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Programmed death as a therapeutic target to reduce myocardial infarction
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Programmed death as a therapeutic target to reduce myocardial infarction Keith A. Webster Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, 1600 NW 10th Avenue, RMSB 6038 Miami, FL 33101, USA
In the United States, angioplasty or bypass surgery to remove coronary occlusions is performed on approximately two million patients each year. Although reperfusion is essential for salvaging ischemic myocardium, it also promotes infarction by activating programmed cell death in the formerly ischemic tissue. Reperfusion injury begins when oxidative stress and calcium accumulation by the mitochondria cause activation of the so-called mitochondrial death channels. These channels have become the focus of evolving strategies to protect the heart from infarction. Preclinical and preliminary clinical studies indicate that agents with diverse modes of action can reduce infarct size by 50% or more and significantly preserve myocardial functions. This article reviews the most advanced pharmacological approaches for their ability to reduce infarct size by inhibiting the mitochondrial death pathways. Introduction Cell death during myocardial infarction (MI) occurs by necrosis and apoptosis, both regulated by stress signals that originate in the mitochondria (reviewed in [1]). Apoptosis is a programmed process that requires energy. It is initiated by the activation of cell-surface receptors (the extrinsic pathway) or by permeability changes of mitochondria (the intrinsic pathway). The Bcl-2 family of proteins plays important roles in the intrinsic pathways by determining the release of cytochrome c and other pro-apoptotic factors from the mitochondria into the cytoplasm, where they initiate death pathways [2,3]. Two channels have been proposed to accommodate this release: the mitochondrial permeability transition pore (mPTP) and the mitochondrial apoptosis channel (mAC, or Bax channel) (reviewed in [4,5]). There is controversy over the properties, structure and regulation of these channels but strong evidence that Bcl-2 proteins contribute to the regulation of both [5,6]. Until recently, apoptosis and necrosis during MI were considered to be mechanistically and physically distinct; the former was thought to be largely a consequence of oxidative damage that affects energy-competent cells and takes place principally during reperfusion, whereas the latter was thought to involve a collapse of ion gradients across the plasma membrane caused by energy depletion during ischemia. These distinctions have now been blurred by demonstrations that death can simultaneously involve Corresponding author: Webster, K.A. ([email protected]). Available online 10 August 2007. www.sciencedirect.com
features of both necrosis and apoptosis and that signals from the mitochondria regulate both pathways [7]. One of the most radical changes in our understanding of programmed death during MI involves the role of the mPTP, a complex that was formerly thought to include at least five proteins [4]. Two of these proteins, the outermembrane voltage-dependent anion channel (VDAC) and the inner-membrane adenine nucleotide translocator (ANT), have essential physiological functions in transporting metabolites across the mitochondrial membranes. Calcium-induced mitochondrial swelling, facilitated by oxidative stress, opens the pore. Normal opening requires a regulatory component, cyclophilin D (CypD) [4]. CypD is a protein isomerase that interacts functionally and possibly physically with the mPTP from the matrix side of the inner membrane (Figure 1). CypD is inhibited by the immunosuppressant agent cyclosporine-A (CyA), and we have known for many years that CyA is powerfully protective against MI [4]. Recently, genetic studies in which the CypD gene was ablated have revealed that the CyA-sensitive mPTP primarily drives a pathway of necrosis – not apoptosis, as was formerly thought [7,8]. CypD / cells respond normally to classical inducers of apoptosis, but mPTP opening requires fivefold more calcium, and the hearts of CypD / mice are resistant to MI. These studies do not eliminate the possibility of dual roles for the mPTP in apoptosis, perhaps in combination with a mAC/Baxchannel-like activity [7]. Previous studies support a role for Bcl-2 proteins in the regulation of the mPTP, and it is widely believed that both necrosis and apoptosis contribute significantly to infarction [7–9]. One possibility that is consistent with most of the available information is that ischemia reperfusion opens both of the mitochondrial death channels and that mPTP opening influences the activity of the mAC/Bax channel. Cells subjected to chronic mPTP opening are destined to die by necrosis irrespective of other channels, but cells in which mPTP reverses before Glossary MI: Myocardial Infarction or heart attack. CABG: Coronary artery bypass grafting. The procedure whereby a section of a blood vessel (usually from the limb) is excised and transplanted into the coronary artery to bypass an occlusion. PTCA: Percutaneous transluminal coronary angioplasty. Percutaneous refers to needle catheter access to a blood vessel via puncture of the skin. Transluminal is a procedure that is performed within the blood vessel. Angioplasty means ‘to reshape’ the blood vessel, usually by inflation of a balloon contained in the catheter. The procedure is used for removing arterial occlusions that result from atherosclerosis.
0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.07.004
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Figure 1. The mitochondrial death channels. Proteins associated with the mPTP include the voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), cyclophilin-D (CypD), the benzodiazepine receptor (BDR) and hexokinase. The mAC has not been defined structurally, but channel activity is regulated by interactions of Bax, Bak and tBid with the outer mitochondrial membrane (OM); Bax might be a structural component of the mAC. The mPTP and mAC (or Bax channel) are both regulated by Bcl-2 proteins and are opened by calcium ionophores and oxidative stress. Some reports suggest that VDAC and ANT bridge the inner (IM) and outer mitochondrial membranes by interacting at contact points. Other reports [10,11] suggest that neither ANT nor VDAC is required for mPTP function (illustrated by a double arrow and a dashed line). The mPTP opens under conditions of ischemia reperfusion when matrix calcium levels increase above a threshold. CypD and ROS lower the threshold. The mitochondrial membrane potential (D c) is lost when the mPTP opens. Cytochrome c release triggers apoptosis; 15% of total cytochrome c is in the intermembrane space (IMS), and the rest is in the intercristal space (ICS). The latter might become available for release during matrix swelling or by incorporation of tBid into the IM. Low matrix pH inhibits the opening of the mPTP when D c is dissipated, but low pH induces mPTP opening in energized mitochondria.
outer-membrane rupture could either avoid necrosis and die by apoptosis or survive. Several studies have shown that the permeability transition is reversible, and up to 50% of the mitochondria that undergo the transition during early reperfusion subsequently close the pore and can recover function [9]. The mAC/Bax channel opens at levels of calcium that are significantly lower than those required for causing and sustaining mPTP opening. Therefore, conditions that favor opening of the mAC/Bax channel might occur in cells that are unloading calcium. Recent gene-ablation studies have also raised questions about essential roles for ANT and VDAC in the mitochondrial permeability transition [10,11]. The regulation and relative contributions of the different death pathways is clearly of great importance in determining the conditions for optimal protection against MI. Bcl-2 proteins are universally viewed as regulators of apoptosis, so it was not anticipated that mPTP opening (previously thought to be regulated by Bcl-2) causes necrosis. From a therapeutic perspective, this distinction is important because apoptosis-blocking agents, such as caspase inhibitors, are not expected to directly block infarction caused by necrosis. However, these results do not exclude apoptosis from important roles in infarction, neither do they refute roles for Bcl-2 proteins in the regulation of the mPTP and necrosis, as well as apoptosis. Preclinical studies reveal an extensive list of interventions and pharmacological agents that reduce infarction when applied at the time of ischemia or reperfusion. In these models, the degrees of protection afforded www.sciencedirect.com
by agents with distinct mechanisms of action are remarkably similar, conferring 50%–70% reduction of infarct size. Such agents include CsA that selectively targets the mPTP [12], pan-caspase inhibitors that selectively block apoptosis [13], and interventions that respectively activate or inhibit pro- and anti-apoptotic Bcl-2 proteins [14]. The equivalent degrees of protection conferred by these interventions reflect the heterogeneous nature of the infarct process and the close interrelationships between the different death pathways. Each intervention targets the mitochondria, either downstream or at the level of the death channels. Some of the leading targets and strategies for blocking programmed cell death and infarction in the heart will be reviewed in the following sections. Indirect targeting of the death channels The patient groups most likely to benefit from infarct protection are those that have severely occluded coronary arteries and are undergoing angioplasty (percutaneous transluminal coronary angioplasty [PTCA]) or coronary artery bypass grafting (CABG) [15]. It is well established that myocardial infarction in these patients continues to develop for extended periods after successful reperfusion [16]. Preclinical studies and early clinical data provide a range of options that could prevent loss of myocardium during and after these procedures. Pharmacological mimics of the procedure known as ischemic preconditioning (IPC) are presently at the most advanced stages of development.
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Ischemic pre- and post-conditioning IPC involves exposing the heart to several short episodes of ischemia reperfusion before prolonged ischemia. It was first reported almost two decades ago and subsequently confirmed by multiple investigators to be one of the most effective cardioprotective strategies ever discovered (reviewed in [17,18]). Intense study over the intervening 20 years has led to a dissection of the mechanisms of IPC, descriptions of three interrelated strategies and the identification of pharmacological mimics that, like the physical procedures, can reduce infarct size by 40%–60% in animal models of MI. Post-ischemic conditioning differs from early and late IPC in that the conditioning stimulus (physical or pharmacological) is applied after the index ischemia, immediately before reperfusion. The discovery of postconditioning is important because the treatment of patients undergoing PTCA or CABG also requires that the intervention be administered after the ischemic episode, at the time of reperfusion [19]. Late IPC refers to protection that appears 24–72 h after the initiation of IPC.
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It is critically dependent on nitric oxide (NO) signaling and involves transcriptional as well as activity changes of both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) (reviewed in [18]). The targets of IPC have not been fully resolved, but there is compelling evidence that one target is the mPTP (Figure 2). Signals from cell-surface receptors are transmitted by protein kinases, including protein kinase C (PKC), protein kinase B (Akt) and the extracellular-signal-regulated kinase (ERK), and converge on the mPTP through a mitochondrial ATP-sensitive potassium channel (mK-ATP) and/or the signaling intermediate glycogen synthase kinase-3b (GSK-3b) [20]. Some of the most promising IPC mimics are already approved to treat other conditions but have not undergone rigorous clinical tests in the setting of MI. These include statins, PDE-5 inhibitors and volatile anesthetics. Statins 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors are widely prescribed for their anti-
Figure 2. Pathways of protection by pharmacological IPC mimics. IPC is initiated by agonists of multiple transmembrane G-protein-coupled receptors (GPCR) that activate PKC and/or PI3 kinase. PKCd and PKCe have been associated with the IPC response; PKCd is pro-apoptotic, whereas e is protective. PKC activity is increased by dual signaling pathways: (1) an early receptor-initiated pathway that generates diacylglycerol by activating phospholipases (PLC/PLD) and (2) ROS generated by the mitochondrial electron transport chain and via the mtK-ATP channel. Mitogen-activated protein kinase pathways, including p38 and ERK1/2, are downstream of GPCR and PKC; p38a promotes apoptosis, and p38b is protective. The late phase of protection involves new protein synthesis and enhanced NO production from iNOS and eNOS. COX, antioxidants, heatshock proteins and PKCe might contribute to late IPC. Several of these components are activated by PDE-5 inhibitors (sildenafil) and interventions that increase NO production. The targets for IPC include mitochondrial death channels, and the signals might be channeled through GSK-3b and the mtK-ATP channel. Akt phosphorylates multiple substrates that can block mPT and the activation of Bax. The lag phase before late IPC involves increased expression of iNOS, HSPs, COX and anti-oxidant genes. www.sciencedirect.com
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hypercholesterolemic and anti-hypertensive actions. These agents have been shown to possess additional therapeutic properties that are independent of their lipid-lowering actions. Statins are potent modulators of eNOS activity; they block the inhibitory actions of oxidized low-density lipoprotein on eNOS expression and stimulate synthesis by selectively stabilizing eNOS mRNA. Statins also activate Akt and thereby increase eNOS activity by direct phosphorylation. Statins have been shown to reduce vascular inflammation and oxidative stress, increase coronary blood flow and reduce myocardial necrosis associated with acute MI [211]. A recent retrospective clinical study involving 1663 patients found that acute statin treatment improved the 30-day outcome and independently reduced the mortality of patients undergoing CABG by 50% [22]. The generally accepted mechanism of cardioprotection by statins is to mimic IPC through NO-mediated activation of mtK-ATP channels and inhibition of the mPTP [23] (Figure 2). These agents are being heralded as one of the most powerful pharmacological strategies available for the treatment of cardiovascular disease. Because of their demonstrated safety and pleiotropic properties, statins administered acutely are strongly recommended for patients undergoing cardiac surgery. However, at the present time it is not clear how the protection conferred by statins compares with that of other IPC mimics. Further studies of statins in large-animal models of MI is warranted, perhaps in combination with other IPC mimics (see below). The proven safety and exceptional therapeutic profiles of statins make them excellent candidates as infarct-sparing agents for patients undergoing CABG or percutaneous transluminal coronary angioplasty (PTCA). Phosphodiesterase type-5 (PDE-5) inhibitors Sildenafil, varenafil and tadalafil are PDE-5 inhibitors that are approved for the treatment of erectile dysfunction (ED) (reviewed in [24]). They work by increasing cyclic guanosine monophosphate and activating protein kinase G (PKG). PKG lowers intracellular calcium by phosphorylating Ca2+-dependent K+ channels, phospholamban and the 1,4,5-inositol triphosphate receptor, thus causing smoothmuscle relaxation (Figure 2). Sildenafil (Viagra) and its derivatives mimic both early and late IPC and reduce infarction in animal models by 70% in the early phase and 40% in the late phase. The dose of Viagra required for mimicking IPC is less than that required for treating ED, and an attractive aspect of these agents is that chronic administration of a low dose might produce sustained preconditioning. Other beneficial effects of PDE-5 inhibitors on cardiovascular disease have also been described [25]. Sildenafil was shown to prevent pressure-overload hypertrophy in rabbits; this effect was attributed to inhibition of ERK, Akt and GSK-3b phosphorylation [26]. The latter property raises questions about the mechanisms of action and targets of PDE-5 inhibitors in the heart. Activation of Akt and ERK and phosphorylation of GSK-3b are central to IPC, and inhibition of these should antagonize IPC. A recent report demonstrated that sildenafil and atorvastatin conferred protection that was additive, implying different mechanisms of action [27]. Statins might complement sildenafil by activating Akt and ERK [28]. www.sciencedirect.com
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These results support the implementation of clinical trials testing combinations of acute sildenafil and statin for patients undergoing PTCA and CABG. Anesthetic-induced preconditioning (APC) Opioids and volatile halogenated anesthetics also mimic both early and late pre- and post-conditioning by stimulating receptors on the cell surface (reviewed in [29]) (Figure 2). The pathway shares the same intermediates and targets as IPC; these include inhibitory G-proteins, PKC, Akt, ERK [30], mK-ATP channels, GSK-3b and the mPTP (reviewed in [31]). An increase of reactive oxygen species (ROS), mediated by a partial inhibition of the mitochondrial electron transport chain at complex I, initiates APC (reviewed in [32]). Preclinical studies demonstrate that the degree of infarct reduction conferred by volatile anesthetics is independent of their hypotensive actions and comparable in magnitude to that reported for IPC (reviewed in [29,33]). Recently, the results of the first multicenter randomized controlled trial of APC were reported. In this trial, 112 patients undergoing off-pump CABG were randomized to receive desflurane or propofol (intravenous anesthetic) [34]. This study reported a 50% reduction in postoperative troponin I (reflecting proportionally less cardiac injury), a reduced number of patients requiring postoperative inotropes and fewer patients requiring prolonged hospitalization in the volatile anesthetic group. These results are consistent with a previous trial of 320 coronary surgery patients that were also randomized to receive intravenous anesthetic regimen or a volatile anesthetic agent maintained throughout surgery [35]. APC is presently the most advanced of the IPC mimics, in that it has been tested in two large trials that have confirmed its safety and efficacy [34,35]. This wealth of positive data from both preclinical and clinical studies supports the implementation of a large-scale multicenter trial to determine the impact of this strategy on cardiac morbidity and mortality. Unresolved questions include whether the protection mediated by APC is maximal, whether the protection is consistent across patient groups with different etiologies and whether combinations with other IPC mimics (statins, PDE-5 inhibitors) will provide additional protection. Novel strategies based on IPC Gene therapy to permanently precondition the hearts of patients at high risk for MI has been pioneered by Bolli et al. (reviewed in [16]). Delivery of iNOS or eNOS genes by direct injection of adenoviral vectors has demonstrated sustained protection from infarction two months after gene therapy, at levels equivalent to IPC and without side effects [36,37]. The next phase in the development of this strategy is to use a more permanent gene-delivery vehicle, such as an adenovirus-associated virus (AAV), in largeanimal models of MI to confirm efficacy and determine the duration of protection. Gene-delivery procedures are invasive and expensive, so a substantial period of protection needs to result (one year or more) for them to be worthwhile. It might be possible to improve the safety profile of gene therapy by using tissue-specific regulated promoters that respond to the conditions of MI (reviewed in [38]). The
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protective value of enhancing NO availability was recently confirmed in experiments where the infusion of sodium nitrite directly into the left ventricle was shown to reduce infarct size by more than 50% [39]. The authors postulate that the hypoxic-acidic environment of the post-ischemic myocardium promotes the reduction of nitrite to NO and triggers an IPC response. Both the gene-therapy and biochemical approaches to enhancing NO bioavailability represent attractive strategies for cardioprotection, and further development of these approaches should be encouraged. An unresolved question for the future of IPC mimics is whether the use of volatile anesthetics during cardiac surgery provides the maximal protection that can be obtained by IPC stimuli. The two most recent trials support this possibility and, if confirmed, will obviate the use of additional mimics, at least in the acute setting [34,35]. The results support the general use of volatile anesthetics for the surgical procedures required for CABG and PTCA. Further preclinical and clinical studies are required for determining whether mimics such as statin and PDE-5 inhibitors should be added to APC. Direct targeting of the death channels Akt is at the crossroads of multiple signaling pathways that collectively regulate cell growth and survival. The targets of Akt include the FOXO transcription factors, caspases, GSK-3b and the pro-apoptotic Bcl-2 family protein BAD [7]. These properties allow Akt to contribute to the regulation of mitochondria-mediated necrosis as well as apoptosis and position it as one of the most important targets to protect the heart. Additionally, as a central regulator of insulin signaling, Akt regulates the activities of glucose-transport proteins and the mTOR complex. A disadvantage of Akt signaling is that the pathway can be desensitized. For example, animal studies indicate that ageing and diabetes reduce the IPC response, and this effect can be associated with mitochondrial dysfunction, insulin resistance and a decreased ability to activate Akt and generate NO [40,41]. Although increasing the IPC stimulus can improve these responses [41], it is possible that age and disease will blunt even an optimized IPC stimulus. If these pathways are indeed defective in diseased and/or aged hearts, a more effective approach will be to target the death pathways more directly. One approach that takes advantage of a target more proximal to the mitochondria involves inhibitory peptides that block the activity of PKC-d. Studies in rodents and pigs have shown that PKC-d is activated during MI and causes the inhibition of Akt and activation of BAD [42,43]. Inhibitory peptides block this effect and reduce infarction. Results of DELTA-MI, a phase I/II clinical trial involving 154 patients randomized (2:1) to receive one of four escalating doses of the PKC-d inhibitory peptide KAI-9803 or a placebo, were released recently (Roe, M.T. et al., presented at the American College of Cardiology Annual Scientific Session, New Orleans, LA, March 2007). The authors reported favorable trends of efficacy with less cardiac creatine kinase release post-angioplasty and smaller infarct size, although these reductions were not significant because of the small sample size. There was no indication www.sciencedirect.com
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that the intracoronary delivery of peptide was a safety hazard. The results support the implementation of a larger trial using intermediate doses of the peptide. An even more direct approach is to target the Bcl-2 proteins themselves. Direct inhibition or genetic ablation of the BH3-only proteins Bid, BAD, PUMA and Bnip3 reduce infarction by 50% or more in animal models, and this result is equivalent to that obtained with most IPC stimuli. BH3-only proteins Bcl-2 proteins regulate programmed death by controlling calcium release from intracellular stores and regulating the mitochondrial death channels. Members of this family have different numbers of a specific peptide sequence, known as a Bcl-2 homology (BH) domain, that is required for oligomerization between self and other Bcl-2 family members. The physical location and activity of each Bcl-2-family protein are largely determined by its binding to other Bcl-2-family proteins and scaffolds [44–46]. Bax and Bak are the principal effector proapoptotic Bcl-2 family members and contain three BH domains (BH1–BH3). Cells lacking these two proteins are resistant to death pathways initiated by the mitochondria [47]. Bax is either loosely attached to the outer mitochondrial membrane or sequestered in the cytosol through interactions with protein chaperones [48]. Activation of Bax involves translocation into the mitochondrial membrane, where it homo-oligomerizes or interacts with other proteins to regulate the permeability of the membranes to apoptogenic proteins [48] (Figure 3). The activation of Bax and Bak is regulated by the third class of Bcl-2 proteins that contain single BH3 domains; these proteins are known as BH3-only proteins. They sense and transmit the death stimulus and represent highly specific targets for inhibiting MI. BID (Bcl-2 Interacting Domain) is one of the most abundant and widespread mammalian BH3-only proteins. It is strongly expressed during development, and remains abundant in many adult tissues, including the heart [49]. In healthy cells, Bid is cytosolic or loosely membrane associated and functionally inert. It is cleaved at the N terminus by caspase 8, granzyme B or calpain to give the active form known as tBid (truncated Bid) [7]. tBid translocates to the mitochondria, where it mobilizes cytochrome c and other apoptogenic proteins and facilitates their release through the outer membrane, possibly by interacting with Bax [50,51]. Bid-mediated apoptosis has been shown to contribute significantly to both neuronal and myocardial ischemic injury [52], and blocking Bid cleavage with a calpain inhibitor has been shown to reduce infarct size by more than 50% in an ex-vivo model [53]. Calpain inhibitors have also been shown to slow the progression of heart failure in rats [54,55]. Because of its roles in both extrinsic and intrinsic death pathways, Bid is an attractive therapeutic target. Inhibition of Bid cleavage by selective calpain and caspase-8 inhibitors presents a strategy for acute and possibly chronic protection against infarction and adverse remodeling. Such inhibitors might be effective when delivered acutely, both before and at intervals after PTCA or CABG procedures. Calpain inhibitors are currently under development for the treatment of other
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Figure 3. Induction and activation of Bcl-2 proteins during ischemia and reperfusion. Ischemia causes hypoxia, inhibition of mitochondrial electron transport, and transcriptional induction of Bnip3 and Noxa. During ischemia ATP levels and intracellular pH decrease, cytosolic calcium increases, and the MAPKs ERK and p38 are activated. The Bnip3 death pathway is activated by hypoxia-acidosis and can be blocked by neutralization of pH or inhibition of the mitochondrial pore (mPTP). Reperfusion activates electron transport and stimulates ROS. Post-ischemic stunning and cardiac myocyte death are both linked to elevated ROS. Multiple kinases are stimulated by reperfusion calcium accumulates in the mitochondria. BH3-only proteins activated during ischemia reperfusion include Bnip3, tBid, BAD and PUMA. Green symbols indicate targets that reduce ischemic damage when inhibited (T-bars) or induced (arrows).
chronic conditions, including Parkinson’s disease and Duchene muscular dystrophy [55]. Bnip3 (Bcl-XL or E1B 19K-binding protein) is expressed at low levels in most organs under normal (non-ischemic) conditions but is induced by hypoxia and ischemia (reviewed in [56]). Bnip3 is an atypical BH3-only protein that drives an atypical death pathway. The BH3 domain has only a low homology with other BH3 domains and is not required for the death function. Deletion of the transmembrane domain eliminates the death function and converts the remaining N-terminal portion into a dominant-negative that is protective [57]. The components of the Bnip3-mediated death pathway are controversial; caspase dependent and independent pathways have been reported [58,59]. Bnip3 has the dubious distinction of promoting cell death during both phases of ischemia reperfusion injury. It is induced by hypoxia and activated by acidosis or reperfusion. Our laboratory described the dual roles of hypoxia and acidosis in activating Bnip3 and promoting a CsA-dependent death pathway with apoptosis-like DNA fragmentation, but no caspase activation [45]. Recently, it was reported that Bnip3 is activated by ischemia reperfusion and mediates a classical intrinsic death pathway [59]. We also found that Bnip3 is induced and activated by hypoxia reoxygenation by a pathway that might involve the activation of protein kinase c [60]. The Bnip3 N-terminal deletion fragment inhibits both acidosisand reperfusion-mediated death pathways, and infusion of the peptide at the time of reperfusion confers more than www.sciencedirect.com
50% reduction of infarct size. The ability of Bnip3 to promote cell death under conditions of ischemia alone as well as reperfusion singles it out as a potentially important therapeutic target. Work is in progress in this laboratory to obtain small inhibitory peptides that block the Bnip3 pathway. PUMA (p53 modulated upregulator of apoptosis) is activated transcriptionally by all p53-activating stimuli, including DNA damage and oxidative stress [61]. It can also promote death independently of p53 in response to conditions that induce endoplasmic reticulum (ER) stress [61,62]. PUMA can bind and sequester all of the prosurvival Bcl-2 proteins and promotes death independently of other BH3-only proteins [2]. PUMA is induced in models of MI, and mice with ablation of the PUMA gene display 50% reduced infarction compared with wild-type littermates [63]. Pharmacological strategies to selectively target PUMA are not yet available. Conclusions and perspectives Necrotic- and apoptotic-death programs contribute to tissue loss during MI; the relative contributions of these programs to the overall damage are still unknown. Preclinical as well as preliminary clinical results indicate that 50% or more of the myocardial damage that occurs during MI could be prevented by pharmacological intervention at the time of reperfusion. Statins and PDE-5 inhibitors with established safety profiles are among the leading agents that could be most rapidly adopted for the treat-
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ment of patients undergoing angioplasty or coronary artery bypass. Perhaps the most attractive option involves the use of volatile anesthetics during surgery; two large clinical trials now confirm the efficacy of these agents in protecting the heart. An unresolved question is whether volatile anesthesia alone provides optimal protection or whether it should be combined with other protective strategies. Molecular approaches that target the mitochondrial death channels reproducibly lower infarct size in preclinical models, but these approaches have not yet been translated into clinical applications. The strong performance of the PKCd inhibitor peptide in the porcine infarct model combined with an attractive mechanism of action provided the driving force for the pioneering DELTA-MI trial. Although the results of this trial are only suggestive, they support further testing of this and other molecular approaches, perhaps in combination with IPC mimics. The death signals generated by ischemia reperfusion dichotomize on their way into and out of the mitochondria, and although a single pharmacological agent might intercept more than one signal, separate agents might be necessary to optimize efficacy and retain specificity and safety. Great advances have been made in our knowledge of the death pathways and avenues of intervention. The combination of infarct reduction with stem cell and possibly gene therapies presents a promising future for new treatments in the fight against cardiovascular disease. Acknowledgements Supported by National Institutes of Health grants HL44578 and HL72924 and by a Walter G. Ross Chair in vascular biology.
References 1 Webster, K.A. et al. (2006) Redox stress and the contributions of BH3only proteins to infarction. Antioxid. Redox Signal. 8, 1667–1676 2 Willis, S.N. and Adams, J.M. (2005) Life in the balance: how BH3-only proteins induce apoptosis. Curr. Opin. Cell Biol. 17, 617–625 3 Danial, N.N. and Korsmeyer, S.J. (2004) Cell death: critical control points. Cell 116, 205–219 4 Tsujimoto, Y. et al. (2006) Mitochondrial membrane permeability transition and cell death. Biochim. Biophys. Acta 1757, 1297–1300 5 Dejean, L.M. et al. (2006) 2006 Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? Cell Death Differ. 13, 1387–1395 6 Nutt, L.K. et al. (2002) Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores. J. Biol. Chem. 277, 9219–9225 7 Nakagawa, T. et al. (2005) Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434, 652–658 8 Baines, C.P. et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658–662 9 Kerr, P.M. et al. (1999) Reversal of permeability transition during recovery of hearts from ischemia and its enhancement by pyruvate. Am. J. Physiol. 276, H496–H502 10 Kokoszka, J.E. et al. (2004) The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427, 461–465 11 Baines, C.P. et al. (2007) 2007 Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat. Cell Biol. 9, 550–555 12 Halestrap, A.P. (2006) Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem. Soc. Trans. 34, 232–237 13 Bott-Flugel, L. et al. (2005) Gene transfer of the pancaspase inhibitor P35 reduces myocardial infarct size and improves cardiac function. J. Mol. Med. 83, 526–534 www.sciencedirect.com
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14 Hochhauser, E. et al. (2003) Bax ablation protects against myocardial ischemia reperfusion injury in transgenic mice. Am. J. Physiol. Heart Circ. Physiol. 284, H2351–H2359 15 Downey, J.M. and Cohen, M.V. (2006) Reducing infarct size in the setting of acute myocardial infarction. Prog. Cardiovasc. Dis. 48, 363–371 16 Hofstra, L. et al. (2000) Visualisation of cell death in vivo in patients with acute myocardial infarction. Lancet 356, 209–212 17 Downey, J.M. and Cohen, M.V. (2005) Unraveling the mysteries of classical preconditioning. J. Mol. Cell. Cardiol. 39, 845–848 18 Jones, S.P. and Bolli, R. (2006) The ubiquitous role of nitric oxide in cardioprotection. J. Mol. Cell. Cardiol. 40, 16–23 19 Staat, P. et al. (2005) Postconditioning the human heart. Circulation 112, 2143–2148 20 Juhaszova, M. et al. (2004) Glycogen synthase kinase-3b mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J. Clin. Invest. 113, 1535–1549 21 Efthymiou, C.A. et al. (2005) Atorvastatin and myocardial reperfusion injury: new pleiotropic effect implicating multiple prosurvival signaling. J. Cardiovasc. Pharmacol. 45, 247–252 22 Pan, W. et al. (2004) Statins are associated with a reduced incidence of perioperative mortality after coronary artery bypass graft surgery. Circulation 110 (Suppl 1), II45–II49 23 Jones, S.P. et al. (2003) Simvastatin attenuates oxidant-induced mitochondrial dysfunction in cardiac myocytes. Circ. Res. 93, 697–699 24 Kukreja, R.C. et al. (2005) Pharmacological preconditioning with sildenafil: basic mechanisms and clinical implications. Vascul. Pharmacol. 42, 219–232 25 Opie, L.H. et al. (2006) Controversies in ventricular remodeling. Lancet 367, 356–367 26 Takimoto, E. et al. (2005) Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat. Med. 11, 214–222 27 Rosanio, S. et al. (2006) Enhanced cardioprotection against ischemia reperfusion injury with combining sildenafil with low-dose atorvastatin. Cardiovasc. Drugs Ther. 20, 27–36 28 Fung, E. et al. (2005) The potential use of type-5 phosphodiesterase inhibitors in coronary artery bypass graft surgery. Chest 128, 3065– 3073 29 De Hert, S.G. (2006) Volatile anesthetics and cardiac function. Semin. Cardiothorac. Vasc. Anesth. 10, 33–42 30 Raphael, J. et al. (2005) Isoflurane-induced myocardial preconditioning is dependent on phosphatidylinositol-3-kinase/Akt signalling. Br. J. Anaesth. 95, 756–763 31 Bienengraeber, M.W. et al. (2005) Cardioprotection by volatile anesthetics. Vascul. Pharmacol. 42, 243–252 32 Kevin, L.G. et al. (2005) Reactive oxygen species as mediators of cardiac injury and protection: the relevance to anesthesia practice. Anesth. Analg. 101, 1275–1287 33 De Hert, S.G. (2004) Cardioprotection with volatile anesthetics: clinical relevance. Curr. Opin. Anaesthesiol. 17, 57–62 34 Guarracino, F. et al. (2006) Myocardial damage prevented by volatile anesthetics: a multicenter randomized controlled study. J. Cardiothorac. Vasc. Anesth. 20, 477–483 35 De Hert, S.G. (2006) Anesthetic preconditioning: how important is it in today’s cardiac anesthesia? J. Cardiothorac. Vasc. Anesth. 20, 473–476 36 Li, Q. et al. (2006) Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences. Am. J. Physiol. Heart Circ. Physiol. 290, H584–H589 37 Smith, R.S., Jr et al. (2005) Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction. Life Sci. 76, 2457–2471 38 Webster, K.A. (2003) Therapeutic angiogenesis: a complex problem requiring a sophisticated approach. Cardiovasc. Toxicol. 3, 283–298 39 Duranski, M.R. et al. (2005) Cytoprotective effects of nitrite during in vivo ischemia reperfusion of the heart and liver. J. Clin. Invest. 115, 1232–1240 40 Juhaszova, M. et al. (2005) Protection in the aged heart: preventing the heart-break of old age? Cardiovasc. Res. 66, 233–244 41 Tsang, A. et al. (2005) Preconditioning the diabetic heart: the importance of Akt phosphorylation. Diabetes 54, 2360–2364 42 Inagaki, K. et al. (2003) Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo. Circulation 108, 2304–2307
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43 Murriel, C.L. et al. (2004) Protein kinase Cdelta activation induces apoptosis in response to cardiac ischemia and reperfusion damage: a mechanism involving BAD and the mitochondria. J. Biol. Chem. 279, 47985–47991 44 Regula, K.M. et al. (2002) Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ. Res. 91, 226–231 45 Vande Velde, C. et al. (2000) BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol. Cell. Biol. 20, 5454–5468 46 Adams, J.M. and Cory, S. (2001) Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 26, 61–66 47 Degli Esposti, M. and Dive, C. (2003) Mitochondrial membrane permeabilisation by Bax/Bak. Biochem. Biophys. Res. Commun. 304, 455–461 48 Antignani, A. and Youle, R.J. (2006) How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr. Opin. Cell Biol. 18, 685–689 49 Espost, M.D. (2002) The roles of Bid. Apoptosis 7, 433–440 50 Scorrano, L. et al. (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2, 55–67 51 Dejean, L.M. et al. (2005) Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel. Mol. Biol. Cell 16, 2424–2432 52 Plesnila, N. et al. (2001) BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia. Proc. Natl. Acad. Sci. U. S. A. 98, 15318–15323 53 Chen, M. et al. (2002) Calpain and mitochondria in ischemia/ reperfusion injury. J. Biol. Chem. 277, 29282–29286
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54 Takahashi, M. et al. (2006) Possible involvement of calpain activation in pathogenesis of chronic heart failure after acute myocardial infarction. J. Cardiovasc. Pharmacol. 47, 413–421 55 Saez, M.E. et al. (2006) The therapeutic potential of the calpain family: new aspects. Drug Discov. Today 11, 917–923 56 Webster, K.A. et al. (2005) BNip3 and signal-specific programmed death in the heart. J. Mol. Cell. Cardiol. 38, 35–45 57 Ray, R. et al. (2000) BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J. Biol. Chem. 275, 1439–1448 58 Kubasiak, L.A. et al. (2002) Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc. Natl. Acad. Sci. U. S. A. 99, 12825–12830 59 Hamacher-Brady, A. et al. (2007) Response to myocardial ischemia/ reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 14, 146–157 60 Graham et al (2007) Regulation of Bnip3 death pathways by calcium, phosphorylation and hypoxia-reoxygenation. Jul 13; [Epub ahead of print] 61 Erlacher, M. et al. (2005) BH3-only proteins Puma and Bim are ratelimiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo. Blood 106, 4131–4138 62 Reimertz, C. et al. (2003) Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway. J. Cell Biol. 162, 587–597 63 Toth, A. et al. (2006) Targeted deletion of Puma attenuates cardiomyocyte death and improves cardiac function during ischemia/ reperfusion. Am. J. Physiol. Heart Circ. Physiol. 291, H52–H60
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Programmed death as a therapeutic target to reduce myocardial infarction Keith A. Webster Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, 1600 NW 10th Avenue, RMSB 6038 Miami, FL 33101, USA
In the United States, angioplasty or bypass surgery to remove coronary occlusions is performed on approximately two million patients each year. Although reperfusion is essential for salvaging ischemic myocardium, it also promotes infarction by activating programmed cell death in the formerly ischemic tissue. Reperfusion injury begins when oxidative stress and calcium accumulation by the mitochondria cause activation of the so-called mitochondrial death channels. These channels have become the focus of evolving strategies to protect the heart from infarction. Preclinical and preliminary clinical studies indicate that agents with diverse modes of action can reduce infarct size by 50% or more and significantly preserve myocardial functions. This article reviews the most advanced pharmacological approaches for their ability to reduce infarct size by inhibiting the mitochondrial death pathways. Introduction Cell death during myocardial infarction (MI) occurs by necrosis and apoptosis, both regulated by stress signals that originate in the mitochondria (reviewed in [1]). Apoptosis is a programmed process that requires energy. It is initiated by the activation of cell-surface receptors (the extrinsic pathway) or by permeability changes of mitochondria (the intrinsic pathway). The Bcl-2 family of proteins plays important roles in the intrinsic pathways by determining the release of cytochrome c and other pro-apoptotic factors from the mitochondria into the cytoplasm, where they initiate death pathways [2,3]. Two channels have been proposed to accommodate this release: the mitochondrial permeability transition pore (mPTP) and the mitochondrial apoptosis channel (mAC, or Bax channel) (reviewed in [4,5]). There is controversy over the properties, structure and regulation of these channels but strong evidence that Bcl-2 proteins contribute to the regulation of both [5,6]. Until recently, apoptosis and necrosis during MI were considered to be mechanistically and physically distinct; the former was thought to be largely a consequence of oxidative damage that affects energy-competent cells and takes place principally during reperfusion, whereas the latter was thought to involve a collapse of ion gradients across the plasma membrane caused by energy depletion during ischemia. These distinctions have now been blurred by demonstrations that death can simultaneously involve Corresponding author: Webster, K.A. ([email protected]). Available online 10 August 2007. www.sciencedirect.com
features of both necrosis and apoptosis and that signals from the mitochondria regulate both pathways [7]. One of the most radical changes in our understanding of programmed death during MI involves the role of the mPTP, a complex that was formerly thought to include at least five proteins [4]. Two of these proteins, the outermembrane voltage-dependent anion channel (VDAC) and the inner-membrane adenine nucleotide translocator (ANT), have essential physiological functions in transporting metabolites across the mitochondrial membranes. Calcium-induced mitochondrial swelling, facilitated by oxidative stress, opens the pore. Normal opening requires a regulatory component, cyclophilin D (CypD) [4]. CypD is a protein isomerase that interacts functionally and possibly physically with the mPTP from the matrix side of the inner membrane (Figure 1). CypD is inhibited by the immunosuppressant agent cyclosporine-A (CyA), and we have known for many years that CyA is powerfully protective against MI [4]. Recently, genetic studies in which the CypD gene was ablated have revealed that the CyA-sensitive mPTP primarily drives a pathway of necrosis – not apoptosis, as was formerly thought [7,8]. CypD / cells respond normally to classical inducers of apoptosis, but mPTP opening requires fivefold more calcium, and the hearts of CypD / mice are resistant to MI. These studies do not eliminate the possibility of dual roles for the mPTP in apoptosis, perhaps in combination with a mAC/Baxchannel-like activity [7]. Previous studies support a role for Bcl-2 proteins in the regulation of the mPTP, and it is widely believed that both necrosis and apoptosis contribute significantly to infarction [7–9]. One possibility that is consistent with most of the available information is that ischemia reperfusion opens both of the mitochondrial death channels and that mPTP opening influences the activity of the mAC/Bax channel. Cells subjected to chronic mPTP opening are destined to die by necrosis irrespective of other channels, but cells in which mPTP reverses before Glossary MI: Myocardial Infarction or heart attack. CABG: Coronary artery bypass grafting. The procedure whereby a section of a blood vessel (usually from the limb) is excised and transplanted into the coronary artery to bypass an occlusion. PTCA: Percutaneous transluminal coronary angioplasty. Percutaneous refers to needle catheter access to a blood vessel via puncture of the skin. Transluminal is a procedure that is performed within the blood vessel. Angioplasty means ‘to reshape’ the blood vessel, usually by inflation of a balloon contained in the catheter. The procedure is used for removing arterial occlusions that result from atherosclerosis.
0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.07.004
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Figure 1. The mitochondrial death channels. Proteins associated with the mPTP include the voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), cyclophilin-D (CypD), the benzodiazepine receptor (BDR) and hexokinase. The mAC has not been defined structurally, but channel activity is regulated by interactions of Bax, Bak and tBid with the outer mitochondrial membrane (OM); Bax might be a structural component of the mAC. The mPTP and mAC (or Bax channel) are both regulated by Bcl-2 proteins and are opened by calcium ionophores and oxidative stress. Some reports suggest that VDAC and ANT bridge the inner (IM) and outer mitochondrial membranes by interacting at contact points. Other reports [10,11] suggest that neither ANT nor VDAC is required for mPTP function (illustrated by a double arrow and a dashed line). The mPTP opens under conditions of ischemia reperfusion when matrix calcium levels increase above a threshold. CypD and ROS lower the threshold. The mitochondrial membrane potential (D c) is lost when the mPTP opens. Cytochrome c release triggers apoptosis; 15% of total cytochrome c is in the intermembrane space (IMS), and the rest is in the intercristal space (ICS). The latter might become available for release during matrix swelling or by incorporation of tBid into the IM. Low matrix pH inhibits the opening of the mPTP when D c is dissipated, but low pH induces mPTP opening in energized mitochondria.
outer-membrane rupture could either avoid necrosis and die by apoptosis or survive. Several studies have shown that the permeability transition is reversible, and up to 50% of the mitochondria that undergo the transition during early reperfusion subsequently close the pore and can recover function [9]. The mAC/Bax channel opens at levels of calcium that are significantly lower than those required for causing and sustaining mPTP opening. Therefore, conditions that favor opening of the mAC/Bax channel might occur in cells that are unloading calcium. Recent gene-ablation studies have also raised questions about essential roles for ANT and VDAC in the mitochondrial permeability transition [10,11]. The regulation and relative contributions of the different death pathways is clearly of great importance in determining the conditions for optimal protection against MI. Bcl-2 proteins are universally viewed as regulators of apoptosis, so it was not anticipated that mPTP opening (previously thought to be regulated by Bcl-2) causes necrosis. From a therapeutic perspective, this distinction is important because apoptosis-blocking agents, such as caspase inhibitors, are not expected to directly block infarction caused by necrosis. However, these results do not exclude apoptosis from important roles in infarction, neither do they refute roles for Bcl-2 proteins in the regulation of the mPTP and necrosis, as well as apoptosis. Preclinical studies reveal an extensive list of interventions and pharmacological agents that reduce infarction when applied at the time of ischemia or reperfusion. In these models, the degrees of protection afforded www.sciencedirect.com
by agents with distinct mechanisms of action are remarkably similar, conferring 50%–70% reduction of infarct size. Such agents include CsA that selectively targets the mPTP [12], pan-caspase inhibitors that selectively block apoptosis [13], and interventions that respectively activate or inhibit pro- and anti-apoptotic Bcl-2 proteins [14]. The equivalent degrees of protection conferred by these interventions reflect the heterogeneous nature of the infarct process and the close interrelationships between the different death pathways. Each intervention targets the mitochondria, either downstream or at the level of the death channels. Some of the leading targets and strategies for blocking programmed cell death and infarction in the heart will be reviewed in the following sections. Indirect targeting of the death channels The patient groups most likely to benefit from infarct protection are those that have severely occluded coronary arteries and are undergoing angioplasty (percutaneous transluminal coronary angioplasty [PTCA]) or coronary artery bypass grafting (CABG) [15]. It is well established that myocardial infarction in these patients continues to develop for extended periods after successful reperfusion [16]. Preclinical studies and early clinical data provide a range of options that could prevent loss of myocardium during and after these procedures. Pharmacological mimics of the procedure known as ischemic preconditioning (IPC) are presently at the most advanced stages of development.
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Ischemic pre- and post-conditioning IPC involves exposing the heart to several short episodes of ischemia reperfusion before prolonged ischemia. It was first reported almost two decades ago and subsequently confirmed by multiple investigators to be one of the most effective cardioprotective strategies ever discovered (reviewed in [17,18]). Intense study over the intervening 20 years has led to a dissection of the mechanisms of IPC, descriptions of three interrelated strategies and the identification of pharmacological mimics that, like the physical procedures, can reduce infarct size by 40%–60% in animal models of MI. Post-ischemic conditioning differs from early and late IPC in that the conditioning stimulus (physical or pharmacological) is applied after the index ischemia, immediately before reperfusion. The discovery of postconditioning is important because the treatment of patients undergoing PTCA or CABG also requires that the intervention be administered after the ischemic episode, at the time of reperfusion [19]. Late IPC refers to protection that appears 24–72 h after the initiation of IPC.
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It is critically dependent on nitric oxide (NO) signaling and involves transcriptional as well as activity changes of both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) (reviewed in [18]). The targets of IPC have not been fully resolved, but there is compelling evidence that one target is the mPTP (Figure 2). Signals from cell-surface receptors are transmitted by protein kinases, including protein kinase C (PKC), protein kinase B (Akt) and the extracellular-signal-regulated kinase (ERK), and converge on the mPTP through a mitochondrial ATP-sensitive potassium channel (mK-ATP) and/or the signaling intermediate glycogen synthase kinase-3b (GSK-3b) [20]. Some of the most promising IPC mimics are already approved to treat other conditions but have not undergone rigorous clinical tests in the setting of MI. These include statins, PDE-5 inhibitors and volatile anesthetics. Statins 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors are widely prescribed for their anti-
Figure 2. Pathways of protection by pharmacological IPC mimics. IPC is initiated by agonists of multiple transmembrane G-protein-coupled receptors (GPCR) that activate PKC and/or PI3 kinase. PKCd and PKCe have been associated with the IPC response; PKCd is pro-apoptotic, whereas e is protective. PKC activity is increased by dual signaling pathways: (1) an early receptor-initiated pathway that generates diacylglycerol by activating phospholipases (PLC/PLD) and (2) ROS generated by the mitochondrial electron transport chain and via the mtK-ATP channel. Mitogen-activated protein kinase pathways, including p38 and ERK1/2, are downstream of GPCR and PKC; p38a promotes apoptosis, and p38b is protective. The late phase of protection involves new protein synthesis and enhanced NO production from iNOS and eNOS. COX, antioxidants, heatshock proteins and PKCe might contribute to late IPC. Several of these components are activated by PDE-5 inhibitors (sildenafil) and interventions that increase NO production. The targets for IPC include mitochondrial death channels, and the signals might be channeled through GSK-3b and the mtK-ATP channel. Akt phosphorylates multiple substrates that can block mPT and the activation of Bax. The lag phase before late IPC involves increased expression of iNOS, HSPs, COX and anti-oxidant genes. www.sciencedirect.com
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hypercholesterolemic and anti-hypertensive actions. These agents have been shown to possess additional therapeutic properties that are independent of their lipid-lowering actions. Statins are potent modulators of eNOS activity; they block the inhibitory actions of oxidized low-density lipoprotein on eNOS expression and stimulate synthesis by selectively stabilizing eNOS mRNA. Statins also activate Akt and thereby increase eNOS activity by direct phosphorylation. Statins have been shown to reduce vascular inflammation and oxidative stress, increase coronary blood flow and reduce myocardial necrosis associated with acute MI [211]. A recent retrospective clinical study involving 1663 patients found that acute statin treatment improved the 30-day outcome and independently reduced the mortality of patients undergoing CABG by 50% [22]. The generally accepted mechanism of cardioprotection by statins is to mimic IPC through NO-mediated activation of mtK-ATP channels and inhibition of the mPTP [23] (Figure 2). These agents are being heralded as one of the most powerful pharmacological strategies available for the treatment of cardiovascular disease. Because of their demonstrated safety and pleiotropic properties, statins administered acutely are strongly recommended for patients undergoing cardiac surgery. However, at the present time it is not clear how the protection conferred by statins compares with that of other IPC mimics. Further studies of statins in large-animal models of MI is warranted, perhaps in combination with other IPC mimics (see below). The proven safety and exceptional therapeutic profiles of statins make them excellent candidates as infarct-sparing agents for patients undergoing CABG or percutaneous transluminal coronary angioplasty (PTCA). Phosphodiesterase type-5 (PDE-5) inhibitors Sildenafil, varenafil and tadalafil are PDE-5 inhibitors that are approved for the treatment of erectile dysfunction (ED) (reviewed in [24]). They work by increasing cyclic guanosine monophosphate and activating protein kinase G (PKG). PKG lowers intracellular calcium by phosphorylating Ca2+-dependent K+ channels, phospholamban and the 1,4,5-inositol triphosphate receptor, thus causing smoothmuscle relaxation (Figure 2). Sildenafil (Viagra) and its derivatives mimic both early and late IPC and reduce infarction in animal models by 70% in the early phase and 40% in the late phase. The dose of Viagra required for mimicking IPC is less than that required for treating ED, and an attractive aspect of these agents is that chronic administration of a low dose might produce sustained preconditioning. Other beneficial effects of PDE-5 inhibitors on cardiovascular disease have also been described [25]. Sildenafil was shown to prevent pressure-overload hypertrophy in rabbits; this effect was attributed to inhibition of ERK, Akt and GSK-3b phosphorylation [26]. The latter property raises questions about the mechanisms of action and targets of PDE-5 inhibitors in the heart. Activation of Akt and ERK and phosphorylation of GSK-3b are central to IPC, and inhibition of these should antagonize IPC. A recent report demonstrated that sildenafil and atorvastatin conferred protection that was additive, implying different mechanisms of action [27]. Statins might complement sildenafil by activating Akt and ERK [28]. www.sciencedirect.com
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These results support the implementation of clinical trials testing combinations of acute sildenafil and statin for patients undergoing PTCA and CABG. Anesthetic-induced preconditioning (APC) Opioids and volatile halogenated anesthetics also mimic both early and late pre- and post-conditioning by stimulating receptors on the cell surface (reviewed in [29]) (Figure 2). The pathway shares the same intermediates and targets as IPC; these include inhibitory G-proteins, PKC, Akt, ERK [30], mK-ATP channels, GSK-3b and the mPTP (reviewed in [31]). An increase of reactive oxygen species (ROS), mediated by a partial inhibition of the mitochondrial electron transport chain at complex I, initiates APC (reviewed in [32]). Preclinical studies demonstrate that the degree of infarct reduction conferred by volatile anesthetics is independent of their hypotensive actions and comparable in magnitude to that reported for IPC (reviewed in [29,33]). Recently, the results of the first multicenter randomized controlled trial of APC were reported. In this trial, 112 patients undergoing off-pump CABG were randomized to receive desflurane or propofol (intravenous anesthetic) [34]. This study reported a 50% reduction in postoperative troponin I (reflecting proportionally less cardiac injury), a reduced number of patients requiring postoperative inotropes and fewer patients requiring prolonged hospitalization in the volatile anesthetic group. These results are consistent with a previous trial of 320 coronary surgery patients that were also randomized to receive intravenous anesthetic regimen or a volatile anesthetic agent maintained throughout surgery [35]. APC is presently the most advanced of the IPC mimics, in that it has been tested in two large trials that have confirmed its safety and efficacy [34,35]. This wealth of positive data from both preclinical and clinical studies supports the implementation of a large-scale multicenter trial to determine the impact of this strategy on cardiac morbidity and mortality. Unresolved questions include whether the protection mediated by APC is maximal, whether the protection is consistent across patient groups with different etiologies and whether combinations with other IPC mimics (statins, PDE-5 inhibitors) will provide additional protection. Novel strategies based on IPC Gene therapy to permanently precondition the hearts of patients at high risk for MI has been pioneered by Bolli et al. (reviewed in [16]). Delivery of iNOS or eNOS genes by direct injection of adenoviral vectors has demonstrated sustained protection from infarction two months after gene therapy, at levels equivalent to IPC and without side effects [36,37]. The next phase in the development of this strategy is to use a more permanent gene-delivery vehicle, such as an adenovirus-associated virus (AAV), in largeanimal models of MI to confirm efficacy and determine the duration of protection. Gene-delivery procedures are invasive and expensive, so a substantial period of protection needs to result (one year or more) for them to be worthwhile. It might be possible to improve the safety profile of gene therapy by using tissue-specific regulated promoters that respond to the conditions of MI (reviewed in [38]). The
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protective value of enhancing NO availability was recently confirmed in experiments where the infusion of sodium nitrite directly into the left ventricle was shown to reduce infarct size by more than 50% [39]. The authors postulate that the hypoxic-acidic environment of the post-ischemic myocardium promotes the reduction of nitrite to NO and triggers an IPC response. Both the gene-therapy and biochemical approaches to enhancing NO bioavailability represent attractive strategies for cardioprotection, and further development of these approaches should be encouraged. An unresolved question for the future of IPC mimics is whether the use of volatile anesthetics during cardiac surgery provides the maximal protection that can be obtained by IPC stimuli. The two most recent trials support this possibility and, if confirmed, will obviate the use of additional mimics, at least in the acute setting [34,35]. The results support the general use of volatile anesthetics for the surgical procedures required for CABG and PTCA. Further preclinical and clinical studies are required for determining whether mimics such as statin and PDE-5 inhibitors should be added to APC. Direct targeting of the death channels Akt is at the crossroads of multiple signaling pathways that collectively regulate cell growth and survival. The targets of Akt include the FOXO transcription factors, caspases, GSK-3b and the pro-apoptotic Bcl-2 family protein BAD [7]. These properties allow Akt to contribute to the regulation of mitochondria-mediated necrosis as well as apoptosis and position it as one of the most important targets to protect the heart. Additionally, as a central regulator of insulin signaling, Akt regulates the activities of glucose-transport proteins and the mTOR complex. A disadvantage of Akt signaling is that the pathway can be desensitized. For example, animal studies indicate that ageing and diabetes reduce the IPC response, and this effect can be associated with mitochondrial dysfunction, insulin resistance and a decreased ability to activate Akt and generate NO [40,41]. Although increasing the IPC stimulus can improve these responses [41], it is possible that age and disease will blunt even an optimized IPC stimulus. If these pathways are indeed defective in diseased and/or aged hearts, a more effective approach will be to target the death pathways more directly. One approach that takes advantage of a target more proximal to the mitochondria involves inhibitory peptides that block the activity of PKC-d. Studies in rodents and pigs have shown that PKC-d is activated during MI and causes the inhibition of Akt and activation of BAD [42,43]. Inhibitory peptides block this effect and reduce infarction. Results of DELTA-MI, a phase I/II clinical trial involving 154 patients randomized (2:1) to receive one of four escalating doses of the PKC-d inhibitory peptide KAI-9803 or a placebo, were released recently (Roe, M.T. et al., presented at the American College of Cardiology Annual Scientific Session, New Orleans, LA, March 2007). The authors reported favorable trends of efficacy with less cardiac creatine kinase release post-angioplasty and smaller infarct size, although these reductions were not significant because of the small sample size. There was no indication www.sciencedirect.com
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that the intracoronary delivery of peptide was a safety hazard. The results support the implementation of a larger trial using intermediate doses of the peptide. An even more direct approach is to target the Bcl-2 proteins themselves. Direct inhibition or genetic ablation of the BH3-only proteins Bid, BAD, PUMA and Bnip3 reduce infarction by 50% or more in animal models, and this result is equivalent to that obtained with most IPC stimuli. BH3-only proteins Bcl-2 proteins regulate programmed death by controlling calcium release from intracellular stores and regulating the mitochondrial death channels. Members of this family have different numbers of a specific peptide sequence, known as a Bcl-2 homology (BH) domain, that is required for oligomerization between self and other Bcl-2 family members. The physical location and activity of each Bcl-2-family protein are largely determined by its binding to other Bcl-2-family proteins and scaffolds [44–46]. Bax and Bak are the principal effector proapoptotic Bcl-2 family members and contain three BH domains (BH1–BH3). Cells lacking these two proteins are resistant to death pathways initiated by the mitochondria [47]. Bax is either loosely attached to the outer mitochondrial membrane or sequestered in the cytosol through interactions with protein chaperones [48]. Activation of Bax involves translocation into the mitochondrial membrane, where it homo-oligomerizes or interacts with other proteins to regulate the permeability of the membranes to apoptogenic proteins [48] (Figure 3). The activation of Bax and Bak is regulated by the third class of Bcl-2 proteins that contain single BH3 domains; these proteins are known as BH3-only proteins. They sense and transmit the death stimulus and represent highly specific targets for inhibiting MI. BID (Bcl-2 Interacting Domain) is one of the most abundant and widespread mammalian BH3-only proteins. It is strongly expressed during development, and remains abundant in many adult tissues, including the heart [49]. In healthy cells, Bid is cytosolic or loosely membrane associated and functionally inert. It is cleaved at the N terminus by caspase 8, granzyme B or calpain to give the active form known as tBid (truncated Bid) [7]. tBid translocates to the mitochondria, where it mobilizes cytochrome c and other apoptogenic proteins and facilitates their release through the outer membrane, possibly by interacting with Bax [50,51]. Bid-mediated apoptosis has been shown to contribute significantly to both neuronal and myocardial ischemic injury [52], and blocking Bid cleavage with a calpain inhibitor has been shown to reduce infarct size by more than 50% in an ex-vivo model [53]. Calpain inhibitors have also been shown to slow the progression of heart failure in rats [54,55]. Because of its roles in both extrinsic and intrinsic death pathways, Bid is an attractive therapeutic target. Inhibition of Bid cleavage by selective calpain and caspase-8 inhibitors presents a strategy for acute and possibly chronic protection against infarction and adverse remodeling. Such inhibitors might be effective when delivered acutely, both before and at intervals after PTCA or CABG procedures. Calpain inhibitors are currently under development for the treatment of other
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Figure 3. Induction and activation of Bcl-2 proteins during ischemia and reperfusion. Ischemia causes hypoxia, inhibition of mitochondrial electron transport, and transcriptional induction of Bnip3 and Noxa. During ischemia ATP levels and intracellular pH decrease, cytosolic calcium increases, and the MAPKs ERK and p38 are activated. The Bnip3 death pathway is activated by hypoxia-acidosis and can be blocked by neutralization of pH or inhibition of the mitochondrial pore (mPTP). Reperfusion activates electron transport and stimulates ROS. Post-ischemic stunning and cardiac myocyte death are both linked to elevated ROS. Multiple kinases are stimulated by reperfusion calcium accumulates in the mitochondria. BH3-only proteins activated during ischemia reperfusion include Bnip3, tBid, BAD and PUMA. Green symbols indicate targets that reduce ischemic damage when inhibited (T-bars) or induced (arrows).
chronic conditions, including Parkinson’s disease and Duchene muscular dystrophy [55]. Bnip3 (Bcl-XL or E1B 19K-binding protein) is expressed at low levels in most organs under normal (non-ischemic) conditions but is induced by hypoxia and ischemia (reviewed in [56]). Bnip3 is an atypical BH3-only protein that drives an atypical death pathway. The BH3 domain has only a low homology with other BH3 domains and is not required for the death function. Deletion of the transmembrane domain eliminates the death function and converts the remaining N-terminal portion into a dominant-negative that is protective [57]. The components of the Bnip3-mediated death pathway are controversial; caspase dependent and independent pathways have been reported [58,59]. Bnip3 has the dubious distinction of promoting cell death during both phases of ischemia reperfusion injury. It is induced by hypoxia and activated by acidosis or reperfusion. Our laboratory described the dual roles of hypoxia and acidosis in activating Bnip3 and promoting a CsA-dependent death pathway with apoptosis-like DNA fragmentation, but no caspase activation [45]. Recently, it was reported that Bnip3 is activated by ischemia reperfusion and mediates a classical intrinsic death pathway [59]. We also found that Bnip3 is induced and activated by hypoxia reoxygenation by a pathway that might involve the activation of protein kinase c [60]. The Bnip3 N-terminal deletion fragment inhibits both acidosisand reperfusion-mediated death pathways, and infusion of the peptide at the time of reperfusion confers more than www.sciencedirect.com
50% reduction of infarct size. The ability of Bnip3 to promote cell death under conditions of ischemia alone as well as reperfusion singles it out as a potentially important therapeutic target. Work is in progress in this laboratory to obtain small inhibitory peptides that block the Bnip3 pathway. PUMA (p53 modulated upregulator of apoptosis) is activated transcriptionally by all p53-activating stimuli, including DNA damage and oxidative stress [61]. It can also promote death independently of p53 in response to conditions that induce endoplasmic reticulum (ER) stress [61,62]. PUMA can bind and sequester all of the prosurvival Bcl-2 proteins and promotes death independently of other BH3-only proteins [2]. PUMA is induced in models of MI, and mice with ablation of the PUMA gene display 50% reduced infarction compared with wild-type littermates [63]. Pharmacological strategies to selectively target PUMA are not yet available. Conclusions and perspectives Necrotic- and apoptotic-death programs contribute to tissue loss during MI; the relative contributions of these programs to the overall damage are still unknown. Preclinical as well as preliminary clinical results indicate that 50% or more of the myocardial damage that occurs during MI could be prevented by pharmacological intervention at the time of reperfusion. Statins and PDE-5 inhibitors with established safety profiles are among the leading agents that could be most rapidly adopted for the treat-
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ment of patients undergoing angioplasty or coronary artery bypass. Perhaps the most attractive option involves the use of volatile anesthetics during surgery; two large clinical trials now confirm the efficacy of these agents in protecting the heart. An unresolved question is whether volatile anesthesia alone provides optimal protection or whether it should be combined with other protective strategies. Molecular approaches that target the mitochondrial death channels reproducibly lower infarct size in preclinical models, but these approaches have not yet been translated into clinical applications. The strong performance of the PKCd inhibitor peptide in the porcine infarct model combined with an attractive mechanism of action provided the driving force for the pioneering DELTA-MI trial. Although the results of this trial are only suggestive, they support further testing of this and other molecular approaches, perhaps in combination with IPC mimics. The death signals generated by ischemia reperfusion dichotomize on their way into and out of the mitochondria, and although a single pharmacological agent might intercept more than one signal, separate agents might be necessary to optimize efficacy and retain specificity and safety. Great advances have been made in our knowledge of the death pathways and avenues of intervention. The combination of infarct reduction with stem cell and possibly gene therapies presents a promising future for new treatments in the fight against cardiovascular disease. Acknowledgements Supported by National Institutes of Health grants HL44578 and HL72924 and by a Walter G. Ross Chair in vascular biology.
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