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The Hatter Institute for Cardiovascular Studies, University College London Hospital and Medical School, London, United Kingdom; and Department of Physiology, University of South Alabama College of Medicine, Mobile, Alabama
ABSTRACT I. INTRODUCTION: MYOCARDIAL PRECONDITIONING II. NATURAL HISTORY OF CLASSICAL ISCHEMIC PRECONDITIONING A. Triggers Versus Mediators B. Duration of Effect C. End Points of Preconditioning Studies 1. Infarct size 2. Stunning 3. Recovery of mechanical function 4. Arrhythmias 5. Electrocardiographic changes D. The Preconditioning Stimulus E. Preconditioning in Other Organs F. Remote Preconditioning III. CELLULAR MECHANISMS OF CLASSICAL PRECONDITIONING A. Trigger Mechanisms B. Mediators: Signal Transduction Pathways 1. PKC 2. Tyrosine kinase and the mitogen-activated protein kinases 3. Phosphatidylinositol 3-kinase C. KATP Channels 1. What are KATP channels? 2. The mitoKATP channels 3. How mitoKATP channels could be protective 4. Trigger role of mitoKATP channels 5. Free radicals and mitoKATP channels 6. Prostaglandins D. Possible End-Effectors 1. Metabolic effects 2. The mitoKATP channel 3. Mitochondrial permeability transition pore 4. The sodium/hydrogen exchanger 5. Osmotic swelling 6. Decreased cytoskeletal fragility 7. Apoptosis 8. Gap junctions 9. Free radicals 10. Tumor necrosis factor-{alpha} E. Summary of Classic Preconditioning IV. NATURAL HISTORY OF THE SECOND WINDOW OF PROTECTION A. What is SWOP? B. Triggers 1. Adenosine 2. NO 3. Bradykinin 4. Opioids 5. Other proposed triggers C. Mediators: Signal Transduction Pathways 1. PKC 2. Tyrosine kinases and MAPKs D. Possible End-Effectors 1. Heat stress proteins 2. Antioxidant enzyme systems 3. Cyclooxygenase 4. The mitoKATP channel 5. NO V. PRECONDITIONING HUMAN MYOCARDIUM A. In Vitro Studies 1. Human cell preparations 2. Human muscle preparations B. In Vivo Studies 1. Exercise-induced preconditioning 2. Preinfarction angina 3. Angioplasty studies 4. Surgical studies C. Therapeutic Implications D. Summary VI. CONCLUSION AND PERSPECTIVES
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2 h after the preconditioning ischemia followed a day later by a second window of protection lasting 3 days. Both types of preconditioning share similarities in that the preconditioning ischemia provokes the release of several autacoids that trigger protection by occupying cell surface receptors. Receptor occupancy activates complex signaling cascades which during the lethal ischemia converge on one or more end-effectors to mediate the protection. The end-effectors so far have eluded identification, although a number have been proposed. A range of different pharmacological agents that activate the signaling cascades at the various levels can mimic ischemic preconditioning leading to the hope that specific therapeutic agents can be designed to exploit the profound protection seen with ischemic preconditioning. This review examines, in detail, the complex mechanisms associated with both forms of preconditioning as well as discusses the possibility to exploit this phenomenon in the clinical setting. As our understanding of the mechanisms associated with preconditioning are unravelled, we believe we can look forward to the development of new therapeutic agents with novel mechanisms of action that can supplement current treatment options for patients threatened with acute myocardial infarction. |
I. INTRODUCTION: MYOCARDIAL PRECONDITIONING |
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In the early 1970s, attention was first focused on this problem in the animal laboratory. Maroko et al. (215) proposed that a variety of interventions at the time of coronary occlusion could reduce the size of the resulting infarct in the open-chest dog. At that time the models for evaluating infarct size were very crude, and none of the original interventions proposed, such as beta blockers (215), glucose-insulin-potassium (217), or hyaluronidase (216), ever proved to be effective. Nevertheless, those early studies initiated the search for interventions that could limit infarct size in the clinical setting. Jennings and Reimer (159) demonstrated that reperfusion was essential to protecting the ischemic myocardium, and soon thereafter, the introduction of thrombolytic therapies became routinely available. While reperfusion therapy clearly was reducing infarct size, as demonstrated by the extensive NIH-sponsored Thrombin Inhibition in Myocardial Ischemia (TIMI) trials, it also became clear that reperfusion had not eliminated infarction. Unfortunately, myocardium begins to die in minutes; however, dissolution of the offending thrombus and subsequent reperfusion usually requires hours to accomplish so there was still a need for an infarct sparing intervention. The quest was hampered by the fact that scientists did not (and still do not) know what the lethal event is in the ischemic heart. A number of candidates such as anti-inlammatory agents and free radical scavengers were examined through the early 1980s but all with mixed results (285).
It is interesting to note that until 1986 it was not even known whether therapeutic infarct size limitation was possible. While a rather large number of drugs were touted to limit infarct size in the setting of ischemia/reperfusion, none of these studies could be consistently reproduced in the animal laboratories. Much of the confusion at the time derived from the fact that the amount of infarct size limitation claimed by any of these studies was modest (10-20%) and that the animal models were still quite poor. Many of the baseline variables affecting infarct size such as temperature (65), risk zone size (409), and collateral flow (284) were poorly appreciated at that time and were seldom adequately controlled. In retrospect, most of those studies, including some published by the authors of this review, were probably simply false positives.
Perhaps the single greatest advance in our understanding of the cell survival machinery was the discovery in 1986 by Murry et al. (239) of an intrinsic mechanism of profound protection, which they termed ischemic preconditioning (see Fig. 1). In this seminal paper, they showed that four cycles of 5 min of ischemia with intermittent reperfusion were shown to limit infarct size by 75%, an amount of protection heretofore unheard of. More importantly in the subsequent years that followed, the anti-infarct effect of preconditioning could be reproduced by all who tried it (for a review of the early studies, see Ref. 192). For the first time it was shown that infarct size limitation was theoretically possible. Indeed, so powerful was the observed protection that this phenomenon has been recognized as "the strongest form of in vivo protection against myocardial ischemic injury other than early reperfusion" (175). It appeared therefore that all that remained was to investigate the mechanism associated with the profound protection in order for pharmacological mimetics to be developed that could ultimately be used in the clinical setting. At the time of writing this review there have been in excess of 2,000 papers published on this subject.
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II. NATURAL HISTORY OF CLASSICAL ISCHEMIC PRECONDITIONING |
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When considering preconditioning it is useful to think in terms of triggers, mediators, memory, and end-effectors. The preconditioning ischemia triggers a change in the physiology of the heart, rendering it very resistant to infarction. We know that a series of signal transduction pathways carry the signal for protection, and those presumably terminate on one or more end-effectors. The end-effectors actually cause the protection during the lethal ischemic insult (index ischemia) and/or the subsequent reperfusion period. Somewhere in the signal transduction pathways between the trigger signal and the end-effector is a memory element that is set by the preconditioning protocol and keeps the heart in a preconditioned state. All steps distal to the memory step including the end-effector can be classified as mediators since they exert their activity only after the index ischemia has begun, and those proximal to the memory element can be considered triggers because they exert their effect only prior to the index ischemia.
Ischemic preconditioning has been demonstrated in all animal species studied to date including the chicken (202), dog (239), mouse (322), pig (301), rabbit (72), rat (201), and sheep (53). All of our knowledge about preconditioning is empirically derived from studies in a variety of species including humans. Although it is assumed that preconditioning's mechanism is common to all species, there have been obvious mechanistic discrepancies among the various reports, and some of them could well be related to species differences. Some of these differences are obvious such as the role of xanthine oxidase as discussed below, but the reader should note the species being studied and consider the possibility that any reported mechanism may be species specific and more importantly may not be relevant to human heart. In terms of the extent of the protection observed, it must be noted that preconditioning's protection is lost when the ischemic insult was extended to 3 h, indicating that reperfusion after the lethal ischemic insult is an absolute requirement (239).
These observations indicate that ischemic preconditioning delays rather than prevents cell death. Most animal studies to date would suggest that preconditioning acts as if it had reduced the duration of the ischemic insult by 20-30 min. In primate myocardium, which for unknown reasons infarcts much more slowly, that benefit may be much longer (309). Murry and colleagues also found that the protection seen with preconditioning was independent of collateral flow indicating that the ability of the heart to withstand ischemia had been directly modified. The cardio-protection described by Murry et al. (239) has become known as "classic" or "early" ischemic preconditioning.
The preconditioned state is very transient following a preconditioning protocol and lasts for only 1-2 h in anesthetized animals (240, 287, 363) and is lost somewhere between 2 and 4 h in conscious rabbits (52). How the heart remembers that it is preconditioned is another mystery that has resisted laboratory investigation. Thornton et al. (340) reported that protein synthesis inhibition with either actinomycin D or cycloheximide did not block preconditioning's protection seemingly eliminating gene expression as a possible mechanism of the memory. Matsuyama et al. (219) also obtained the same result with actinomycin D; however, using a much higher concentration of cycloheximide, they were able to block protection from ischemic preconditioning. They suggested that because cycloheximide only blocks translation of message that preconditioning causes the translation of a prexisting mRNA coding for a protective protein. Since the initial study by Thornton et al. directly confirmed protein synthesis inhibition, the most likely explanation is that the latter study may have suffered from a nonspecific effect due to the high cycloheximide concentration. Also, because the preconditioned state can be achieved within 10 min, it is unlikely that any protein could be expressed in such a short time period. The memory information is probably carried as a reversible posttranslational modification of some preexisting protein (such as a phosphorylation or translocation), but the site of that modification is unknown.
Although the initial window of protection is quite transient, a delayed form of the protection reappears within 24 h of the preconditioning stimulus, which has been referred to as the second window of protection (SWOP) (213). The less robust, although more prolonged, SWOP occurs between 12 and 72 h after a preconditioning stimulus (26) (see Fig. 2). Both classical and SWOP preconditioning share some similarities. In both cases the preconditioning ischemia provokes the release of a number of trigger substances that interact with cell surface receptors, thereby initiating a signaling cascade of events. These triggers appear to be the same in both forms of preconditioning. The time course within which the SWOP confers protection allows for the possibility of new protein synthesis, posttranslational protein modification, and a change in the compartmentalisation of existing proteins. The mechanisms of SWOP will be discussed in greater detail in section IVB.
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C. End Points of Preconditioning Studies
The original end point used for the assessment of protection from classical preconditioning was infarct size (239) expressed as a percentage of the risk zone. Many animals have coronary collateral vessels that provide a low level of irrigation during ischemia. Collateral flow opposes infarction causing an inverse relationship between collateral flow and infarct size. In animals with preformed collaterals such as the dog, collateral flow must be taken into account (284). Infarct size is commonly assessed by staining viable tissue with tetrazolium salts. The tetrazolium salts react with NADH and dehydrogenase enzymes staining the tissue (174). Dead cells lose enzyme and cofactor due to membrane failure and thus do not stain. To sufficiently wash out these components 2-3 h of reperfusion are required. The gold standard for infarct size, however, is histological examination of tissue slices after 3 or more days of reperfusion as was used by Murry et al. in their original description of preconditioning (239). Under these conditions, classical preconditioning has limited infarct size in every species tested.
Whether preconditioning attenuates stunning has not been totally resolved. Stunning refers to a loss of contractility that immediately follows a sublethal ischemic insult. Unlike the infarcted heart, the stunned myocardium recovers fully in a day or two. For a review on the stunned myocardium, see Reference 48. In most species an ischemic insult of 15 min or less stuns the heart but does not cause infarction. Ovize et al. (258) studied the dog heart which, unlike the human heart, is rich in xanthine oxidase. They preconditioned hearts with either a 2.5- or a 5-min period of ischemia before the 15-min index ischemia. Although recovery of function in the preconditioned hearts was twice that in the control group, the authors concluded that prior preconditioning did not influence the degree of recovery. This conclusion was based on an analysis in which postischemic function was related to the level of collateral blood flow in each heart. The improved recovery in preconditioned hearts was attributed to better perfusion during ischemia rather than preconditioning. Examination of those data reveals, however, that there was no significant correlation between collateral flow and recovery of function in two of the three experimental groups, indicating that the assumption regarding the influence of collateral flow could not be supported. Hence, the authors' conclusion may not be firm, and preconditioning rather than higher collateral flow may well have protected against stunning in that study. Jenkins et al. (158) noted that preconditioning did not affect stunning in rabbits, a species whose hearts are deficient in xanthine oxidase. Landymore et al. (190) noted that preconditioning prevented stunning after cardiac transplantation in sheep, but it is not known whether sheep hearts contain xanthine oxidase. Finally, Sekili et al. (307) noted that when dog hearts were pharmacologically preconditioned with a transient infusion of adenosine that no protection against stunning could be observed. The pharmacological preconditioning would not have attenuated adenosine release on a subsequent coronary occlusion (121). If classical preconditioning has an antistunning effect it must be small. That is surprising because SWOP reportedly has a robust antistunning effect in both rabbits (47) and pigs (334).
3. Recovery of mechanical function
Postischemic recovery of contractile function is a commonly used end point for ischemic preconditioning in the isolated rat heart (22, 61, 380). However, recovery of mechanical function after an ischemic insult is influenced by both a combination of the number of surviving myocytes and the degree to which they have been stunned. It is well appreciated that the effect of preconditioning on recovery of function is much less pronounced in species other than the rat (318).
Gelpi et al. (117) proposed that the antistunning effect in the rat might be the result of altered adenosine metabolism in the rat's heart. Free radicals have been shown to strongly contribute to stunning of reperfused myocardium (48), and rat hearts are rich in xanthine oxidase (97). Adenosine released during ischemia is converted to inosine and then hypoxanthine. Upon reperfusion, hypoxanthine is oxidized by xanthine oxidase producing injurious free radicals that stun the heart (51, 62, 64). Preconditioning the heart with a brief period of ischemia followed by reperfusion will greatly attenuate the amount of adenosine released during the next ischemic episode (364), which in turn would reduce the amount of xanthine oxidase-mediated free radical production and thus stunning. Indeed, Gelpi et al. (117) found that the xanthine oxidase inhibitor allopurinol improved postischemic function and greatly attenuated the improvement from preconditioning in the rat model.
In rabbit hearts, which do not contain xanthine oxidase, preconditioning had no effect on postischemic function. To further complicate the situation, the mechanism by which preconditioning attenuates purine release seems to be unrelated to that used by the anti-infarct effect (121) so that extrapolation of data from the rat recovery of function model to preconditioning in humans may be very misleading.
The overall aim of myocardial salvage is to improve ventricular function. Oddly enough few studies have tested whether preconditioning really does yield a stronger heart in the animal laboratory. Cohen et al. (76) measured ventricular wall motion in conscious rabbits subjected to regional ischemia/reperfusion. Not only did preconditioning reduce infarct size, but it also improved wall motion in the ischemic zone. It took at least a day for stunning to subside however before the benefit could be appreciated.
Ischemic preconditioning has also been reported to alter the incidence of ischemia and reperfusion-induced arrhythmias in dogs (367) and rats (310). The experience with most investigators studying other species has been that preconditioning either has little effect on arrhythmias or actually exacerbates it (256). Free radicals are also known to lead to the genesis of arrhythmias in the heart (187), and it is likely that antiarrhythmic effect of preconditioning in dog and rat may again be related to attenuated purine release, although that hypothesis has not been directly tested.
5. Electrocardiographic changes
It must be emphasized that infarct size testing is the only established end point for preconditioning at this time, and extrapolated findings from recovery of contractile function or arrhythmias need to be interpreted with caution. Unfortunately, studies in humans have forced investigators to examine surrogate end points such as electrocardiographic changes. Cribier et al. (80) noted that in patients undergoing coronary angioplasty that the S-T segment voltage was much lower during the second coronary occlusion than on the first. He concluded that this reflected the protection of preconditioning from the first balloon inflation. A number of drugs were subsequently tested in this setting to see if they could mimic or block preconditioning in humans (for a review, see Ref. 344). One criticism of the technique was that the reduced S-T segment voltage might only have reflected opening of collateral vessels between the inflations. Shattock et al. (308) disproved that hypothesis by showing that the same response could be seen in pig heart, which is collateral deficient. It was later shown that the S-T segment changes were influenced by surface ATP-sensitive potassium (KATP) channels while protection from preconditioning was influenced by those in the mitochondria (41). It would appear that preconditioning opens both populations of channels while only the mitochondrial population acts to protect (see below). Thus S-T segment changes may not be a reliable end point for preconditioning studies either.
D. The Preconditioning Stimulus
Ischemic preconditioning requires a brief period of ischemia followed by reperfusion to trigger the response. The minimum period of reperfusion required giving protection after the preconditioning ischemia lies between 30 s and 1 min (5). Preconditioning was originally reported to be an "all or none" phenomenon. Li et al. (200) compared 1, 6, and 12 cycles of 5-min coronary occlusions in the dog and found no differences in the protection. Another report (363) found no differences between one and two 5-min cycles of preconditioning in the rabbit. A similar observation was made in ex vivo human cardiac tissue (236). On the other hand, studies using in vivo rat (24a) or pig (304) models found that the resulting infarct size varied with the strength of the preconditioning stimulus, suggesting a graded response. Off-on systems are rare in nature, so more than likely preconditioning is merely following a very steep dose-response curve. Once a maximal response is achieved, further stimulation has no additional effect, giving the impression of an all or none system. Many studies take advantage of this very steep dose-response relationship. A single 2-min coronary occlusion will not precondition the rabbit as it is below threshold (363). However, in the presence of an intervention that potentiates the triggers of preconditioning, such as angiotensin-converting enzyme (ACE) inhibitors which augment interstitial bradykinin levels (155, 227) or agents that augment adenosine such as acadasine (52), preconditioning will occur.
E. Preconditioning in Other Organs
Preconditioning was first described in the heart but since then it has been seen in various forms in a variety of organs. Classical preconditioning is seen in skeletal muscle (262), and its mechanism seems to be virtually identical to that seen in the heart directly protecting the parenchymal cells (261). Classical preconditioning has also been described in the gut (152) and in the kidney (49). In the intestine, the microcirculation is the primary target for ischemic injury while in the kidney it is the proximal tubular cells. Yet the result is the same in all three tissues, a rapid protection against cell death during ischemia. In the brain (171), preconditioning is only protective in a second window-type setting a day after the preconditioning stimulus. Thus it would appear that preconditioning represents a generalized adaptation to protect a wide variety of cells against stressful stimuli such as ischemia.
One form of preconditioning that is poorly understood is remote preconditioning. In 1993 Przyklenk et al. (278) reported that preconditioning one region of the dog heart caused protection in a remote region. They hypothesized that a circulating humor or perhaps a neural reflex triggered protection in the remote region. Similarly mesenteric artery occlusion was also seen to result in protection of the rat heart, further supporting the hypothesis (368), and it was subsequently found that blockade of opioid receptors in the rat blocked that response (266), suggesting either a circulating endorphin or neural link. Nakano et al. (246) tried to duplicate the effect in a rabbit model where a region of the heart was preconditioned in situ and then the heart was removed and exposed to global ischemia. The amount of infarction was measured in both the preconditioned and the nonpreconditioned regions, but no differences were seen. It was concluded that preconditioning one region of the heart does not necessarily precondition the remote regions in all species.
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III. CELLULAR MECHANISMS OF CLASSICAL PRECONDITIONING |
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In 1991 Downey and colleagues (206) found that the adenosine A1 receptor acts to trigger ischemic preconditioning's protection in the rabbit heart, revealing that ischemic preconditioning is receptor mediated. Blockers of the A1 receptor eliminated preconditioning's protection while transient exposure to an A1 agonist would confer the protection. Shortly thereafter, Banerjee et al. (22) reported a similar situation with norepinephrine through the 
-receptors in the rat heart. We now know that any Gi-coupled receptor can trigger the preconditioned state, and in fact, multiple receptors work in parallel to provide redundancy to the preconditioning stimulus. During a brief ischemic period, the heart appears to release adenosine, bradykinin, norepinephrine, and opioids. Population of their respective receptors then triggers the preconditioned state through activation of Gi protein. As a result, blockade of the bradykinin receptor in the rabbit blocks protection from a single cycle of preconditioning but not from multiple cycles (122). Thus blockade of a single receptor type acts only to raise the ischemic threshold required to trigger protection rather than completely block it (see Fig. 3). The cardiac myocytes express other Gi receptors such as the angiotensin AT1, the endothelin ET1, and the muscarinic receptors that can also trigger a preconditioned state but do not seem to participate in ischemic preconditioning simply because agonists to those receptors are not produced in the ischemic myocardiaum. For a detailed review of this receptor interplay, see Reference 71.
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Free radicals also act as trigger mechanisms to preconditioning. Treatment with a free radical scavenger can raise the threshold of preconditioning, and a free radical generator can trigger a preconditioned state (17, 354). It is thought that the free radicals act to directly activate protective kinases (120, 369). In species whose hearts are rich in xanthine oxidase, the free radicals may derive directly from xanthine oxidase's action on purine catabolism. This conceivably could lead to a nonreceptor-mediated triggering of protection. In other species, such as the rabbit heart, the free radicals seem to have a much more complex origin, as will be discussed later in this review.
Several other non-receptor-triggered forms of preconditioning have been described. Ashraf's group (230) has found that a short period of elevated Ca2+ in the coronary perfusate will put the isolated rat heart into a protected state that appears to be the same as that from ischemic preconditioning as it is protein kinase C (PKC) dependent (see sect. IIIB1). Furthermore, they found that the calcium channel blocker verapamil could block not only Ca2+-induced preconditioning in the rat heart but ischemic preconditioning as well (229). A similar observation was made in an in situ dog model where intracoronary calcium chloride triggered protection and ischemic preconditioning could be blocked with a sodium/calcium exchanger blocker (279).
A transient period of hyperthermia has also seemed to be cardioprotective with both a transient early phase and a prolonged late phase (395). Whether the early phase uses the preconditioning mechanism is unknown. Stretch of the myocardial fibers is reported to precondition the dog heart (257), and the protection could be blocked by gadolinium, a blocker of stretch-activated channels. Transient exposure to ethanol can trigger the preconditioned state, but interestingly, if it continues to be present during the index ischemia, it will block its own protection (185). Other forms of preconditioning such as pacing (183, 263) and hypoxia (372) likely induce their protection through release of receptor ligands due to the negative energy balance.
Although nitric oxide (NO) has been linked to the trigger and end-effector phases of delayed preconditioning (see later), evidence for its role in early preconditioning is limited if not controversial (210, 247, 277, 379, 384). It appears that in early preconditioning, NO may lower the threshold for the protection observed, even though in itself it may not be a direct trigger of early preconditioning (36). Importantly, because exogenous (not endogenous) NO has been shown to trigger preconditioning both in rabbits (247) as well as in the endothelial NO synthase (eNOS) knock-out mouse (36), it appears as if the downstream targets for NO remain intact and as such may be pivotal in mediating the protection.
B. Mediators: Signal Transduction Pathways
The role of PKC in preconditioning was codiscovered by Mitchell et al. (228) and Ytrehus et al. (408) in 1994. It can be shown that blockade of PKC eliminates the protection from a preconditioned heart but has no effect on a nonpreconditioned heart. Similarly, activation of PKC by phorbol esters will put the heart into a preconditioned state. PKC is a serine/threonine kinase that is activated by lipid cofactors derived from breakdown of membrane lipids by phospholipase C. There are multiple isoforms of PKC in the heart, each having a similar substrate specificity. They can be broken down into the classical isoforms , 
, and 
, which are dependent on both the lipid cofactor diacylglycerol (DAG) and calcium. The novel isoforms, 
, 
, and 
, are calcium independent needing only DAG. Finally, the atypical isoform 
requires neither DAG nor calcium. The PKC isoforms are thought to achieve specificity by their peculiar physical translocation to docking sites. Mochly-Rosen and colleagues (161) discovered that each isoform will dock on a unique binding protein called a receptor for activated C kinase (RACK) when activated. These RACKS are strategically located only on certain organelles within the cell to bring the PKC isoform in proximity to a specific substrate protein. Binding to the RACK completes the activation and causes the isoform to phosphorylate any nearby substrate. It is thought that only certain isoforms participate in preconditioning. The (125, 203, 274), the (412), and the (374) isoforms of PKC have all been proposed to be responsible for preconditioning's protection. The intracellular targets of PKC have not been established.
One of the great mysteries of preconditioning is its memory. Transient activation of the Gi-coupled receptors puts the heart into a preconditioned state lasting 1 h. Receptor occupation is a trigger of the preconditioned state, and thus the critical time for receptor occupation is during the preconditioning ischemia before the index ischemia (204). This is in contrast to administration of a PKC blocker. If staurosporine, a potent PKC blocker, is given with the same schedule, then protection persists. It would be logical that PKC would phosphorylate substrate during preconditioning and then as long as the substrate remained phosphorylated the heart would be protected. Waning of protection would result from dephosphorylation by phosphatases. Unfortunately, the facts do not fit the theory. The kinase activity of PKC can be completely blocked with staurosporine during preconditioning, and the heart still enters a preconditioned state (398). Only if the staurosporine is present during the 30-min index ischemia is protection aborted. Thus PKC is a mediator rather than a trigger of protection, and the memory step must reside upstream of PKC activity. One theory of preconditioning's memory that has never been proven nor disproved is the translocation of PKC hypothesis. Activation of PKCs requires the physical translocation of the enzymes to their docking sites. It was proposed early on that such translocations would position the kinase for early phosphorylation of substrate with a subsequent occlusion (209).
Kitakaze et al. (173) proposed that the memory in preconditioning is due to the activation of 5'-nucleotidase by PKC. When activated the heart would generate more adenosine from the breakdown of ATP during ischemia, and the adenosine would directly protect the heart. In that case the PKC would be the trigger and adenosine the mediator. The evidence supporting this was that blockade of 5'-nucleotidase blocked the protection of preconditioning in a dog model (173). There are several arguments to this hypothesis however. Although a PKC inhibitor could block protection triggered by adenosine in rabbits, an adenosine receptor blocker could not block protection from a PKC activator (149). That would put adenosine upstream of PKC rather than downstream. Subsequently, Silva et al. (315) found that augmenting interstitial adenosine manyfold during ischemia with an adenosine deaminase inhibitor offered no limitation of infarct size in the dog heart, indicating that elevated adenosine during ischemia alone does not protect. Protection must be triggered by adenosine receptor occupation before ischemia. More than likely the blockade of 5'-nucleotidase in Kitakaze's experiment simply removed the adenosine trigger for preconditioning.
2. Tyrosine kinase and the mitogen-activated protein kinases
Other kinases have been identified. Using genistein, a broad spectrum tyrosine kinase inhibitor, Maulik et al. (221) found that it could block protection from ischemic preconditioning and proposed that at least one tyrosine kinase is in the overall pathway. Baines et al. (20) provided evidence that a tyrosine kinase was downstream of PKC; however, several other studies suggest that it (or another one) may be in parallel with PKC in both pig (358) and rat (111). The dynamics of the parallel arrangement are interesting. When a mild preconditioning stimulus such as a single 5-min coronary occlusion is given, either a PKC or a tyrosine kinase blocker on its own will block protection, suggesting that both pathways must be activated to achieve threshold for protection. When a more robust stimulus is used, however, then blocking either pathway alone will not block protection, and both inhibitors must be present. This suggests that either pathway can be protective on its own if stimulated enough (336).
Maulik et al. (221) proposed that the tyrosine kinase in question was the 38-kDa stress-activated mitogen-activated protein kinase (MAPK) (p38 MAPK). What followed has to be one of the most confusing chapters in the ischemic preconditioning stories. Each subfamily of the MAPK family, the 42/44-kDa extracellular receptor kinase (ERK), the 46/54-kDa c-jun kinase (JNK), and the 38-kDa p38 MAPK, has been suggested to play a role in the cardioprotection achieved by ischemic preconditioning (for review, see Refs. 226, 270). All of the MAPKs are activated by dual phosphorylation of a serine and a threonine by a MAPK kinase. The MAPK kinase is a tyrosine kinase and at least the ones targeting p38 MAPK can be blocked by genistein (244).
In isolated rabbit hearts, ERK1 activity reportedly increases only in ischemically preconditioned myocardium (170), but no difference in ERK1 and ERK2 phosphorylation between nonpreconditioned and preconditioned myocardium is detectable in pigs in vivo (33). Like p38 MAPK (see below), ERK can activate MAPKAP kinase 2 and , leading to phosphorylation of the small heat shock protein, hsp27 (300).
A causal role of ERK activation in the cardioprotection achieved by ischemic preconditioning is controversial. While PD 98059, an inhibitor of ERK, fails to block the infarct size reduction seen after ischemic preconditioning in isolated rabbit and rat (233) hearts (170), its intramyocardial infusion appeared to abolish ischemic preconditioning's protection in pigs in vivo (321). It is of interest that ERK1 forms a signaling complex with PKC- in the heart along with other MAPK kinases (21). Thus translocations of MAPKs may be involved in the signaling in addition to their phosphorylation.
Both JNK46 and JNK54 are present in the heart (67) and are strongly activated during reperfusion after ischemia. Their activation/phosphorylation during ischemia has been suggested by some studies (33), but in another, a reduction in JNK phosphorylation during ischemia was reported (244). JNK46 activation during no-flow ischemia is most likely mediated by PKC, since activation of JNK46 is completely blocked by chelerythrine, a PKC inhibitor (273). Anisomycin, an activator of both JNK and p38 MAPK, reduces infarct size in rabbits (18) and pigs (23). In isolated rat hearts, blockade of JNK46 with curcumin blocks the infarct size reduction of ischemic preconditioning to a similar extent as blockade of p38 MAPK using SB203580 (294).
Most attention has focused on the p38 MAPK cascade. At least five isoforms of p38 MAPK have been identified, although only p38 - and -MAPK are expressed to any degree within the heart (298). Different isoforms of p38 MAPK appear to mediate different biological functions (for review, see Ref. 226). In neonatal rat cardiomyocytes, p38 -MAPK mediates apoptosis, whereas p38 -MAPK is antiapoptotic (375).
The activation patterns of p38 MAPK in ischemic heart vary widely between reports. p38 MAPK, like all of the MAPKs, has two amino acids that must be phosphorylated for activation: a threonine residue at amino acid 180 and a tyrosine residue at site 182. This kinase is activated by MAPK kinase (MEK) 3 and 6, which in turn are activated by MAPK kinase kinases (MKK). The phosphorylation of p38 MAPK can be measured with phosphospecific antibodies. p38 MAPK is phosphorylated within minutes during global or regional no-flow ischemia in isolated rat hearts (43, 105, 221), as well as in rat (311, 407), dog (291), and pig hearts in vivo (24, 33).
With prolongation of ischemia, however, the phosphorylation of p38 MAPK may be reduced toward preischemic values, whereas phosphorylation is once again increased upon reperfusion (311, 407). The transient p38 MAPK activation during prolonged ischemia might be related to decreased phosphorylation or increased dephosphorylation by phosphatases (for review, see Ref. 299). In contrast to the above studies, no activation of p38 MAPK by ischemia per se is seen in nonpreconditioned isolated rabbit hearts (211, 244, 377). Following ischemic preconditioning, phosphorylation of p38 during the index ischemia is reported to be increased in isolated rat and rabbit hearts (220, 221, 244, 377), unaltered in pig hearts in vivo (33), and even decreased in dog hearts in vivo (291). Ischemic preconditioning reportedly prevents the ischemia-induced activation of p38 -MAPK in rat cardiomyocytes (298). At present, it is difficult to see any consistent pattern in the data. Much of the variability in the above reports may stem from technical problems in the processing of the tissue as phosphatases remove these phosphate groups quickly and dephosphorylation can occur with freezing and thawing in the presence of even the best phosphatase inhibitors.
The importance of p38 activation for cardioprotection also is controversial. Rat cardiomyocytes transfected with a dominant negative p38 isoform, which prevents ischemia-induced p38 activation, are more resistant to lethal simulated ischemia (298). Similarly, in isolated rat hearts (300) and pig hearts in vivo (24), blockade of p38 with SB 203580 did not affect the infarct size reduction achieved by ischemic preconditioning, but reduced infarct size in nonpreconditioned hearts. In total contrast, SB 203580 effectively blocked preconditioning's protection in other cell models (13, 242, 377) and abolished the infarct size reduction of ischemic preconditioning in isolated rat heart (221, 232) and the isolated rabbit heart (245) and dog hearts in vivo (291).
Explanations for the controversial findings might relate to the relative balance between different isoforms of p38 in different species and experimental models as well as the selectivity of different inhibitors in a given dose range. SB 203580, for example, not only inhibits p38 MAPK (81), but also dose-dependently inhibits JNK (68), tyrosine kinases such as p56 lck and c-src (370), and cyclooxygenase (50), and it activates c-raf (105). To further complicate matters, some of the above kinases have been implicated in ischemic preconditioning by various investigators.
3. Phosphatidylinositol 3-kinase
Recent evidence has implicated phosphatidylinositol 3-OH kinase (PI 3-kinase) in the signaling of classical preconditioning. Tong et al. (352) were first to report that the PI 3-kinase inhibitor wortmannin could block protection from preconditioning using contractile dysfunction as the end point. That was subsequently confirmed by Mocanu et al. (233) using an infarct size model. PI 3-kinase has been implicated as protective in other cell systems (90). The question with respect to preconditioning is, where is PI 3-kinase located in the signaling system? This is discussed in more detail in section IIIC5.
KATP channels have been shown over the last 10 years to be an important mediator of cardioprotection, and their role in ischemic preconditioning has been demonstrated in whole animals, isolated hearts, and cardiac myocytes. KATP channels were first described by Noma (249) in cardiac ventricular myocytes. These potassium channels are termed ATP sensitive because they are normally inhibited by physiological levels of ATP. KATP channels are modulated by pH, fatty acids, NO, SH-redox state, various nucleotides, G proteins, and various ligands (adenosine, acetylcholine, etc.) (102, 172).
With regard to preconditioning, there is general consensus that KATP channel plays a key role in preconditioning. Gross et al. (129) were the first to propose that opening of the KATP channel was involved in preconditioning's protection. Studies have shown that not only do KATP channel openers mimic preconditioning, but that blockers abolish the ischemic preconditioning's protection (7, 129, 305, 362). Further studies have shown that the preconditioning mimicked by adenosine A1 receptor stimulation can also be abolished by glibenclamide, suggesting adenosine receptor activation to be upstream of KATP channel activation (131, 362).
It was initially assumed that the sarcolemmal KATP channel was the end-effector of preconditioning's protection, and this protection was originally ascribed to shortening of the action potential (for review, see Ref. 130). At the time of these early observations it was not appreciated that cardiomyocytes contained two different types of KATP channels, sarcolemmal (surface KATP) and mitochondrial (mitoKATP), and that each had a distinct pharmacological profile. However, Garlid et al. (115) and Liu et al. (208) subsequently provided convincing evidence in a recovery of function models and a cardiomyocyte model, respectively, that it was not the surface but the mitoKATP channel that was responsible for the protection. Although some data, particularly studies using HMR 1098, a selective sarcolemmal KATP channel blocker (342), still suggest a critical role for the sarcolemmal KATP channel, most evidence is consistent with the mitochondrial rather than the surface channel as being most important. It should be kept in mind, of course, that virtually all of that theory hinges on pharmacological evidence that could still prove to be flawed.
The KATP channel consists of an inward rectifying potassium channel (Kir) in association with a sulfonylurea binding protein (Sur). Several isoforms of each exist, and the channels can assemble in different combinations. The mitoKATP channel is thought to have a similar structure to the sarcolemmal KATP channel with both a Kir and Sur subunit, although the exact composition of the mitoKATP channel has not been resolved. However, there are differential pharmacological responses of the cardiac sarcolemmal and mitoKATP channels. 5-Hydroxydecanoate (5-HD) inhibits mitoKATP channels in the micromolar range, but not sarcolemmal KATP channels under any concentration. Diazoxide, a KATP channel opener, has been shown to be 1,000 times more potent in opening mitoKATP channels than sarcolemmal KATP channels (116).
In addition, diazoxide-induced cardioprotection has been demonstrated in the micromolar range without any action potential duration (APD) shortening, excluding sarcolemmal KATP channel involvement (115). Baines et al. (18) were the first to show that diazoxide could limit infarct size and that 5-HD could block the anti-infarct effect of both diazoxide and ischemic preconditioning. Not all data are supportive of the mitoKATP role in preconditioning. Recently, Kir6.2 knock-out mice were found to have no functioning sarcolemmal KATP channels and also could not be preconditioned despite the fact that their mitoKATP were still intact (325).
3. How mitoKATP channels could be protective
The mitochondria make ATP by allowing H+ extruded by the electron transport apparatus to reenter along a strong electrochemical gradient through the F1 apparatus. In so doing ADP is phosphorylated to ATP. Opening the KATP channel will cause potassium to enter mitochondria along its favorable electrochemical gradient. A potassium/hydrogen exchanger on the inner mitochondrial membrane allows intramitochondrial potassium to exchange for extramitochondrial H+. Entering H+ would theoretically uncouple the mitochondrion because it bypasses F1 and hence reduces ATP production. In actuality, however, the amount of uncoupling resulting from potassium entry is very small (estimated to be 5 mV), assumed to be caused by a low density of channels in the inner membrane (114). Terzic's group has reported the greatest change, which was 10 mV with a baseline of -180 mV in isolated mitochondria (143). These data were confirmed in the intact cell by Minners et al. (227a). The potassium that enters is, however, osmotically active and will cause the matrix to swell.
There are several theories that seek to explain why opening the mitoKATP channels should be protective. Terzic and co-workers (144) found that opening mitoKATP channels made isolated mitochondria more resistant to Ca2+ entry. Garlid and co-workers (95, 189) suggest that mitochondrial swelling subsequent to potassium entry causes preservation of the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase on the outer membrane through which ADP traditionally enters the intermembrane space. That juxtaposition effectively keeps ADP out of the intermembrane space and forces the mitochondria to phosphorylate only creatine, which is the most efficient means of transferring energy to the cytoplasm. Of course all of these theories assume that the mitoKATP channel is the end-effector of preconditioning's protection.
4. Trigger role of mitoKATP channels
Recent experiments have reexamined the assumption that mitoKATP channels are only the end-effectors of protection. Liu et al. (208) introduced a cardiomyocyte model in which FADH fluorescence was monitored. The slight uncoupling with mitoKATP channel opening was proposed to slightly oxidize the flavoproteins and increase their fluorescence. These fluorescence studies showed that the effects of both diazoxide and 5-HD are readily reversible when the drugs are washed out. Yet Ashraf's group (376) reported that a 5-min pulse of diazoxide followed by washout put the rat heart into a preconditioned state even though the mitoKATP channels should have reclosed when the index ischemia began. Pain et al. (260) repeated the above experiment in the isolated rabbit heart and found that a 5-min pulse of diazoxide indeed protected the heart against infarction and that the drug could be washed out for as long as 30 min without loss of the protection. Pinacidil, a nonselective KATP channel opener, had the same effect. These data suggested that transient opening of mitoKATP channels puts the heart into a preconditioned state that continued long after the channel should have closed again.
Pain et al. (260) further studied the timing of channel opening required to protect ischemic hearts. They set out to determine whether mitoKATP channel opening was a trigger or mediator of protection. As explained above, triggers act before the index ischemia while mediators act during the index ischemia and therefore must be down-stream events. If KATP opening is the end-effector of preconditioning, then it would be expected to fall into the mediator category. To investigate whether the mitoKATP channel is a trigger or mediator, both Pain et al. (260) and Wang et al. (373) used isolated rabbit hearts and administration of 5-HD either only during the preconditioning stimulus (early) or only during the index ischemia (late). In both studies, early 5-HD blocked protection supporting a trigger role. Pain et al. (260) were unable to block protection with 5-HD given in the late protocol just before the index ischemia and concluded that mitoKATP channels were only triggers. Wang et al. (373) however could abort protection if the concentration of 5-HD was increased fourfold over that required to prevent protection in the early protocol. They theorized that a higher concentration may have been required because channel phosphorylation reduced the potency of 5-HD for channel blockade (295). The other possibility, of course, is that the higher concentration of 5-HD introduced a nonspecific effect. Thus, while both investigative groups agree that mitoKATP channels act as a trigger of preconditioning, Wang et al. (373) suggest that these channels may have a dual role as both triggers and mediators.
Further support for mitoKATP channels as a mediator comes from Gross and Auchampach (129) who infused glibenclamide in dogs between the time of PC and the index ischemia and blocked protection. Gres et al. (126) recently addressed both of these issues in their pig model. In the above study (129), glibenclamide was given right at the onset of reperfusion after the preconditioning ischemia. The reperfusion, of course, would be the critical time for reactive oxygen species formation and requires open mitoKATP channels. When Gres et al. (126) gave the glibenclamide with the onset of reperfusion, it indeed blocked protection. However, if they delayed administration of the glibenclamide for 5 min but still included it during the index ischemia, protection was unaffected, arguing against any mediator role of KATP channels. When they tested a higher concentration of glibenclamide, infarcts were larger but, unfortunately, this concentration of glibenclamide also increased infarct size in nonpreconditioned hearts by a similar increment. On the other hand, infarcts were not increased in nonpreconditioned hearts with the high dose of 5-HD used by Wang et al. (373). Yao et al. (399) also noted in their chick cell model that protection from a PKC activator could be blocked when 5-HD was introduced only during the prolonged simulated ischemia. Thus the current weight of evidence supports both a trigger and a mediator role for the channel. An attractive explanation of this dual role would be a scenario in which channel opening triggers a kinase cascade that feeds back in a positive manner to keep the channel open during the index ischemia.
If mitoKATP channel opening is an upstream event, then where in the pathway are the signaling kinases located? Ashraf's group (376) showed that the PKC blocker chelerythrine could block protection from a pulse of diazoxide in the isolated rat heart. While Pain et al. (260) could not show a similar result in the rabbit heart, they were able to block diazoxide's protection with the tyrosine kinase blocker genistein, indicating that there was at least one tyrosine kinase downstream from mitoKATP channel opening in the rabbit model. Thus mitoKATP channel opening protects by activating kinases. This further indicates that mitoKATP channel opening acts as an upstream link in a signal transduction chain leading to kinase activation. But how could channel opening be a signal?
5. Free radicals and mitoKATP channels
Steenbergen and co-workers (108) provided the solution to this puzzle. They found that diazoxide's protection could be blocked by a free radical scavenger, N-acetylcysteine. Their observation was then confirmed by Pain et al. (260) who used the scavenger N-2-(mercaptopropionyl)glycine (MPG) in a similar experiment. Yao et al. (400) found that pharmacological preconditioning of chick cardiomyocytes with the muscarinic agonist acetylcholine caused the cells to produce a small burst of free radicals. Yao et al. (407) used the probe 2',7'-dichlorofluorescin diacetate (DCFH) which fluoresces when oxidized by free radicals. This burst could be blocked by myxothiazol (31, 186), indicating that the increased radical production was the result of electron transport within the mitochondria, probably from site III of the electron transport chain where myxothiazol blocks the flow of electrons (for review of free radicals and their cellular origins, see Ref. 98). Furthermore, the burst could be blocked by 5-HD. These observations led Pain et al. (260) to propose a new paradigm incorporating mitoKATP channels and free radicals in PC's signaling pathway leading to protection. In this model receptor occupancy leads to mitoKATP channel opening, which then causes the mitochondria to produce reactive oxygen species (ROS). The free radicals would then activate the downstream kinases that ultimately modulate the end-effector.
Support for the free radical hypothesis was gained by the observation that diazoxide increased free radical production in isolated cardiomyocytes (108) in a human atrial-derived cell line (60) and in vascular smooth muscle cells (186). In all cases the increase in radical production could be blocked by 5-HD. More recently, Oldenburg et al. (254) have shown that exposure of smooth muscle cells to acetylcholine, an agonist known to trigger preconditioning, causes a similar burst of radicals that is dependent on a muscarinic receptor, a pertussis toxin-sensitive G protein, PI 3-kinase, and mitoKATP channels. Finally, Cohen et al. (75) found that protection triggered by acetylcholine, bradykinin, norepinephrine, or morphine could be blocked by either 5-HD or a free radical scavenger applied during the trigger phase. The study of Cohen et al. (75) was strong evidence that all of the above receptors couple through the mitoKATP channel/free radical pathway. Interestingly, adenosine was different as neither the KATP blocker nor the scavenger could affect its protection when applied during the trigger phase. Downey and colleagues (75) proposed that adenosine must have had a parallel coupling to the kinases. See Figure 4 for a diagram of the proposed signaling pathways for classical preconditioning.
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Qin et al. (282) found that the PI 3-kinase inhibitor wortmannin could block protection from acetylcholine but not from adenosine. Furthermore, it only blocked when the wortmannin bracketed the drug infusion, suggesting that PI 3-kinase acted as a trigger. Unpublished studies reveal that wortmannin does block the acetylcholine-induced burst of ROS in isolated cardiomyocytes and that it is upstream of the mitoKATP channels. Based on that, we would propose that it links the surface receptors to the mitoKATP channel.
The hypothesis that free radicals participate in the trigger signal for preconditioning may answer one of the mysteries of preconditioning: why receptor stimulation during a simple occlusion does not protect the heart. During a prolonged occlusion receptor agonists would be released, populate their receptors, and open the mitoKATP channels. However, the oxygen would be lacking to fuel the burst of ROS so that the signal would die out at that point. Only with reperfusion could ROS be produced so that the signal transduction pathway could be completed. In support of that observation, several investigators found that a period of total occlusion protected both pig (303) and rabbit (107) hearts from a subsequent period of lowflow ischemia. Receptor-mediated opening of mitoKATP channels during the total occlusion period would result in ROS formation during the low-flow period when oxygen was again available. Not all investigators agree on when the ROS are formed, however. Becker et al. (31) report significant ROS formation during simulated ischemia in chick cardiomyocytes. Whether there is enough residual oxygen available during a preconditioning ischemia to make the ROS signal without reperfusion is currently unknown.
Several studies have looked at prostaglandin pathways in ischemic preconditioning. Murphy et al. (237) found that preconditioning's preservation of postischemic function in the rat model could be blocked by a 12-lipoxygenase inhibitor. More recently, Gres et al. (127) found that indomethacin, a cyclooxygenase blocker, could abolish protection from a weak but not a strong preconditioning protocol in open-chest pigs. Whether cyclooxygenase was acting in the trigger or mediator phase was not investigated. Indomethacin could not block preconditioning in rabbits (205), and cyclooxygenase 1 and 2 knock-out mice could still be preconditioned (54). Clearly the role of prostaglandin pathways in preconditioning warrants further investigation.
The end-effector of preconditioning has been amazingly elusive. After more than a decade and a half of intensive research, the actual mechanism whereby the cell is protected against lethal injury is an enigma. There have been many theories over the years. The oldest theory was that of Murry et al. (241), who suggested an improved energy balance in the preconditioned ischemic myocardium. They proposed that perhaps the mitochondrial ATP-ase activity had been inhibited in these hearts. Although energetics are improved in preconditioned hearts, there is convincing evidence that that may not be the mechanism. In some protocols the favorable energy balance during ischemia may not be great enough to overcome the initial deficit caused by the preconditioning ischemia itself. Kolocassides et al. (182) found that their ischemically preconditioned rat hearts actually went into contracture earlier and had consistently lower ATP levels during the index ischemia despite obvious protection. The preconditioned hearts recovered from contracture during reperfusion, whereas the nonpreconditioned hearts did not.
The mitoKATP channel remains a viable candidate. Particularly in light of compelling evidence that it must reopen during the index ischemia, the likely time where the end-effector is exerting its effect. How the opening would protect the ischemic heart is not so clear as it will cause a slight uncoupling of the mitochondria and swelling (for a review, see Ref. 114); neither effect would be expected to protect. Terzic's group (144) finds that KATP channel openers make the mitochondria resistant to calcium overload. Perhaps opening mitoKATP channels prevent opening of the mitochondrial transition pore (156) during deep ischemia. Indeed, in a paper recently published, Hausenloy et al. (137) describes a new paradigm for preconditioning in which opening the mitoKATP channel could act to prevent opening of the mitochondrial transition pore described below.
3. Mitochondrial permeability transition pore
Halestrap et al. (135) proposed that ROS generation at reperfusion plus calcium entering the cell could cause adenine nucleotide translocase (ANT) to open a large-diameter pore within the mitochondrial membranes. The pore formation involves the binding of mitochondrial cyclophilins to the ANT and can be prevented by treating the hearts with cyclosporin A which binds the cyclophilins. The transition pore disrupts mitochondrial function and allows foreign substances into the matrix, which effectively destroys the mitochondria. It has long been recognized that pretreatment with cyclosporin A is cardioprotective. However, it was not known whether the protection derived from its inhibitory effect on phosphatases (378) or its inhibition of transition pore opening (128). Yellon's group (137) recently proposed that ischemic preconditioning might act to prevent opening of this pore. They demonstrated that protection from either ischemic preconditioning, diazoxide or an adenosine agonist could be blocked by actractyloside, an opener of the transition pore. Actractyloside had no effect on infarct size in non-preconditioned hearts. Diazoxide also inhibited calcium-induced pore opening in isolated mitochondria. This is persuasive evidence supporting the transition pore as the end-effector of preconditioning. Arguing against inhibition of the transition pore as an end-effector of preconditioning is a recent study by Garlid's group (95). In that study mitochondria isolated from ischemic hearts showed no difference in state 2 respiration compared with those from nonischemic hearts. That would not be expected if a transition pore had opened in these mitochondria. In that study the outer mitochondrial membranes did show an increased permeability to proteins which preconditioning prevented, however.
4. The sodium/hydrogen exchanger
Xaio and Allen (386) have suggested that the sodium/proton exchanger might be the end-effector of ischemic preconditioning and that its inhibition might lead to protection of the ischemic heart. Certainly pharmacological inhibition of the exchanger is one of the most potent protectors of the ischemic heart yet discovered (165). Xaio and Allen noted that the sodium/proton exchanger appeared to be blocked at reperfusion only when rat hearts had been preconditioned. Addition of HOE 642, a highly selective blocker of the sodium/proton exchanger, shortly before reperfusion to a nonpreconditioned heart preserved postischemic function by an amount equal to that achieved by ischemic preconditioning. HOE 642 had no additive effect when combined with ischemic preconditioning, further suggesting a common mechanism. Similarly, 5-(N-ethyl-N-isopropyl)amiloride, another blocker of the sodium/proton exchanger, limited infarct size in rabbits by an amount equal to that of preconditioning, and it could not augment preconditioning's anti-infarct effect (293). Neither kinase inhibitors nor 5-HD could abolish amiloride's protection, suggesting that the sodium/proton exchanger must be protective as an end-effector. Probably the most serious criticism of the hypothesis is the PKC, a key step in preconditioning acts to activate rather than inhibit the exchanger (164).
Cells are in osmotic equilibrium and cannot tolerate an osmotic imbalance. Osmotic balance is maintained by matching the osmotic pull of proteins and nucleotides within the cell primarily by sodium outside the cell. Because the conductance of the sarcolemma to sodium is very low, extracellular sodium is an efficient osmolyte. That is why virtually every cell type excludes sodium and maintains a negative resting membrane potential. During ischemia ATP is broken down to AMP and two inorganic phosphates, thus tripling the osmotic pull of the nucleotides. Similarly, failure of the sodium-potassium pumps leads to sodium leak into the cell and thus a collapse of the vital sodium gradient. Each millimolar increase in osmolyte concentration exerts an additional 19 mmHg transmembrane pressure. Indeed, Jennings and co-workers (381) proposed that osmotic swelling was the cause of membrane failure and cell death in reperfused myocardium in 1974. During ischemia, movement of water into the cell concentrates the extracellular fluid and thus limits swelling. Upon reperfusion however, the extracellular fluid is replaced with isotonic fluid and explosive swelling ensues.
Preconditioning makes cardiomyocytes very resistant to membrane failure when they are challenged with hypotonic media (12). In ischemically preconditioned rat (169) and pig (292) hearts, the extent of myocardial edema formation, along with infarct size, is reduced. Alterations in channels involved in cell volume regulation therefore might be involved in the cardioprotection achieved by ischemic preconditioning. Chloride channels are involved in moment-to-moment volume regulation, and Diaz et al. (93) hypothesized from experiments in rabb
