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Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary (P.F.); Pharmahungary Group, Szeged, Hungary (P.F.); Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitasklinikum Essen, Universität Duisburg-Essen, Essen, Germany (R.S.); and Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cardiff, UK (G.F.B.)
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Abstract |
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I. Introduction |
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Ischemic heart disease is the leading cause of death in the industrialized world. The treatment of acute ischemic heart disease has entered a new era in which mortality can be approximately halved by procedures that allow the rapid return of blood flow to the ischemic zone of the myocardium, i.e., reperfusion therapy. Reperfusion, however, may lead to further complications such as diminished cardiac contractile function (stunning) and arrhythmia. Moreover, there is experimental evidence that irreversible cell injury leading to necrosis and apoptosis may be precipitated by reperfusion. Therefore, development of cardioprotective agents to improve myocardial function, decrease the incidence of arrhythmias, delay the onset of necrosis, and limit the total extent of infarction during ischemia/reperfusion is of great clinical importance. Earlier pharmacological approaches to attenuate the consequences of ischemia/reperfusion injury have been of limited experimental efficacy or have failed to translate into useful clinical treatments. However, more recently the heart has been shown to possess a remarkable ability to adapt to ischemia/reperfusion stress, and this molecular plasticity of the heart in ischemia/reperfusion has been the focus of intense research in the hope that the underlying mechanisms may be amenable to therapeutic exploitation. Ischemic preconditioning is a well described adaptive response in which brief exposure to ischemia/reperfusion markedly enhances the ability of the heart to withstand a subsequent ischemic injury. Moreover, brief cycles of ischemia/reperfusion applied after a longer period of ischemia also confer cardioprotection against the consequences of myocardial ischemia/reperfusion, a phenomenon called ischemic postconditioning (Fig. 1). The discovery of these two major forms of endogenous cardioprotective mechanisms has encouraged the exploration of new ways to protect the ischemic/reperfused myocardium and has amplified our knowledge of the molecular basis of injury and survival during ischemia/reperfusion.
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B. Risk Factors for Ischemic Heart Disease
Ischemic heart disease develops as a consequence of a number of etiological risk factors and always coexists with other disease states. These include systemic arterial hypertension and related left ventricular hypertrophy, hyperlipidemia, and atherosclerosis, diabetes and insulin resistance, heart failure, as well as aging. These systemic diseases with aging as a modifying condition, exert multiple biochemical effects on the heart that can potentially affect the development of ischemia/reperfusion injury per se and interfere with responses to cardioprotective interventions (Fig. 1). Therefore, the development of rational therapeutic approaches to protect the ischemic heart requires preclinical studies that examine cardioprotection specifically in relation to complicating disease states and risk factors.
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II. Introduction to Endpoints of Ischemia/Reperfusion Injury and Experimental Approaches to Cardioprotection |
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; Hearse, 1996; Ganz and Braunwald, 1997). In experimental models and in clinical situations, ischemia may be followed by reperfusion, that is, the re-admission of oxygen and metabolic substrates with washout of ischemic metabolites. The process of reperfusion is associated with further biochemical, structural, and functional changes in myocardium and may determine cell survival and cell death. It is common for commentators to consider ischemia/reperfusion injury as a composite entity with distinct components of injury associated specifically with ischemia and with reperfusion since it is a truism that reperfusion can never occur independently of ischemia. A. Clinical and Experimental Endpoints of Injury
Irreversible cell injury leading to infarction, the development of arrhythmias, and the loss of myocardial contractility are all relevant as clinical consequences of occlusive coronary disease and they are also important as experimental correlates and endpoints. The following is a synoptic description of the major endpoints, providing an introductory context for the experimental studies that we review subsequently. For the most part in this review, we refer to studies using markers of irreversible cell injury and tissue infarction.
1. Irreversible Cellular Injury and Infarction.
Ultrastructural changes occur in myocardium rapidly after the onset of ischemia. These may be considered reversible alterations if reperfusion of the tissue can be effected promptly. However, ischemia lasting more than 20 to 30 min (without collateral flow or residual flow through the infarct-related artery) results in a transition from a state of reversible ultrastructural alterations to a state of irreversible tissue injury that is ultimately characterized as coagulative necrosis (Herdson et al., 1965; Reimer and Jennings, 1979; Jennings et al., 1981). A number of factors that influence the onset and extent of irreversible injury in experimental models have been identified. These include the size of the area at risk (Reimer and Jennings, 1979; Ytrehus et al., 1994), the extent of collateral blood flow or residual flow through the infarct-related artery (Reimer and Jennings, 1979), the duration of ischemia (Reimer and Jennings, 1979; Ytrehus et al., 1994), and myocardial temperature (Miki et al., 1998). There is also some evidence that systemic hemodynamic influences during ischemia, including heart rate, may contribute to the rate of development of irreversible injury (Schulz et al., 1995b).
In the absence of reperfusion, no intervention is able to limit infarct development, and it is clear that reperfusion is the sine qua non for tissue salvage. Reperfusion and revascularization therapies in acute myocardial infarction (fibrinolysis, percutaneous coronary intervention, and emergency coronary artery bypass grafting) have the primary aim of salvaging viable tissue, which may be reversibly injured, within the ischemic risk zone and thereby limiting the extent of necrosis. In clinical myocardial infarction, both early and late mortality are closely related to the duration of unrelieved coronary occlusion. This philosophy of prompt reperfusion/revascularization in acute myocardial infarction is succinctly defined in the axiom: "time is muscle and muscle is life" (Simoons et al., 1997).
Some cells subjected to ischemia/reperfusion display hallmarks of apoptosis. However, there is controversy as to the extent of apoptosis in ischemia/reperfusion injury, when apoptosis occurs, and the relationships between apoptosis and necrosis (Kajstura et al., 1996; Misao et al., 1996; Olivetti et al., 1996; Saraste et al., 1997; Baliga, 2001; Bishopric et al., 2001). Early studies of apoptosis in experimental infarction using permanent coronary artery occlusion in the rat (i.e., ischemia without reperfusion) suggested that apoptosis represented the major form of myocyte death (Kajstura et al., 1996). Subsequently, the majority of evidence suggests that the number of cells undergoing apoptosis is likely to be relatively few compared with the number of necrotic cells. However, it is not clear at what stage apoptosis occurs in relation to necrosis or how these two processes are related. Although the mechanisms initiating apoptosis are still unknown, it seems likely that opening of the mitochondrial permeability transition pore (mPTP1) during reperfusion, after ischemia of sufficient duration, serves as a key mechanism of cell death, amplifying or accelerating cell death to produce a pattern of reperfusion-induced necrosis (section II.B.2.) (Crompton, 1999; Hajnóczky et al., 2000; Bishopric et al., 2001; Di Lisa et al., 2001; Pacher and Hajnóczky, 2001; Pacher et al., 2001; Halestrap et al., 2004).
2. Contractile Dysfunction and Ventricular Arrhythmias.
Depression of myocardial contractility is an early consequence of myocardial ischemia (Tennant and Wiggers, 1935) and may result in acute cardiac failure. Impairment of contractility leading to left ventricular dysfunction and chronic cardiac failure may occur over extended periods after the development of infarction. In the absence of necrosis and with full reperfusion, myocardial contractility will recover completely. However, full recovery may take several hours or days in vivo. This condition of delayed, but ultimately complete, recovery of contractile function after reperfusion of viable tissue is called "myocardial stunning" (Braunwald and Kloner, 1982; Kloner and Jennings, 2001). The multifactorial mechanisms underlying myocardial stunning are complex and beyond the scope of the present survey. However, ROS generation and intracellular calcium overload as a direct result of reperfusion are pivotal aspects of this pathology (Kusuoka et al., 1987; Bolli et al., 1989a,b; Opie, 1989). It is relevant to note here that the rate and/or extent of recovery of postischemic function is widely used as an injury index in experimental ischemia/reperfusion studies; in many such cases, contractile recovery is a mixed endpoint, representing both loss of contractility due to irreversible injury and delayed recovery of viable myocardium due to stunning.
In myocardium subjected to reduced perfusion, a further pattern of depression of contractility is termed myocardial "hibernation" (Rahimtoola, 1999; Schulz and Heusch, 2000). The essential features of hibernating myocardium are that metabolism and contractility are reduced in response to a reduction in coronary blood flow but that reperfusion can restore contractile function to normal. However, hibernation is a complex pathology, exhibiting alterations of cellular metabolism, myocardial structure, myocardial perfusion, and subendocardial flow reserve. The interested reader is referred to Canty and Fallavollita (2005) and Heusch (1998) for detailed reviews of the condition.
During ischemia, arrhythmias may develop, ranging in severity from isolated ventricular premature beats, through runs of ventricular tachycardia, to ventricular fibrillation (Tennant and Wiggers, 1935; Curtis et al., 1987; Carmeliet, 1999). Early arrhythmias (phase I arrhythmias) after coronary artery occlusion may contribute to sudden cardiac death following coronary occlusion (Janse and Wit, 1989). In experimental models of coronary occlusion, the incidence and duration of arrhythmias has been used as an injury index although it is important to note that arrhythmias develop before the onset of irreversible tissue injury. Reperfusion of myocardium after relatively brief periods of ischemia may also precipitate a pattern of arrhythmia ranging in severity (Manning and Hearse, 1984; Carmeliet, 1999). Clinically reperfusion-induced arrhythmia may be observed during thrombolysis (Goldberg et al., 1983) and after percutaneous coronary intervention (Holdright et al., 1996).
B. Experimental Approaches to Infarct Size Limitation
1. Historical Background.
Experimental research in the field of cardioprotection, which dates from the early 1970s when the concept of therapeutic infarct size limitation was first promoted by Eugene Braunwald and colleagues (Maroko et al., 1971), has resulted in a large and complex body of literature. Although a review of all the pharmacological approaches to infarct size limitation is impossible here, the quest during the 1970s and 1980s for drugs and other agents that could slow the development of or prevent myocardial necrosis was largely unsuccessful. It is possible now to sketch the major historical developments and to discern the conceptual and technical obstacles to the successful development of infarct-limiting treatments.
First of all, although experimental models of coronary artery occlusion provided extensive descriptive accounts of the morphological changes associated with the development of necrosis (Herdson et al., 1965; Jennings et al., 1965, 1978; Jennings and Ganote, 1974; Whalen et al., 1974; Schaper, 1979, 1986; Schaper et al., 1992), they provided relatively few insights into the molecular mechanisms underlying cell death. As a consequence, the early conceptual approaches to infarct limitation in the 1970s centered on agents to reduce myocardial oxygen demand or vasodilators to increase oxygen and metabolic substrate delivery (Maroko et al., 1971; Braunwald and Maroko, 1975). Hence, agents such as β-adrenoceptor antagonists (Burmeister et al., 1981; Reynolds et al., 1981; Downey et al., 1982); calcium channel blockers (Reimer and Jennings, 1984; Reimer et al., 1985; Wende et al., 1975); and glyceryl trinitrate (Bleifeld et al., 1973; Malm et al., 1979; Fukuyama et al., 1980) were extensively investigated with no consistently reproducible evidence of infarct limitation.
Second, the fact that reperfusion was essential to halt the progressive wave front of necrosis and salvage ischemic myocardium—a concept so obvious to us now—was largely unrecognized until the late 1970s. In numerous studies, including some of those cited above, drugs were administered in animal models of infarction with permanent coronary occlusion, with the assumption that tissue could be saved from necrosis within a small but important "border zone" between normal and ischemic tissue. Although reperfusion with fibrinolytic agents became rapidly established in the early 1980s as a primary approach in the therapy of acute myocardial infarction, numerous experimental studies in the 1980s continued to use models with permanent coronary artery occlusion. The controversial concept of an infarct border zone was almost certainly incorrect (Hearse, 1983).
Third, the recognition that reperfusion was associated with specific patterns of injury, collectively termed reperfusion injury, identified reperfusion arrhythmias and stunning as clear therapeutic targets. However, the concept of irreversible injury occurring as a result of reperfusion proved very controversial. Several pathological mechanisms associated with reperfusion, including ROS generation, intracellular calcium overload, and the recruitment of inflammatory cells, became the foci of basic studies in which agents were administered as adjuncts to reperfusion. Examples include the application at reperfusion of SOD (Jolly et al., 1984; Uraizee et al., 1987; Przyklenk and Kloner, 1989; Downey et al., 1991), adenosine and adenosine receptor agonists (for a comprehensive account of the early experimental literature, see Baxter et al., 2000), nonsteroidal anti-inflammatory drugs (Romson et al., 1982; Mullane et al., 1984; Allan et al., 1985; Reimer et al., 1985; Crawford et al., 1988), and antineutrophil antisera (for a review, see Baxter, 2002a). The resulting literature for nearly two decades was characterized by no clear sense of experimental reproducibility or consistency of interpretation with regard to pharmacological infarct limitation.
In retrospect, the development during the 1970s and 1980s of experimental infarct size limitation as a plausible scientific concept was hampered by the relatively late recognition that reperfusion is an essential requirement, both clinically and experimentally; and by limited understanding of appropriate molecular targets. The recognition of ischemic preconditioning from the late 1980s onward proved to be the most significant development in the quest to identify rational approaches to infarct size limitation. An explosion of research effort has identified a number of molecular mechanisms pertinent to cell death and cytoprotection that form the basis of contemporary experimental approaches.
2. The Reperfusion Injury Paradigm of Irreversible Injury. There is little question that unrelieved (i.e., permanent) ischemia causes cell death by coagulative necrosis (section II.A.1.). However, controversy has surrounded the destructive role played by reperfusion (Fig. 2, a and b). Until recently the predominating view was that cell death occurred largely during ischemia, fundamentally as a result of ATP depletion and its multiple consequences. In this older paradigm, reperfusion is essential to restore ATP synthesis and thereby to salvage those cells uninjured or reversibly injured by ischemia. Some argued that any cells that died during reperfusion were already irreversibly injured and doomed to die and that the death of cells irreversibly injured by ischemia was merely accelerated by reperfusion.
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). A key component of mitochondrial response to stress is the formation of the mPTP, a nonspecific, multimeric pore structure spanning the mitochondrial inner and outer membranes (Halestrap and Brenner, 2003; Bernardi et al., 2006). The precise molecular composition of the pore is currently unknown and the subject of intense debate. However, a key component is cyclophilin-D, a peptidyl-prolyl cis-trans isomerase that binds and is inhibited by cyclosporin A (Crompton et al., 1988). mPTP serves as a high-conductance, nonselective, voltage-dependent channel and is closed under normal physiological conditions when the inner membrane is impermeable to most solutes. Under conditions of cell stress, however, the formation of the open pore in the inner membrane results in the loss of membrane impermeability and rapid collapse of the mitochondrial membrane potential. The factors that determine whether and when mPTP opens during myocardial ischemia/reperfusion and evidence for mPTP opening contributing to ischemia/reperfusion injury have been studied extensively during the last two decades (Crompton et al., 1987; Duchen et al., 1993; Lemasters et al., 1997; Halestrap et al., 1998; Di Lisa et al., 2001; Halestrap et al., 2004; Di Lisa and Bernardi, 2006). The consensus of current opinions is that conditions during early reperfusion, but not during ischemia, favor the formation of the open pore and that inhibition of pore opening in reperfusion protects against cell death.
In the currently developing paradigm of ischemia/reperfusion injury and cardioprotection, formation or inhibition of mPTP at reperfusion is the primary determinant of cell death or survival. Evidence favors mPTP opening causing cell death by necrosis (Di Lisa et al., 2001), although it has been proposed that either apoptosis or necrosis might be precipitated, depending on the extent of mPTP opening (Halestrap et al., 2004). Although the molecular events occurring during ischemia determine whether mPTP opens during reperfusion, the corollary of this new view is that specific manipulation during reperfusion of conditions that inhibit mPTP opening offers the potential to attenuate cell death through reperfusion-specific cardioprotective strategies. As we shall discuss subsequently, ischemic preconditioning, pharmacological pretreatments that mimic preconditioning, ischemic postconditioning, and selected agents given at reperfusion may protect through a common mechanism of attenuating mPTP opening in early reperfusion.
In summary, the current view of reperfusion is that it is essential to salvage ischemic tissue. However, reperfusion has the potential to cause further irreversible cell injury, largely dependent on the duration of preceding ischemia, and this is closely linked to the extent of mPTP opening in early reperfusion. None of the recent information detracts from the proven therapeutic value of reperfusion, but it has prompted a reassessment of reperfusion injury and its mechanisms and the potential for therapeutic intervention to maximize the benefits of reperfusion in acute myocardial infarction.
3. Cardioprotection through Classic Preconditioning.
The formal description by Murry et al. (1986) of ischemic preconditioning presented an experimental phenomenon that was the most markedly protective intervention able to limit infarct size in a consistent and reproducible manner. They showed in the anesthetized dog that four 5-min periods of left anterior descending coronary artery occlusion, interspersed with 5-min reperfusion periods, before a 40-min occlusion of the same artery resulted in profound limitation of infarct size. This cardioprotective effect of ischemic preconditioning was independent of changes in transmural myocardial blood flow and Murry et al. proposed that the effect was a result of rapid metabolic adaptation of the ischemic myocardium. The wide reproducibility of this phenomenon using a variety of preconditioning protocols in a number of species and experimental preparations and with a number of endpoints of protection (section III.A.), rapidly led to ischemic preconditioning being established as a "gold standard" for cardioprotection. Various workers recognized that the cardioprotective potential of ischemic preconditioning is transient. For example, Van Winkle et al. (1991) showed that in rabbit myocardium, the protection against infarction afforded by a single 5-min preconditioning period was lost when the interval between the preconditioning stimulus and the infarct protocol was extended beyond 60 min. However, in 1993, two groups independently reported a recrudescence of protection 24 h after preconditioning in canine (Kuzuya et al., 1993) and rabbit (Marber et al., 1993) myocardium. Subsequently, the protection against infarction during this phase was shown to be present between 24 and 72 h after the preconditioning stimulus. The terms "second window of protection," "delayed preconditioning," or "late-phase preconditioning" have been applied to distinguish this late-onset, long-lasting phenomenon from the "classic" or "early" preconditioning effect originally described by Murry et al. (1986).
Since 1990, more than 3000 original studies have been published addressing various aspects of the molecular mechanisms of ischemic preconditioning in myocardium and other tissues; the majority of these have related to classic preconditioning. A comprehensive review of all of the investigated mechanisms is beyond the scope of this review. It is possible here to provide a broad summary of the major mechanisms that have been investigated and to synthesize the developments within the major schools of thought that have influenced the extrapolation of preconditioning mechanisms to potential therapeutic strategies. The molecular adaptation that underlies the remarkably potent but short-lasting protective effect seen in classic preconditioning is not fully understood and is undoubtedly complex (Fig. 3). The interested reader is referred to comprehensive reviews published elsewhere (Yellon et al., 1998; Schulz et al., 2001; Yellon and Downey, 2003).
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; Armstrong, 2004; Hausenloy and Yellon, 2006). The principal kinases that have been identified to exert obligatory roles in classic preconditioning are protein kinase C (PKC), the relevant isoforms of which may vary from one species to another (Liu et al., 1994; Meldrum et al., 1997; Budas et al., 2007), phosphatidylinositol 3-kinase (PI3K) and its substrate kinase Akt (also known as protein kinase B) (Tong et al., 2000; Mocanu et al., 2002; Mocanu and Yellon, 2007), p38 mitogen-activated protein kinase (MAPK) (Weinbrenner et al., 1997; Mocanu et al., 2000; Schulz et al., 2001; Steenbergen, 2002), p42/p44 MAPK/ERK (Ping et al., 1999; Strohm et al., 2000), receptor tyrosine kinases of the Src family (Maulik et al., 1996; Vahlhaus et al., 1998; Oldenburg et al., 2003), and the JAK/STAT pathway (Hattori et al., 2001; Barry et al., 2007). More recent interest has focused on glycogen synthase-3β (GSK-3β) as a distal kinase, phosphorylated (and hence inactivated) by other kinases including Akt and p42/p44 MAPK/ERK (Tong et al., 2002; Juhaszova et al., 2004). Much of the evidence supporting involvement of these protein kinases has relied on the demonstration that they are phosphorylated, translocated, or display increased activity in preconditioned myocardium or that pharmacological inhibitors of their activation blunt or abrogate the protection afforded by preconditioning.
The upstream triggers for activation of these kinases and the sequence of their activation in a multistep cascade are not clear; interpretation of numerous experimental studies is complicated by variations in species and endpoints. However, with regard to infarct size limitation, it seems likely that adenosine, bradykinin, opioid peptides, and prostaglandins released or accumulating in ischemic myocardium during the preconditioning stimulus, bind to G-protein-coupled receptors, namely adenosine A1 and A3, kinin B2, opioid 
1 and EP3, respectively (Gross and Gross, 2006). The relative importance of these autacoids may vary among species and according to the intensity of the preconditioning stimulus (Schulz et al., 1998). However, as a general summary, pharmacological blockade of individual receptors blunts or abolishes the protective effect of preconditioning whereas transient preischemic activation of any of the receptors with exogenous autacoid or synthetic receptor ligands induces protection ("pharmacological preconditioning") that is usually quantitatively similar to that seen with ischemic preconditioning.
b. ATP-sensitive potassium channels and classic preconditioning.
An intriguing component of the classic preconditioning mechanism is involvement of the ATP-dependent potassium channels (KATP) (Gross, 1995; Oldenburg et al., 2002; Gross and Peart, 2003; Ardehali and O'Rourke, 2005). KATP, expressed at the sarcolemma of cardiomyocytes, opens during hypoxia, ischemia, or metabolic inhibition, thereby facilitating increased potassium influx and shortening of action potential duration. Increased cellular potassium influx increases osmotic load and shortening of action potential duration is arrhythmogenic. The sarcolemmal KATP consists of pore forming [potassium inward rectifier (Kir)] and receptor subunits [sulfonylurea receptors (SURs)], the latter being blocked by substances such as glibenclamide or more specifically by HMR1098 and HMR1883. Nicorandil, cromakalim, and pinacidil are examples of drugs that open sarcolemmal KATP channels (O'Rourke, 2000).
In most species examined, glibenclamide abolishes the protective effect of preconditioning (Gross and Auchampach, 1992; Ferdinandy et al., 1995), whereas pharmacological openers of KATP confer protection quantitatively similar to that obtained with ischemic preconditioning (Grover et al., 1989; for a review, see Auchampach and Gross, 1994). It has been proposed that a PKC-induced increased trafficking of KATP to the sarcolemma (Budas et al., 2004), which opens sarcolemmal KATP causing shortening of myocyte action potential duration and reduced Ca2+ influx (Rainbow et al., 2004), is responsible for preconditioning-induced protection (Gross, 1995). However, a number of observations are inconsistent with this conjecture, including the demonstration of protection independent of surface current changes in isolated nonbeating myocytes (Garlid et al., 1997; Liu et al., 1998b) and the ability of some KATP openers to protect against ischemia/reperfusion injury at doses or concentrations that do not influence the action potential duration (Yao and Gross, 1994; Grover et al., 1995, 1996). Nevertheless, the importance of sarcolemmal KATP activation for pharmacological preconditioning with diazoxide and pinacidil (Tanno et al., 2001) or desflurane (Toller et al., 2000) as well as a trigger for the delayed phase of ischemic (Patel et al., 2005) or opioid-induced (Chen et al., 2003) preconditioning remains a matter of debate.
At the level of mitochondria, potassium flux across the inner mitochondrial membrane influences mitochondrial membrane potential, volume regulation, energy production, and calcium homeostasis. To allow potassium cycling across the potassium-impermeable inner membrane, mitochondria express antiporter (hydrogen/potassium) and potassium channels, one of these being the calcium-activated potassium channel and the other one being a mitochondrial KATP. Indeed, in mitoplasts using single-channel recordings by means of the patch-clamp technique, a potassium current was measured (15-82 pS), which was blocked by ATP or 5-hydroxydecanoic acid (5-HD) (Dahlem et al., 2004). Under physiological conditions at a mitochondrial membrane potential of approximately -180 mV, however, the channel should express minimal ion conductance (Dahlem et al., 2004). In isolated rat heart mitochondria, openers of the mitochondrial KATP such as pinacidil, cromakalim, and levcromakalim reduced mitochondrial membrane potential and increased mitochondrial respiration and radical formation, matrix volume, and calcium release from calcium-preloaded mitochondria, with all effects being dependent on the extramitochondrial potassium concentration (Holmuhamedov et al., 1998). Furthermore, in the mitochondria-enriched fraction from PC12 cells, specific antibodies against SUR1 and Kir6.1 detected immunoreactive proteins of the apparent molecular masses of 155 and 50 kDa (Tai et al., 2003), although the latter finding was questioned using rabbit ventricular cardiomyocytes (Seharaseyon et al., 2000). Most recently, fluorescence imaging of isolated mitochondria from rat adult cardiomyocytes expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing KATP were localized to mitochondria and that mitochondrial localization was increased by PKC activation (Garg and Hu, 2007); indeed mice genetically deficient in Kir6.2 display no preconditioning response (Suzuki et al., 2002). In 1997, Keith Garlid and Gary Grover (Garlid et al., 1997) proposed that a mitochondrial KATP might be the molecular target of the cardioprotective KATP opener drug diazoxide. Subsequently, several hundred studies have focused on opening of the mitochondrial KATP as a central mechanism of ischemic preconditioning (Ardehali and O'Rourke, 2005). Furthermore, opening of the calcium-activated potassium channel by NS-1619 before ischemia/reperfusion decreased infarct size in mice (Wang et al., 2004), highlighting the importance of mitochondrial potassium flux for cardioprotection.
Although there is little doubt that KATP opening is both protective and involved in the mechanism of preconditioning, some words of caution need to be made concerning the reliance on some of the pharmacological tools used to study mitochondrial KATP. These include openers of the mitochondrial KATP such as diazoxide, nicorandil, cromakalim, levcromakalim, bimakalim, and aprikalim; and inhibitors such as 5-HD, glibenclamide, and tetraphenylphosphonium (Table 1).
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In summary, the existing literature overwhelmingly supports a central role of sarcolemmal and mitochondrial KATP for the cardioprotection obtained by classic ischemic preconditioning as well as certain pharmacological strategies based on the preconditioning paradigm. Not surprisingly, however, many drugs used to open or inhibit mitochondrial KATP elicit significant additional effects relevant to cardioprotection, such as augmented NO release or mitochondrial uncoupling (Brennan et al., 2006), making a definitive conclusion on the underlying mechanism difficult.
c. The evolving model of signal transduction in classic preconditioning.
Jim Downey and Michael Cohen (Cohen et al., 2000a; Oldenburg et al., 2002; Critz et al., 2005) have proposed that adenosine, acting on A1 or A3 receptor subtypes, couples directly to PKC via phospholipase C and diacylglycerol formation. They proposed a hypothetical scheme wherein bradykinin and opioids, in contrast with adenosine, trigger a complex signal transduction pathway involving transactivation of receptor tyrosine kinase and subsequent activation of PI3K/Akt. Activated (phosphorylated) Akt phosphorylates endothelial NO synthase (eNOS) resulting in NO generation, activation of soluble guanylyl cyclase, cGMP accumulation, and activation of cGMP-dependent protein kinase (PKG). The roles of ROS, NO, reactive nitrogen species, and their downstream cellular targets are somewhat controversial in classic preconditioning (for extensive reviews, see Ferdinandy and Schulz, 2003; Jones and Bolli, 2006). However, it seems that ROS/RNS play a critical role in the signal transduction pathway, leading to activation of PKC (Otani, 2004).
Many potential substrates of PKC have been proposed as contributing to downstream mechanisms of preconditioning-induced protection and although PKC activation is clearly a necessary component of the mechanism of preconditioning in many studies, its functions and targets in classic preconditioning have until recently been unclear. Elegant studies by Garlid's group (Costa et al., 2005, 2006) have provided clearer insights and significant progress. PKG appears to be the terminal cytosolic step in the signal transduction cascade, phosphorylating an unknown target at the mitochondrial outer membrane. It is clear from the work of Costa et al. (2005) that mitoKATP opening is both PKG- and PKC
-dependent. However, PKG cannot phosphorylate mitoKATP channel proteins directly because it is too large to cross the mitochondrial outer membrane. They concluded that PKG phosphorylates an unknown target at the mitochondrial outer membrane that leads to subsequent activation of a PKC pool within the intermembrane space. Subsequently, Costa et al. (2006) demonstrated that PKG inhibits mPTP opening through a mechanism involving activation of two discreet mitochondrial pools of PKC. PKC1 promotes mitoKATP opening, leading to a modest increase in matrix H2O2. H2O2 acts as a signaling intermediate, promoting further PKC1 activation and activating PKC2, which inhibits mPTP formation. This work constitutes a convincing scheme that connects PKG, mitochondrial PKC, mitoKATP opening, ROS generation, and inhibition of mPTP.
Connexin-43 is another protein implicated in classic preconditioning. The protein forms the multimeric hemichannel structure of gap junctions in myocardium and appears to be obligatory for classic preconditioning, as hearts (Schwanke et al., 2002, 2003) or cardiomyocytes (Li et al., 2004) obtained from connexin-43 heterozygous knockout mice display no preconditioning response (Schulz and Heusch, 2004). Connexin-43, however, is, apart from its localization at the sarcolemma, also expressed in the inner membrane of cardiomyocyte mitochondria (Boengler et al., 2005), its transport being mediated by heat shock protein 90 and the translocator of the outer mitochondrial membrane (Rodriguez-Sinovas et al., 2006). Loss of connexin-43 decreases ROS formation secondary to diazoxide, leading to a loss of pharmacological preconditioning-induced protection (Heinzel et al., 2005).
4. Cardioprotection through Late Preconditioning.
Mechanistic investigation of late preconditioning has been far less extensive than that for classic preconditioning, but a picture has emerged of a phenomenon no less complex than classic preconditioning and possibly having some mechanisms in common. At the time of its formal description, late preconditioning was considered to be an adaptive phenomenon, mechanistically distinct from classic preconditioning. The original study by Marber et al. (1993) examined the hypothesis that transient ischemia/reperfusion stress caused the de novo synthesis of the putative cytoprotective protein, inducible 72-kDa heat shock protein (HSP72), and they showed a correlation between HSP72 induction and infarct size limitation 24 h after ischemic preconditioning in the rabbit heart. The study by Kuzuya et al. (1993), detailing the time course of loss and recrudescence of protection after preconditioning in the dog, was complemented by a related study by the same group (Hoshida et al., 1993) describing the time course of induction of the intracellular antioxidant SOD. Induction of cytoprotective factors such as intracellular antioxidants and HSPs had long been recognized as a conserved stress response in eukaryotes subjected to transient oxidant cellular stresses. However, the mechanisms regulating the induction of these factors were not well characterized in mammalian systems although transcriptional regulation of stress response genes was recognized in lower organisms (Mestril and Dillman, 1995; Yellon and Baxter, 1995; Baxter and Yellon, 1996). Nevertheless, HSPs have become emerging molecular targets for cardioprotective drug development (for a review, see Sõti et al., 2005).
A major conceptual development in delayed preconditioning was the recognition that autacoid factors released during preconditioning play an important role in eliciting the late-appearing adaptive response. At the time that late preconditioning was formally described, the important role played by adenosine as a mediator of classic preconditioning was already widely acknowledged. In 1994, Baxter et al. demonstrated in the same rabbit model of delayed preconditioning used by Marber et al. (1993) that pharmacological blockade of adenosine receptors during ischemic preconditioning abolished the development of protection 24 h later. Conversely, administration of 2-chloro-N6-cyclopentyladenosine, a selective A1 receptor agonist to naive rabbits induced a state of cardioprotection against infarction 24 to 72 h later (Baxter et al., 1994; Baxter and Yellon, 1997), mimicking the time course of delayed protection induced by ischemic preconditioning (Baxter et al., 1997). This was the first indication that adenosine, a mediator with a brief biological half-life, could elicit a biological effect evident many hours later, although a delayed and longlasting cardioprotection elicited by prostacyclin had been described several years previously in the work of Szekeres and colleagues (Szekeres, 2005). Bolli's group identified NO as a further obligatory trigger of late preconditioning in rabbit myocardium (Qiu et al., 1997), suggesting the participation of at least two independent autacoid triggers of delayed preconditioning in the rabbit. Subsequent work has identified the involvement of multiple endogenous triggers for delayed preconditioning, including adenosine, NO, bradykinin, cytokines and ROS, with some divergences related to species and experimental endpoint (e.g., infarct size versus myocardial stunning). Moreover, the exogenous administration of selective opioid receptor agonists is able to elicit a delayed cardioprotective response, although participation of endogenous opioid peptides in the late ischemic preconditioning response has not been specifically examined. For fuller descriptions, the reader is referred to focused reviews: Baxter and Yellon, 1998; Bolli, 2000; Baxter and Ferdinandy, 2001; Heusch, 2001; Baxter, 2002b; Dawn and Bolli, 2002; Ferdinandy and Schulz, 2003).
The nexus between upstream triggers of late preconditioning and the transcriptional and post-translational regulation of proteins associated with mediation of late protection involves a complex and poorly defined kinase cascade. Multiple studies using either ischemic preconditioning or pharmacological triggers of delayed protection have highlighted the involvement of PKC, especially PKC (Baxter et al., 1995; Ping et al., 1997; Vondriska et al., 2001), Src and Lck tyrosine kinases, probably downsteam of PKC (Imagawa et al., 1997; Dawn et al., 1999; Ping et al., 1999; Vondriska et al., 2001), the JAK/STAT signaling pathway (Dawn et al., 2004), p38 MAPK (Dana et al., 2000; Lasley et al., 2004; Fryer et al., 2001), PI3K and p70s6 kinase/mammalian target of rapamycin (Kis et al., 2003); and p42/p44 MAPK/ERK (Fryer et al., 2001). Although early activation of these kinases may occur in response to ischemic or pharmacological preconditioning triggers, some studies provide evidence for altered kinase activity 24 h after the preconditioning stimulus (e.g., p38 MAPK activity 24 h after A1 receptor agonist; Dana et al., 2000).
It is clear that delayed preconditioning recruits multiple signaling pathways that are highly dependent on the nature of the priming stimulus, e.g., transient ischemia or application of specific receptor ligands. The most complete account is given in the extensive studies of Bolli and colleagues (reviewed in Dawn and Bolli, 2002), a substantial body of work that has characterized late protection induced by ischemic preconditioning and NO donor compounds. The general scheme involves the interaction of eNOS-derived NO and superoxide to form peroxynitrite anion, which activates PKC. PKC activates Src and Lck tyrosine kinases. The activation of the transcription factor NF-
B occurs by dual serine and tyrosine phosphorylation of the inhibitor protein IKB-
by both PKC and tyrosine kinases. The cytoprotection-related proteins induced by NF-B-regulated gene expression include inducible NOS (iNOS) and cyclooxygenase-2 (COX-2). Inducible NOS-derived NO appears to regulate the activation of COX-2 in the preconditioned myocardium, determining a pattern of prostanoid generation that is critical for the appearance of a cardioprotected phenotype (Bolli et al., 2002). This reliance of late preconditioning on the up-regulation of iNOS and COX-2 proteins is convincingly demonstrated by a series of pharmacological and functional genomic studies involving pharmacological inhibition and genetic deletion of iNOS and COX-2. How COX-2-derived prostanoids ultimately exert their cytoprotective action, and their relationships with other cytoprotective mechanisms such as antioxidant enzymes, heme oxygenase (Ockaili et al., 2005), HSPs, mitoKATP channel opening (Baxter and Yellon, 1999), and mPTP inhibition (Hausenloy and Yellon, 2004) remain unknown. DNA microarray studies have revealed that preconditioning changes the gene expression pattern of rat hearts extensively, which suggests that very complex cellular mechanisms are involved in the evolution of late cardioprotection conferred by preconditioning (Onody et al., 2003). It is likely that delayed preconditioning involves the recruitment of multiple mechanisms of genetic adaptation, many of which have not been studied yet.
5. Cardioprotection through Postconditioning.
Despite the unquestioned need for reperfusion to prevent ischemic necrosis, in the past 5 years there has been accumulating experimental evidence that reperfusion per se is associated with the paradoxical activation of lethal signals that culminate in necrosis/apoptosis. The current development of concepts in this field of investigation is linked to the recent convergence of three major themes of investigation. The first is the promotion by Yellon and colleagues of the concept of antiapoptotic prosurvival kinases targeting against reperfusion injury (Yellon and Baxter, 1999; Hausenloy and Yellon, 2006). The second is a growing interest in and appreciation of the contribution of mPTP as a mediator of injury during reperfusion (Hausenloy et al., 2002). The third is the formal description in 2003 of a phenomenon termed ischemic postconditioning (Zhao et al., 2003). This area of investigation is now evolving rapidly and the current information suggests that reperfusion-induced cell injury contributes to a far greater extent than was previously accepted. Although many experimental studies during the last two decades have suggested that certain interventions administered immediately before or during the early moments of reperfusion could attenuate the extent of cell death and infarct size, these
