In complex tissue such as in atherosclerotic plaques, phagocytocis of apoptotic cells may be inefficient. Moreover, apoptotic cells may undergo a secondary necrosis under some circumstances [1– 8]. A hallmark of apoptosis is fragmentation of DNA into units of about 200 base pairs. Three distinct cellular
Presented at the 3rd International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, June 2– 6, 2002. Address reprint requests to Dr Valen, Crafoord Laboratory L6:00, Karolinska Hospital, 17176 Stockholm, Sweden; e-mail: guro.valen@ cmm.ki.se.
© 2003 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
0003-4975/03/$30.00 PII S0003-4975(02)04687-8
Fig 1. A schematic presentation of the intracellular pathways of apoptosis. Apoptosis can be initiated by stimulation of the membranebound tumor necrosis factor (TNF) family of death receptors and by stress-induced intracellular activation of the Bcl-2 family causing release of cytochrome C from the mitochondria, both leading to activation of caspase cascades. A third pathway, which is caspase independent, is stress-induced release of apoptosis-inducing factor from the mitochondia. See text for details.
large-scale DNA fragmentation. AIF translocation is inhibited by Bcl-2 [4]. In general apoptosis proceeds in two separate phases, the decision phase and the execution phase. In the decision phase integration of proapoptotic and antiapoptotic signals from the cell surface and within the cell takes place. If the balance favors apoptosis, the execution phase is triggered by the activation of the caspase cascades (or nuclear translocation of AIF).
Apoptosis Induction With Special Reference to Cardiac Surgery The signals promoting and inhibiting apoptosis may vary between cell type, and the same signal may have opposing effects [1–3]. For instance nitric oxide may stimulate apoptosis through several routes including upregulation of the proapoptotic transcription factor p53 and downregulation of Bcl-2, while it may inhibit apoptosis through upregulation of nuclear factor kappa B, which induces transcription of the inducible antiapoptotic protein-1, or by inhibiting cleavage of caspase 8 [2, 9]. Based on experimental studies in vitro, in nonhuman (or nonadult) cardiomyocytes, and in extremely controlled experimental situations, stimuli inducing cardiac apoptosis that may be relevant to open heart surgery are thought to be oxidative stress (oxygen free radicals), TNF␣, nitric oxide specially through inducible nitric oxide synthase with peroxynitrite formation, neurohormonal factors such as angiotensin II, and mechanical stress [3, 6, 8, 9]. Cardiopulmonary bypass (CPB) induces a “whole body inflammation” with generation of oxygen free radicals, cytokine release, and altered nitric oxide release due to
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the bloods contact with foreign surfaces [10 –12]. Possibly CPB per se may induce apoptosis, as patients on CPB have a serum increase of Fas and the the Fas ligand [13, 14] whereas serum from patients on CPB induces apoptosis in cultured endothelial cells [15]. This induction is aggravated by deep hypothermic arrest [16]. However, the role of temperature for apoptosis may be dual; a recent gene-array based study suggests that moderate hypothermia may reduce myocardial apoptosis as response to ischemia through modification of several genes in the signaling pathways [17]. CPB-induced apoptosis may be due to a tissue- or cell-specific source; for instance, in pigs on CPB the duodenum appeared more suspeptible to apoptosis than other tissues [16] whereas CPB appears to actually reduce the occurrence of apoptosis in neutrophil granulocytes [18]. The cardioplegicreperfused heart is also a possible source of circulating apoptosis markers, where a specific nuclear factor kappa B-driven inflammatory response partially triggered by ischemia-reperfusion (oxidative stress), mechanical stress, or the hypothermia of the cardioplegic solution is initiated [10 –12, 19].
Detection of Apoptosis A complete review of all methods of apoptosis detection is not included in the present review but rather the most usually employed methods and some limitations of their use. A summary of the apoptosis events is that signals will lead to changes of the mitochondrial membrane permeability, inducing release of soluble mitochondrial proteins. These may directly or indirectly translocate to the nuclei, inducing gene programs leading to DNA fragmentation, chromatin condensation, and ultimately cell death [2– 8]. Changes in the Bcl-2 family of proteins may be the first indicators that apoptosis is induced and are usually detected by immunostaining or immunoblotting. It should be kept in mind that it is not neccessarily an increased amount of protein that is of interest but rather whether there is a subcellular translocation of protein localization, ie, from the cytosol to the mitochondria. Thus extraction of subcellular protein fractions may be preferable to total protein extraction as usually employed for immunoblotting. Other possible targets in this context is the tumor supressor gene p53 but its role for apoptosis in ischemia-reperfusion injury of the heart is uncertain. Permeabilization of the outer mitochondrial membrane can be detected by measuring the mitochondrial membrane potential delta psi [20]. The mitochondrial KATP channel may be involved in apoptosis but whether this is directly or indirectly due to its role in apoptosis-limiting preconditioning is uncertain [20]. Proteins leaked out from the permeabilized mitochondrial membrane include cytochrome C, where again detection of translocated protein is more important than the total cellular protein content. In the cytosol activated as well as nonactivated components in of the caspase cascades, as well as cleavage products of their proteolytic activity such as poly (ADP) ribose, are good targets indicating an ongoing process of apoptosis. The other mitochondrial
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leakage enzyme, AIF, is potentially of great interest for cardiac apoptosis but so far we do not know whether this is an important pathway and there is currently no commercially available kit or antibody. Nuclear translocation of early immediate genes may be a part of an apoptotic signal transduction pathway through mitogen activated protein kinases but may also be unspecific for other intracellular events. Finally, the consequences of apoptosis (chromatin condensation, cell shinkeage, DNA fragmentation) may be employed as indicators. The gold standard of apoptosis is electron microscopy by which morphologic changes can be visualized. However, electron microscopy is a time-consuming technique not available to all laboratories. DNA fragmentation may be a good end-point and can be measured by electrophoresis where fragmented DNA shows up as “ladders.” Another alternative is in situ terminal deoxynucleotidyl transferase-labeled dUTP nick end labeling or TUNEL assay, which will stain fragmented DNA strands in situ and is a quantifiable method. A disadvantage with the TUNEL assay is that is overestimates apoptotic nuclei, as it labels not only fragmented DNA but also RNA or DNA in the process of repair as well as some cells undergoing necrosis [21]. Usually several end-points in different stages of the apoptotic process should be addressed to demonstrate that apoptosis indeed is taking place [1– 8, 20, 21].
Cardiac Apoptosis and Functional Implications
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In the human heart increased apoptosis is seen in clinical conditions such as myocardial infarction, heart failure, and hibernation [3, 22, 23]. Evidence suggests that apoptosis is induced in patients during open heart surgery; in the human cardioplegic/reperfused heart, early immediate genes leading to apoptosis are turned on, cytochrome C is released, DNA fragmentation is present, and chromatin condensation can be seen in the electron microscope [24, 25]. Furthermore soluble Fas and the Fas ligand are increased in patients on cardiopulmonary bypass [13, 14] and apoptosis may contribute to postbypass renal insufficiency [26] as well as to cerebral dysfunction [27]. In cardiomyocytes it appears that apoptosis is induced by reperfusion rather than by ischemia [28, 29]. In animal experiments apoptosis is induced by ischemia-reperfusion injury in parallel with necrosis in all species investigated, including in experimental models mimicking open heart surgery, regionally ischemic hearts in vivo, and isolated regionally or globally ischemic hearts [29 –34]. Apoptosis is a feature of the infarcted myocardium, in which fragmented DNA is frequently found in the area at risk in the infarct border zones. Apoptosis may function to terminate the tissue repair process and may contribute to increasing the bulk of necrotic cells, although this issue is still controversial [22]. Mice with cardiospecific caspase 3 overexpression have increased apoptosis, increased infarctions, and deteriorated heart function when subjected to induced ischemia [35]. Transgenic mice with cardiospecific Bcl-2 overexpression similarily have reduced infarctions as
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well as improved cardiac function when ischemia is induced [36]. Inhibition of apoptosis may protect against ischemiareperfusion injury also in models of stunning and may influence the contractile apparatus per se. In support of this are recent findings where unspecific caspase inhibition (but not caspase 3 or 9 inhibition) [34] or inhibition of caspase 3 improved the contractile recovery of the stunned myocardium [37]. In humans on CPB there appears to be a correlation between degree of apoptosis and loss of cardiac function [25], while in transplanation models apoptosis inhibition in the absence of necrosis is associated with loss of function [38]. However, blockage of apoptosis with an angiotensin receptor II inhibitor in the working rat heart with global ischemia did not influence functional performance during reperfusion [32]. Thus more studies are required to address whether inhibition of apoptosis has direct effects on myocardial contractility.
Apoptosis and Ischemic Preconditioning Brief episodes of ischemia and reperfusion adapt the heart to tolerate a sustained ischemic event. This phenomenon, which is termed ischemic preconditioning, was described first by Murry and associates [39] as an infarct-limiting intervention. Myocardial protection through preconditioning appears to apply to all species investigated including man. Preconditioning may also protect against myocardial stunning although this is a controversial issue as functional protection by preconditioning may be secondary reduced to myocardial necrosis. It has become apparent that preconditioning can be achieved by a range of different stimuli varying from local ischemia and reperfusion in the heart, systemic stimuli such as hypoxia, hyperoxia, or LPS, or ischemia directed toward other organs such as intestine or brain [40 – 42]. The different models may offer immediate or delayed (24 to 72 hours later) protection and probably act through separate signal transduction pathways. The last few years has supplied evidence that reduction of apoptosis is an important feature of the preconditioning response. In experimental models of stunning, heart function improvement as well as apoptosis reduction can be achieved by various preconditioning models [43]. The mechanisms underlying this are not yet fully clarifed but in classic or other immediate models of cardiac preconditioning upregulation of antiapoptotic proteins such as Bcl-2 and survivin and reduction of proapoptotic proteins such as Bax and p53 are involved [29, 44, 45]. Possibly the preconditioning model is important for the signaling pathway of apoptosis. In our laboratory a model of delayed, remote preconditioning targeting the hindlimbs reduced cardiac apoptosis through a caspase 3 dependent pathway without influencing Bcl-2. In rat myocyte culture studies, antiapoptotic signaling by hypoxia involves the opoid delta receptor, the KATP channel, and protein kinase C delta [46]. Overexpression of heat shock proteins of 10 and 60 kDA families, which may be tissue mediators of preconditioning, reduced DNA fragmenta-
tion, cytochrome C release, and caspase 3 activation induced by simulated ischemia and reoxygenation [47].
Summary and Future Directions of Research Apoptosis is induced in the human heart in conditions such as heart failure, myocardial infarction, and hibernation and is induced during open heart surgery where it may contribute to cardiac ischemia-reperfusion injury as well as to postbypass renal failure and cerebral insufficiency. In experimental studies apoptosis is induced during ischemia reperfusion injury in the border zones of myocardial infarction in all species investigated. Apoptosis may potentially be beneficial or detrimental; good in that it is a clean death without causing inflammation to adjacent tissue, bad in that it leads to loss of cells. In experimental studies inhibition of apoptosis contributes to reducing infarct size. Ischemic preconditioning not only protects against cell necrosis but also an antiapoptotic component of protection has become apparent the last years and this may be an important feature of endogenous protection. The functional consequences of apoptosis are currently not fully determined. Furthermore in cardiac surgery with cardiopulmonary bypass or organ preservation for transplantation, hypothermia is a stimulus that may both inhibit and enhance apoptosis. From a clinical point of view, a thorough definition of the temperature limits at which apoptosis is promoted or inhibited should be defined. Cardiopulmonary bypass is accompanied by systemic indices of apoptosis. It is not fully clarified whether activated blood components induces apoptosis in particularly susceptible organs, and if so which, or if a systemic apoptosis of the vasculature and circulating blood cells is the main source. Apoptosis reduction appears to be a goal to reduce the cardiac response to ischemia reperfusion and the systemic inflammatory response to cardiopulmonary bypass. Future therapeutic targets remain to be defined.
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7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23.
Doctor Valen is supported by Grants from the Swedish Medical Research Council (12665), the Swedish Heart-Lung Foundation, the Fredrik O Ingrid Thuring Foundation, and the King Gustav V’s and Queen Victoria’s Foundation.
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25.
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