Pharmacological modulation, preclinical studies, and new clinical features of myocardial ischemic preconditioning

Pharmacology & Therapeutics 88 (2000) 311 ± 331

Associate editor: C.L. Wainwright

Pharmacological modulation, preclinical studies, and new clinical features of myocardial ischemic preconditioning Claudio Napolia,b,*, Aldo Pintoc, Giuseppe Cirinod a

Department of Medicine, Federico II University of Naples, P.O. Box, Naples 80131, Italy b Department of Medicine-0682, University of California, San Diego, CA 92093, USA c Department of Pharmacological Sciences, University of Salerno, Fisciano-Salerno 84100, Italy d Department of Experimental Pharmacology, Federico II University of Naples, Via D. Montesano 49, Naples 80131, Italy

Abstract The term ``ischemic preconditioning (PC)'' was first applied to canine myocardium subjected to brief episodes of ischemia and reperfusion that tolerated a more prolonged episode of ischemia better than myocardium not previously exposed to ischemia. Protective effect of myocardial ischemic PC was demonstrated in several animal species, resulting in the strongest endogenous form of protection against myocardial injury, jeopardized myocardium, infarct size, and arrhythmias other than early reperfusion. New onset angina before acute myocardial infarction, episodes of myocardial ischemia during coronary angioplasty or bypass surgery, and the ``warm-up'' phenomenon may represent clinical counterparts of the PC phenomenon in humans. Here, we have attempted to summarize pharmacological modulation, preclinical studies, and new clinical features of ischemic PC. To date, the pathophysiological basis of the ``chemical PC'' is still not well established, and ``putting PC in a bottle'' for clinical applications still remains a new pharmacological venture. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Ischemic preconditioning; Pharmacology; Myocardial ischemia; Oxygen radicals; Adenosine; KATP channels Abbreviations: AMI, acute myocardial infarction; CGRP, calcitonin gene-related peptide; DOG, 1,2-dioctanoyl-sn-glycerol; 5-HD, 5-hydroxydecanoate; hsp, heat shock protein; IAA-94, indanyloxyacetic acid 94; IB-MECA, N6-(3-iodobenzyl)-adenosine-50-N-methyluronamide; JNK, Jun NH2-terminal kinase; KATP, K+ channel sensitive to adenosine triphosphate; KIR, inward-rectifier K+; 125I-ABA, [125I]N6-4-amino-3-iodobenzyladenosine; MAPK, mitogen-activated protein kinase; MIDCAB, minimally invasive direct coronary artery bypass; MLA, monophosphoryl lipid A; MOR-14, N-methyl-1-deoxynoirimycin; NO, nitric oxide; iNOS, inducible nitric oxide synthase; PC, preconditioning; PIA, N6-(2-phenylisopropyl)adenosine; PKC, protein kinase C; PL, phospholipase; PP, protein phosphatase; TK, tyrosine kinase; TNF, tumor necrosis factor.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological modulation of ischemic preconditioning . . . . . 2.1. Triggers of myocardial ischemic preconditioning . . . . . . 2.1.1. Adenosine receptors . . . . . . . . . . . . . . . . 2.1.2. Opioid receptors . . . . . . . . . . . . . . . . . . 2.1.3. Bradykinin and B2 receptors . . . . . . . . . . . . 2.2. Transducers of myocardial ischemic preconditioning . . . . 2.2.1. Kinase cascade . . . . . . . . . . . . . . . . . . . 2.2.2. Phospholipase D . . . . . . . . . . . . . . . . . . 2.3. End-effectors of myocardial ischemic preconditioning . . . 2.3.1. Sarcolemmal and mitochondrial KATP channels . . 2.4. Glycemic control of myocardial ischemic preconditioning? .

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* Corresponding author. Department of Medicine, Federico II University of Naples, P.O. Box, Naples 80131, Italy. Tel./fax: +39-81-5603990. E-mail addresses: [email protected] or [email protected] (C. Napoli). 0163-7258/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 0 ) 0 0 0 9 3 - 0

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3.

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2.5. 2.6. From 3.1.

A possible role in preconditioning for monophosphoryl lipid A and RC-552 . . . Other possible mechanisms involved in myocardial ischemic preconditioning . . preclinical studies to new clinical features in myocardial ischemic preconditioning Preinfarction angina and ``new-onset'' angina as models of myocardial ischemic preconditioning in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Is the development of myocardial tolerance to ischemia in humans due to ischemic preconditioning or to collateral recruitment? . . . . . . . . . . . . . 3.3. Myocardial stunning and ischemic preconditioning . . . . . . . . . . . . . . . . 3.4. Preconditioning and coronary artery bypass surgery. . . . . . . . . . . . . . . . 3.5. Arrhythmias and myocardial ischemic preconditioning . . . . . . . . . . . . . . 3.6. A loss of preconditioning in the aging heart? . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In 1926, Einstein told Heisenberg that it was nonsense to found a theory on the basis of observable facts alone: ``In reality the very opposite happens. It is theory which decides what we can observe.'' Restating this comment in a somewhat less contentious fashion, we may add that a paradigm, albeit a powerful influence upon what is observed, is basically a complex framework that accumulates new data with increasing time. Data are obtained that represent an anomaly, which does not fit the accepted paradigm. Subsequently, this theory may be replaced by one that becomes the new paradigm; the cycle then continues until a period of anomaly arises again. Pursuing the idea of Einstein, we need to comprehend that solid evidence in some experimental models is only a good beginning of the story. Although a lot of data provides renewed optimism for the inclusive understanding of ``ischemic preconditioning (PC),'' to date, we continue to look for the anomaly(ies) that enables us to resolve the puzzle in clinical conditions. The results of the outstanding study of Murry et al. (1986) undeniably represent a significant advance in our broad understanding of the pathophysiology of tissue ischemia. More recently, ischemic PC has been defined as a powerful endogenous protective mechanism against prolonged ischemic injury that has been shown to occur in a variety of organ systems, including the heart, brain, spinal cord, retina, liver, lung, and skeletal muscle. In particular, myocardial ischemic PC is the phenomenon by which a brief episode(s) of myocardial ischemia increases the ability of the heart to tolerate a subsequent prolonged period of ischemic ± reperfusion injury; PC has both immediate (early phase or first window of protection) and delayed (late phase or second window of protection) protective effects, the importance of which varies between species and organ systems (reviewed in Li et al., 1990; Downey, 1992; Walker & Yellon, 1992; Kloner & Yellon, 1994; Yellon & Baxter, 1995; Bolli, 1996; Connaughton & Hearse, 1996; Kloner et al., 1998a). The distinction in mediators and pathways activated in delayed PC (second

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window) are summarized to the latest acquisition in Table 1. Several mediators involved in the classical early response are also implicated in the second window of protection. While the exact mechanisms of both protective components are still unclear, ischemic PC could be defined as a multifactorial pathophysiological process, requiring the interaction of numerous cellular signals, second messengers, and end-effector mechanisms. Myocardial protection induced by PC develops approximately 7 days after birth, and the inability of neonatal hearts to precondition appears not to be due to insufficient stimulus or extended ischemia (Awad et al., 1998). Stimuli other than ischemia, such as hypoxic perfusion, tachycardia, and pharmacological agents, may have PC-like effects (reviewed in Kloner et al., 1998a). We have examined here two aspects of research primarily in the classical early phase of myocardial ischemic PC. First, we have sought to examine pharmacological studies on the modulation of PC by agonists and antagonists/inhibitors of possible biological pathways involved in this phenomenon. Second, we have focused the remaining part of the review on the new clinical features, in the effort to elucidate which aspects of experimental evidence and pharmacological approach are involved in the human response. 2. Pharmacological modulation of ischemic preconditioning The hallmark of ischemic PC, documented in virtually all species and experimental models evaluated to date in countless laboratories worldwide, is the profound reduction in infarct size and jeopardized myocardium seen in preconditioned groups versus time-matched controls (Przyklenk & Kloner, 1998). Of course, the main objective in recent years has been to identify the possible triggers, transducers, and end-effectors involved in the classical early phase of ischemic PC to allow mimicking of the physiological response by chemical agents. For example, it has been observed that following mild chemical inhibition

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Table 1 Summary of the mechanisms involved in late preconditioning or second window of protection Nuclear factor kB NO NO PKC PKC PP1A, PP2A KATP channels MLA d1 opioid KATP channels A1 receptor activation Hsp72 TK Diacylglycerol Free radicals

Mechanism proposed

Experimental model

Reference

Activation by NO NO hypothesis of late PC iNOS activation iNOS activation Activation by NO Translocation of PKCe from cytosolic to particulate fraction Undetermined

Conscious rabbits

Xuan et al., 1998 Bolli et al., 1998 Imagawa et al., 1999 Takano et al., 1998 Ping et al., 1999a Qiu et al., 1998

Inhibition of MLA-induced late PC by glibenclamide or 5-HD Additive effect with adenosine KATP channel activation ND Glibenclamide and 5-HD blockade Protection against myocardial infarction by repeated A1 stimulation Not involved Not involved Blockade by genisteine of late PC Activation of PKC isoenzymes Reactive oxygen species protect coronary endothelium Blockade of late PC by antioxidant therapy Lack of evidence of increased levels of myocardial antioxidants

Rabbits Conscious rabbits Conscious rabbits Conscious rabbits Ca2 + -tolerant rabbit cardiomyocytes Anesthetized rabbits

Baxter et al., 1997

Chick ventricular myocytes Rat Rat Rabbits Rabbit

Stambaugh et al., 1999 Fryer et al., 1999 Tsuchida et al., 1998 Bernardo et al., 1999 Dana et al., 1998a

Conscious rabbit Rats Rabbit Anesthetized rabbits Anesthetized rats

Maldonado et al., 1997 Qian et al., 1999 Imagawa et al., 1997 Mei et al., 1996 Kaeffer et al., 1997

Conscious pigs

Sun et al., 1996

Conscious pigs

Tang et al., 1997

Armstrong et al., 1998

ND, not determined.

of oxidative phosphorylation, it is possible to mimic PC (Riepe & Ludolph, 1997). Efforts to identify the clinical setting of possible therapeutic applications are all topics of intensive on-going investigation, and this research field is known as ``chemical PC'' (Wainwright, 1992; Riepe & Ludolph, 1997). 2.1. Triggers of myocardial ischemic preconditioning 2.1.1. Adenosine receptors Adenosine receptor involvement has been widely studied, and there is a growing body of evidence for adenosine receptor involvement in PC (for a review, see Miura & Tsuchida, 1999). From the scientific rationale provided from preclinical studies, several clinical approaches are now undertaken using adenosine-mediated PC (see below and Section 3). In anesthetized dogs subjected to 1 hr of coronary artery occlusion, followed by 4 hr of reperfusion, ischemic PC was elicited by 10 min of coronary occlusion, followed by 1 hr of reperfusion before the 1-hr occlusion period (Yao et al., 1997). PC resulted in a marked reduction in infarct size, whereas administration of adenosine or bimakalim, followed by a 1-hr drug-free period, had no effect. However, the simultaneous administration of adenosine and bimakalim resulted in a marked decrease in infarct size. One hour after ischemic PC, administration of glibenclamide blocked the protective effect of ischemic PC, whereas a selective

A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or a nonselective adenosine receptor antagonist PD115199 did not affect it. These data suggested that adenosine and the K + channel sensitive to adenosine triphosphate (KATP) may have a synergistic interaction that is important for the memory phase of PC (Yao et al., 1997). A possible role for the A1 receptor has been further addressed by using PD 81,723, which acts allosterically to increase agonist binding to A1 receptors and to enhance functional A1 receptor-mediated responses in the heart and other tissues (Mizumura et al., 1996). In anesthetized dogs subjected to ischemic PC, PD 81,723 was infused intracoronarily for 17.5 min before the 60-min occlusion period in nonpreconditioned dogs or in dogs preconditioned with 2.5 min of ischemia. Infarct size was not significantly affected by 2.5 min of PC alone or by PD 81,723 alone, but was decreased by PD 81,723 plus PC or by a longer period (5 min) of PC alone. Administration of the selective antagonist of A1 receptors such as 8-cyclopentyl-1,3-dipropylxanthine or the KATP blocker glibenclamide for 15 min before PD 81,723 plus PC blocked the protection. Radioligand-binding studies were conducted using membranes derived from COS-7 cells expressing recombinant canine receptors and agonist radioligands. PD 81,723 enhanced the binding of [125I]N6-4-amino-3-iodobenzyladenosine (125I-ABA) to A1 receptors by increasing the t1/2 for dissociation by 2.18-fold, but PD 81,723 had no effect on the dissociation kinetics of 125 I-ABA from A3 receptors or [125I]-[2-(4-amino-3-iodo-

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phenyl)ethylamino] adenosine from A2 receptors. Glibenclamide, at concentrations up to 10 mmol/L, had no effect on the binding of radioligands to recombinant canine A1, A2, or A3 receptors. These data suggested an important role for the A1 receptor, and suggested that KATP receptor blockade prevented the protection afforded by A1 receptor activation by a mechanism not involving adenosine receptor blockade (Mizumura et al., 1996). Moreover, the A1 receptor agonist N6-(2-phenylisopropyl)adenosine (PIA) treatment reduced myocardial infarction in the rabbit (Hashimi et al., 1998). The anti-infarction effect of ischemic PC and adenosine was significantly blocked by the A1 receptor antagonist 8cyclopentyl-1,3-dipropylxanthine and the KATP-channel blockers Na + 5-hydroxydecanoate (5-HD) and glibenclamide. These observations indicated that adenosine, through A1 receptors, initiates the mechanism of ischemic PC with postreceptor involvement of KATP channels in the heart (Hashimi et al., 1998). The cardioprotective effect of a selective A1 receptor agonist GR79236 also has been tested in a porcine model of myocardial ischemia ± reperfusion injury with controversial results (Louttit et al., 1999; Smits et al., 1998). GR79236 appeared not to reduce infarct size in pigs, which suggests that under these experimental conditions, stimulation of adenosine A2 receptors was important for the cardioprotective effect of the A1/A2 receptor agonist AMP 579 (see below). The adenosine-regulating agent acadesine also failed to reduce infarct size (Smits et al., 1998). However, when pigs were subjected to a 50-min coronary artery occlusion, followed by a 3-hr reperfusion, GR79236 significantly reduced infarct size whether given 10 min before the onset of ischemia or reperfusion. This effect was independent of the bradycardia induced by GR79236, as it was also observed in animals in which heart rate was maintained by electrical pacing (Louttit et al., 1999). However, GR79236 administered 10 min after reperfusion did not reduce infarct size, and it had no effect on the incidence or outcome of ventricular arrhythmias. Also, in this case, the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine abolished the hemodynamic and cardioprotective effects of GR79236 (Louttit et al., 1999). More recently, the effect of different body core temperatures was examined on GR79236- or PC-induced cardioprotection when administered prior to ischemia and on cardioprotection induced by GR79236 administered 10 min prior to the onset of reperfusion (Sheldrick et al., 1999). When rabbits were subjected to a 30-min occlusion of the left coronary artery, followed by a 2-hr reperfusion, GR79236 or PC, administered or applied 10 min prior to the occlusion, significantly limited the development of infarction. The cardioprotective effect was significantly greater than that seen after administration of GR79236. Pretreatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the cardioprotective effect of GR79236, but not that of PC. Maintaining body core

temperature at 38.5°C rather than at 37.0°C did not influence infarct size in controls, but reduced the cardioprotective effect of GR79236 when administered 10 min prior to occlusion or 10 min prior to the onset of reperfusion. The effect of PC was not temperature-dependent. Thus, protection conferred by GR79236 in anaesthetized rabbits is mediated via adenosine A1 receptors (Sheldrick et al., 1999). Myocardial protection was conferred when GR79236 was administered before the onset of ischemia or reperfusion, and it was reduced when body core temperature was maintained at 38.5°C rather than at 37.0°C. In contrast, protection by PC was not reduced by adenosine A1 receptor blockade or by maintaining body core temperature at 38.5°C rather than at 37.0°C. These findings highlight the need for careful control of body core temperature when investigating PC (Sheldrick et al., 1999). The ability of mixed adenosine inhibitors to afford cardioprotection has also been tested in pigs. The mixed agonist AMP 579 was also tested in a model of myocardial infarction in anesthetized pigs induced by a 40-min occlusion of the left coronary artery, followed by 3 hr of reperfusion (Smits et al., 1998). Administration of AMP 579 30 min before ischemia resulted in marked cardioprotection, with a 98% reduction in infarct size. The cardioprotective effect of AMP 579 was a consequence of adenosine receptor stimulation, since it was completely inhibited by pretreatment with the specific adenosine receptor antagonist CGS 15943. Cardioprotection was shown not to be dependent on changes in afterload or myocardial oxygen demand, and was a consequence of adenosine receptor stimulation. The involvement of A3 receptors in PC has been studied by using a Langendorff model of myocardial ischemia ± reperfusion injury (Tracey et al., 1997). Rabbit hearts were exposed to 30 min of regional ischemia and 120 min of reperfusion, and PC by 5 min of global ischemia and 10 min of reperfusion was able to reduce the infarct size. Replacing global ischemia with a 5-min perfusion of the rabbit A1-selective agonist N6-(3-iodobenzyl)-adenosine-50N-methyluronamide (IB-MECA) elicited a concentrationdependent reduction in infarct size. An A1-selective agonist (R-PIA; 25 nM) in this same study reduced infarct size to a similar extent. However, while the cardioprotective effect of R-PIA (A1 selective) was significantly inhibited by the rabbit A1-selective antagonist BWA1433, the IB-MECAdependent cardioprotection was unaffected. However, a nonselective higher dose of BWA1433 significantly attenuated the IB-MECA (A3-selective)-dependent cardioprotection (Tracey et al., 1997). In a model of simulated ``ischemia'' and ``reperfusion'' in quiescent human ventricular cardiomyocytes, cellular injury and metabolic parameters were assessed after various interventions: cells were preconditioned with anoxia, hypoxia, anoxic supernatant, or hypoxic supernatant, with or without an adenosine receptor antagonist or adenosine deaminase (Cohen et al., 1998). Adenosine was applied before, during, or after

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ischemia or continuously, with and without the adenosine receptor antagonist. Cells were treated with the protein kinase C (PKC) agonist phorbol myristate acetate (PMA) and preconditioned cells were incubated with the PKC antagonist calphostin-C. PC with anoxia was most protective. Protection was transferable via anoxic supernatant, which produced the highest concentrations of adenosine, and was lost when adenosine receptor antagonist or adenosine deaminase was used. Exogenous adenosine was most protective when administered before ischemia, whereas if it was administered during ischemia, it was only partially protective. An adenosine receptor antagonist abolished the protective effects of adenosine. Adenosine reproduced the protective effects of PC, preserved ATP, and increased lactate production, perhaps by stimulating glycolysis (Tracey et al., 1997). In a rabbit Langendorff model of myocardial ischemia ± reperfusion injury (Hill et al., 1998), adenosine was shown to be 19-fold selective for inhibition of 125I-ABA binding to recombinant rabbit A1 when compared with rabbit A3 receptors. Ischemic PC and adenosine-mediated cardioprotection were completely blocked in the presence of the rabbit A1-selective antagonist BWA1433 (Hill et al., 1998). Although many agents that were thought to attenuate reperfusion injury have been unsuccessful in clinical investigation, adenosine resulted in a significant reduction in infarct size. These data have indicated that adenosine should be investigated in large multicenter clinical outcome trials. 2.1.2. Opioid receptors The role of opioid receptors in PC has also been widely studied, and there are several lines of evidence for involvement of the d opioid receptor type. The first evidence for a role for opioids in PC was proposed by Schultz et al. (1995, 1996). By using the rat heart, they demonstrated that naloxone pretreatment reduced the protection afforded by PC (Schultz et al., 1995) and that morphine mimicked the cardioprotective effect (Schultz et al., 1996). Similarly, naloxone blocked myocardial ischemic PC in open-chest rabbits (Chien & Van Winkle, 1996). Following this initial evidence, opioid receptor involvement has been studied. Naltrindole, a selective d opioid receptor antagonist, completely abolished the cardioprotective effect induced by PC and morphine in open-chest rats (Schultz et al., 1997). d1 receptors have been shown to be involved in the cardioprotective effect of ischemic PC in rats; however, the mechanism by which d opioid receptor-induced cardioprotection is mediated remains unknown. Several of the known mediators of ischemic PC, such as the KATP channel and Gi/ o proteins, may be involved in the cardioprotective effect produced by d1 opioid receptor activation. Involvement of the d1 receptor has been shown by using a selective d1 agonist. Infusion of TAN-67, a new selective d1 agonist, for 15 min before the long occlusion and reperfusion periods significantly reduced the infarct size, as compared with control (Schultz et al., 1998b). The cardioprotective effect

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of TAN-67 was completely abolished by 7-benzylidenenaltrexone, a selective d1 antagonist; glibenclamide, a KATPchannel antagonist; and pertussis toxin, an inhibitor of Gi/o proteins. These results suggested that stimulating the d1 opioid receptor elicits a cardioprotective effect that is mediated via Gi/o proteins and KATP channels in the intact rat heart (Schultz et al., 1998b). The possible involvement of a m or k opioid receptor has also been studied in rats (Schultz et al., 1998a). Anesthetized, open-chest rats were subjected to 30 min of occlusion and 2 hr of reperfusion. Ischemic PC markedly reduced infarct size, but naltriben, a selective d2 opioid receptor antagonist; b-funaltrexamine, an irreversible m opioid receptor antagonist; and the k opioid receptor antagonist norbinaltorphimine were unable to block the cardioprotective effect of ischemic PC. Similarly the m opioid receptor agonist D-Ala,2N-Me-Phe,4glycerol5-enkephalin had no effect on infarct size. These results also indicated that d1 opioid receptors play an important role in the cardioprotective effect of ischemic PC in the rat heart. There is also some evidence that k opioid receptors could play a role in PC in isolated rat ventricular myocytes by using a selective k opioid receptor antagonist nor-binaltorphimine, or a k opioid receptor agonist (Wu et al., 1999). 2.1.3. Bradykinin and B2 receptors It was shown in 1996 by Miki et al. that captopril potentiates PC without increasing kinin levels, and that the effect of captopril can be reversed by HOE140, a specific bradykinin receptor antagonist. This finding has been further extended using B2 kinin receptor knockout mice, as well as kininogen-deficient rats, demonstrating a loss of the protective effect in these strains. The results obtained in these experiments suggest that activation of prekallikrein may contribute to the effect of PC and that an intact kallikrein± kinin system is necessary for the cardioprotective effect of PC (Yang, X. P. et al., 1997). Similarly, in the human heart, captopril and lisinopril have been shown to increase ischemic PC via B2 receptor activation (Morris & Yellon, 1997). However, in one study, B2 receptor involvement has been ruled out by using isolated rat hearts (Bouchard et al., 1998). A possible cooperation between adenosine receptors (A1/A3) and the B2 receptor has been proposed (Giannella et al., 1997; Kaszala et al., 1997). Using 1,1-diphenyl-2-picryl-hydrazyl to trigger free radical injury in guinea-pig heart and PC to protect the myocardium, it was demonstrated that bradykinin perfusion protected the heart against radical injury (Jin & Chen, 1998). Pretreatment with PC and bradykinin resulted in cardiac protection against free radical injury through the activation of B2 receptors, suggesting that endogenous generation of bradykinin may mediate PC in the guineapig heart. Finally, bradykinin appears to be essential during periods of PC ischemia of shorter duration (i.e., 3 min); adenosine is more important during PC of longer duration (i.e., 10 min) (Schulz et al., 1998).

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2.2. Transducers of myocardial ischemic preconditioning 2.2.1. Kinase cascade The role of PKC in ischemic PC remains controversial (for a review, see Simkhovich et al., 1998). However, there are several studies that have addressed the role of PKC in PC. It has been suggested that kinase activity itself is not required to precondition the rabbit heart, but that some processes upstream of PKC activation are responsible for the triggering and memory of PC in rabbits (Yang X. M. et al., 1997). PKC isoforms, namely a, d, and e, could be implicated in PC (Yoshida et al., 1997). Using rabbit heart, it has been shown that PC can be induced by b-adrenoreceptor stimulation (Yabe et al., 1998). In rat hearts subjected to 40 min of ischemia and 30 min of reperfusion, isoproterenol pretreatment for 2 min followed by 10 min of normoxic perfusion enhanced the recovery of the rate-pressure product of the ischemic/reperfused heart and attenuated the release of creatine kinase during 30 min of reperfusion, similar to a PC stimulus of 5 min of ischemia and 5 min of reperfusion. Cardioprotection and ischemic PC was removed by treatment with polymyxin B. In the same study, similar changes were observed in the subcellular distribution of PKC following b-adrenergic stimulation and PC, suggesting translocation of PKC d, a marker of PKC activation, from the cytosol to the membrane fraction. These results imply that the cardioprotection induced by b-adrenoreceptor stimulation, like ischemic PC, is mediated by PKC activation. The role of PKC in PC has also been studied by using diacylglycerol, an endogenous activator of PKC. Using the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG), Galinanes et al. (1998) have evaluated the possible protective effect against injury during ischemia and reperfusion and if the effect is mediated via PKC activation in rat heart. A cardioprotective effect was achieved with DOG. However, it was less cardioprotective than ischemic PC, and the achieved protection did not appear to necessitate PKC activation prior to ischemia. Recently, a new 1,4-benzothiazepine derivative, JTV519, was investigated on ischemia/ reperfusion injury in isolated rat hearts (Inagaki et al., 2000). The protective effect of JTV519 was completely blocked by pretreatment of the heart with GF109203X, a specific PKC inhibitor. In contrast, the effect of JTV519 was not affected by a1-, A1-, and B2-receptor blockers that couple with PKC in the cardiomyocyte. Immunofluorescence and immunoblots demonstrated that this agent induced concentration-dependent translocation of the d isoform, but not the other isoforms of PKC, to the plasma membrane. Thus, JTV519 may provide a novel pharmacological approach via a subcellular mechanism for mimicking ischemic PC (Inagaki et al., 2000). Tyrosine kinase (TK) inhibitors have also been shown to attenuate ischemic PC. However, it is unclear whether TK is involved in the initiation of and/or the maintenance of this phenomenon. This hypothesis has been tested by using genistein, a nonspecific TK inhibitor; daidzein, an inactive

structural analog of genistein; and lavendustin A, a more specific TK inhibitor (Fryer et al., 1998). In the rat heart, PC-induced cardioprotection was attenuated by genistein pretreatment. However, genistein, administered during the first or third occlusion period of PC, did not significantly attenuate cardioprotection. Lavendustin A pretreatment also attenuated PC, whereas daidzein had no effect, suggesting that activation of a TK was involved in the initiation, but not the maintenance, of PC. TK also appears to be involved in ischemic PC in the rabbit heart (Baines et al., 1998) and in pigs (Valhaus et al., 1998). Recently, it has been shown in conscious rabbits by using the MEK1 inhibitor PD-98059 that selective overexpression of PKC induced activation of both p44 and p42 mitogenactivated protein kinases (MAPKs), accompanied by translocation from the cytosol to the nucleus, and reduced lactate dehydrogenase release during ischemia reperfusion (Ping et al., 1999b). Another recent interesting observation in the rabbit heart suggested the involvement of 12-lipoxygenase metabolism in the cardioprotection induced by PC (Chen et al., 1999), since WEB 2086 administration or PC reduced the incidence of arrhythmias during reperfusion. In another experimental setting, isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion, and hearts were pretreated with genistein to block TK, whereas SB-203580 was used to inhibit p38 MAPK (Maulik et al., 1998). Western blot analysis demonstrated that p38 MAPK is phosphorylated during ischemic stress adaptation. Phosphorylation of p38 MAPK was blocked by genistein, suggesting that activation of p38 MAPK during ischemic adaptation was mediated by a TK signaling pathway. Immunofluorescence microscopy with an anti-p38 antibody revealed that p38 MAPK was localized primarily in perinuclear regions, and moved to the nucleus after ischemic stress adaptation (Maulik et al., 1998). Corroborating these results, myocardial adaptation to ischemia improved left ventricular function and reduced myocardial infarction, both of which were reversed by blocking either TK or p38 MAPK. These results indicate that myocardial adaptation to ischemia triggers a TKregulated signaling pathway, leading to the translocation and activation of p38 MAPK, and implicating a role for MAPK-Activated Protein Kinase-2 (MAPKAPK-2), a kinase immediately downstream from p38 MAPK. Interestingly, small heat shock proteins (hsps) have been implicated in mediation of classic PC in the rabbit; hsp27 is a terminal substrate of the p38 MAPK cascade (Armstrong et al., 1999). p38 MAPK and hsp27 phosphorylation levels, respectively, were also determined during in vitro ischemia in control, calyculin A-treated [protein phosphatase (PP) inhibitor], SB203580-treated, and preconditioned isolated adult rabbit cardiomyocytes (Armstrong et al., 1999). The dual phosphorylation of p38 MAPK was increased by early ischemia (30 ± 60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by PC. Calyculin A

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strongly activated dual phosphorylation of p38 MAPK in oxygenated cells, and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. After 90 min of ischemia, cytoskeletal hsp27 was markedly dephosphorylated. During ischemic incubation, calyculin A blocked ischemic dephosphorylation, SB203580 accelerated ischemic hsp27 dephosphorylation and injury, PC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Thus, the PC effect was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged ( > 60± 90 min) ischemia (Armstrong et al., 1999). Finally, in a very recent study, the regulation of MAPKAPK-2 and the activity of c-Jun NH2-terminal kinase (JNK) were examined in isolated, preconditioned rabbit hearts (Nakano et al., 2000). Ventricular biopsies before treatment and after 20 min of ischemia were homogenized, and the activities of MAPKAPK-2 and JNK were evaluated. For the MAPKAPK-2 experiments, seven groups were studied as follows: control hearts; preconditioned hearts; hearts treated with R-PIA, an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 8-( p-sulfophenyl) theophylline, an adenosine receptor antagonist; preconditioned hearts treated with SB 203580; hearts treated with anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin and the TK inhibitor genistein. There was a 3.8-fold increase in MAPKAPK-2 activity during ischemia in preconditioned hearts. Activation of MAPKAPK-2 in preconditioned hearts was blocked by both 8( p-sulfophenyl) theophylline and SB 203580. MAPKAPK2 activity during ischemia increased 3.5- and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK-2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. There was a comparable, modest decline in JNK activity during 30 min of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These data confirm that the p38 MAPK/MAPKAPK-2 pathway is activated during ischemia, only if the heart is in a preconditioned state (Nakano et al., 2000). 2.2.2. Phospholipase D Phospholipase (PL)D might play a role in myocardial ischemic PC. There is a receptor-dependent PLD present in the myocardium (Eskidilsen-Helmond et al., 1996). Activation of PKC via stimulation of PLC degrades membrane phospholipids to diacylglycerol, an important PKC cofactor. Propranolol, which blocks diacylglycerol production from

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metabolites produced by PLD catalysis, completely abolished the protective effects of PC in isolated rabbit hearts (Cohen et al., 1996). These results were also confirmed in the isolated rat heart (Tosaki et al., 1997). PLD also acts in a synergistic activation by G-protein and PKC in the rat atria (Lindmar & Loffelholz, 1998), suggesting a similar effect during PC in vivo. 2.3. End-effectors of myocardial ischemic preconditioning 2.3.1. Sarcolemmal and mitochondrial KATP channels KATP channels have been shown to play a critical role in ischemic PC, and this hypothesis is supported by several pieces of experimental evidence (Gross & Fryer, 1999; Kevelaitis et al., 1999). Nicorandil, a KATP-channel opener, has been shown to lower the threshold for the infarctreducing PC effect in dogs by activation of myocardial KATP channels (Mizumura et al., 1997). The effects of nicorandil and pinacidil have also been evaluated in isolated and ischemic rabbit cardiomyocytes using the whole-cell recording technique (Critz et al., 1997). Pinacidil increased KATP current  4-fold in isolated cardiomyocytes, and this increase was reversed rapidly after treatment with the KATPchannel blocker glibenclamide. Pinacidil protected cardiomyocytes from simulated ischemia, but the protection achieved from pinacidil was completely eliminated by pretreatment with glibenclamide. In contrast, nicorandil, which opens KATP channels in some tissues, caused no detectable effect on the KATP current. Similarly, nicorandil did not produce cardioprotection in this model of PC. These results indicated that pinacidil and nicorandil have very different effects on rabbit cardiomyocyte KATP channels. Furthermore, because protection correlated with the ability of the agent to open the channel, this supported a role for KATP channels in PC. Mitochondrial KATP channels have also been proposed to be involved in PC (Gross & Fryer, 1999). Diazoxide, a mitochondrial KATP-channel opener, was administered before ischemia was protective, and protection was lost when diazoxide was given after the onset of ischemia. Anisomycin, a p38/Jun kinase activator, also reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-HD, a selective inhibitor of mitochondrial KATP channels (Baines et al., 1999). Moreover, it is possible to simulate ischemia of isolated adult rabbit cardiomyocytes in vitro by centrifuging the cells into an oxygen-free pellet for 3 hr and to induce PC by prior pelleting for 10 min, followed by resuspension for 15 min. Under these experimental conditions, PC delayed the progressive increase in osmotic fragility seen in nonpreconditioned cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. Nicorandil also exerts a direct cardioprotective effect on heart muscle cells, and this effect is believed to

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be mediated by selective activation of mitochondrial KATP channels (Sato et al., 2000). More recently, an interaction between KATP channels and Na + /K + -ATPase, through sarcolemmal ATP, has been shown to modulate the infarct size reducing effect of ischemic PC (Haruna et al., 1998). To further complicate this issue, d opioid receptors appeared to improve recovery of cold-stored hearts to a similar extent as ischemic PC, most likely through an opening of KATP channels (Kevelaitis et al., 1999). 2.4. Glycemic control of myocardial ischemic preconditioning? By using isolated human right atrial trabeculae, it has been shown that human myocardium from patients without long-term exposure to oral hypoglycemic agents is functionally protected by PC, whereas long-term oral hypoglycemic intake blocks the protection by PC (Cleveland et al., 1997). Recent results in rats have shown that the sulfonylurea receptor couples to several types of inward-rectifier K + (KIR) channels, suggesting that glibenclamide blockade of PC may not be the result of this compound selectively blocking the KATP channel (Schultz et al., 1998c). Terikalant, a KIR-channel blocker, did not abolish the cardioprotective effect of ischemic PC at any dose tested, while glibenclamide completely abolished the cardioprotective effect, suggesting that endogenous myocardial KIR channel does not mediate ischemic PC in the rat heart, as opposed to the KATP channel. Another new sulfonylurea recently studied is glimepiride, a drug that is supposed to impact less on extrapancreatic KATP channels than the conventional drug glibenclamide. Glimepiride did not reduce myocardial PC, while glibenclamide was able to prevent it (Klepzig et al., 1999). N-methyl-1-deoxynoirimycin (MOR-14), an a-glucosidase inhibitor, reduces the glycogenolytic rate by inhibiting the a-1,6-glucosidase of the glycogen-debranching enzyme in the liver, in addition to possessing an antihyperglycemic action by blocking a-1,4-glucosidase in the intestine (Arai et al., 1998). MOR-14 dose-dependently decreased the a1,6-glucosidase activity in rabbit heart extract, and also dose-dependently reduced the infarct size without altering the blood pressure or the heart rate (Arai et al., 1998). In addition, MOR-14 decreased a-1,6-glucosidase activity to  20% in vivo, reduced glycogen breakdown, and attenuated lactate accumulation during ischemia. Pre-ischemic treatment with MOR-14 preserved glycogen, attenuated the accumulation of lactate, and reduced infarct size by 69%, indicating that this effect could be associated with a1,6-glucosidase inhibition. It has been suggested that increased mortality after acute myocardial infarction (AMI) is correlated to alteration of protection afforded by ischemic PC. This hypothesis was tested in dogs subjected to a prolonged (60 min) coronary artery occlusion and 3 hr of reperfusion, where glycemic levels were simulated using an intravenous infusion of 15%

dextrose in water (Kersten et al., 1998). Modest degrees of hyperglycemia (300 mg/dL) had no effect on infarct size, but abolished the protective effect of ischemic PC. In contrast, profound hyperglycemia (600 mg/dL) increased infarct size without altering hemodynamic and coronary collateral blood flow, indicating that acute hyperglycemia adversely modulated myocardial injury in response to ischemia in vivo. 2.5. A possible role in preconditioning for monophosphoryl lipid A and RC-552 Monophosphoryl lipid A (MLA) represents a novel agent capable of enhancing myocardial tolerance to ischemia/ reperfusion injury. This cardioprotective activity of MLA manifests itself as a reduction in infarct size, myocardial stunning, and dysrhythmias in multiple animal species (for reviews, see Elliott, 1998; Zhao & Elliott, 1999). The drug was effective in dogs and rabbits at doses of 10 ± 35 mg/kg, with larger doses required in the rat. In the rabbit infarct model, protection appeared 6 hr following drug administration, and lasted for 36 hr. Although multifactorial mechanisms of ischemic tolerance may be induced by MLA, current evidence suggests that the cardioprotective effects of MLA involve myocardial inducible nitric oxide synthase(s) [iNOS(s)] enzyme activation [for the role of nitric oxide [NO] in the cardiovascular system, see Ignarro et al., (1999)] with NO-coupled activation of myocardial KATP channels upon ischemic challenge (Elliott, 1998; Tosaki et al., 1998; Zhao & Elliott, 1999). MLA presently is being evaluated in Phase 2 clinical trials in patients undergoing cardiopulmonary bypass associated with coronary artery bypass engraftment or aortic valve replacement or reconstruction. Severity of lethal and reversible myocardial injury and dysrhythmia are study endpoints. Although further clinical testing will establish the utility of MLA as a cardioprotectant against ischemia ± reperfusion injury in the human, presently this agent is proving very useful in expanding our understanding of mechanisms responsible for delayed cardiac PC against ischemia ±reperfusion injury. The role of opening of KATP channels in MLA-induced myocardial protection after ischemia reperfusion has been evaluated in rabbits using 5-HD to block MLA-stimulated cardiac protection (Janin et al., 1998). Pretreatment with MLA reduced infarct size in rabbits, whereas infarct size increased with 5-HD in MLA-treated rabbits, suggesting that MLA also exerted its protective effect through activation of KATP channels. More recently, it was determined whether RC-552, a novel synthetic glycolipid related in chemical structure to MLA, could afford similar protection (Xi et al., 1999; Zhao & Elliott, 1999). Adult mice were pretreated with RC-552 24 hr before global ischemia and reperfusion in a Langendorff isolated, perfused heart model (Xi et al., 1999). A group of RC-552-treated mice received S-methylisothiourea, a selective inhibitor of iNOS, 30 min before

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heart perfusion. Myocardial infarct size was significantly reduced in the RC-552-treated group, and treatment with S-methylisothiourea abolished the RC-552-induced reduction in infarct size. More importantly, RC-552 failed to reduce infarct size in isolated hearts from iNOS knockout mice compared with that in hearts from control knockout mice without drug treatment. Thus, RC-552 induces delayed cardioprotection via an iNOS-dependent pathway. 2.6. Other possible mechanisms involved in myocardial ischemic preconditioning There are several other mediators whose involvement in PC has been proposed. Here we will comment on some of the most recent acquisitions. It has been shown that endogenous calcitonin generelated peptide (CGRP) may play an important role in the mediation of ischemic PC (Zhou et al., 1999). Whether nitroglycerin provided a PC stimulus and whether the cardioprotective effects of nitroglycerin-induced PC involved endogenous CGRP has also been examined (Hu et al., 1999). Pretreatment with nitroglycerin for 5 min before ischemia improved cardiac function, decreased creatine kinase release, and increased the content of CGRP-like immunoreactivity in the coronary effluent. Isoflurane may have cardioprotective effects that mimic PC. Since KATP channels and adenosine receptors are implicated in ischemic PC, it has been proposed that isoflurane-induced PC and ischemia-induced PC share similar mechanisms, which include activation of KATP channels and adenosine receptors (Ismaeil et al., 1999). Recent data support a cardioprotective effect of isoflurane and, more generally, demonstrate the feasibility of pharmacologically PC the human heart during cardiac bypass surgery (Belhomme et al., 1999). Cl ÿ channel involvement in the myocardial protection afforded by ischemic PC has been shown in isolated rabbit ventricular myocytes using two inhibitors [indanyloxyacetic acid 94 (IAA-94) and 5-nitro-2-(3-phenylpropylamino)benzoic acid] that abolished the protection afforded by ischemic PC (Diaz et al., 1999). Among the signal transduction pathways involved in the mechanism of PC, mention has to be made of the role of cyclic nucleotide phosphodiesterase, as well as of pH, vacuolar proton ATPase, apoptosis, and diacylglycerol. Fluctuations in cyclic nucleotides cyclic GMP and cyclic AMP during PC has been associated with concomitant changes in phosphodiesterase activity (Lochner et al., 1998). Moreover, blockade of vacuolar proton ATPase prevented the effect of PC, suggesting that acidification, even in the absence of Na + /H + exchange, may lead to cell death. This implies that a possible target of PKC in mediating PC could be the activation of vacuolar proton ATPase (Gottlieb et al., 1996). The role of PPs has also been investigated. Fostriecin, a potent inhibitor of PP2A, at a concentration selective for

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the inhibition of PP2A, mimicked PC in both rabbit and pig cardiomyocytes (Armstrong et al., 1997). The role of PPs during ischemic PC in the rabbit heart was further examined. Fostriecin was administered to rabbit hearts, and the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of control and ischemically preconditioned cells were also measured (Weinbrenner et al., 1998). In hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts, but comparable with that in ischemic preconditioned hearts. In isolated myocytes, fostriecin also provided protection comparable with that produced by metabolic PC. PC had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes. Thus, fostriecin protected the rabbit heart from infarction, even when administered after the onset of ischemia. b-adrenoreceptors have been proposed to play a role in PC, and several studies indicate that ischemia-induced activation of the b-adrenergic signaling pathway during PC should also be considered a trigger in eliciting PC (Lochner et al., 1999). A brief period of stimulation of cardiac b-adrenoreceptors with isoproterenol or norepinephrine, but not phenylephrine, caused a PC mimetic effect against postischemic contractile dysfunction in perfused rat heart (Nasa et al., 1997). This effect seems to be mediated in part by activation of PKC (Yabe et al., 1998). Moreover, overexpression of the rat-inducible hsp70 in transgenic mouse increased the resistance of the heart to ischemic injury (Marber et al., 1995). The role of inositol 1,4,5-trisphosphate in PC initially has been proposed by using neomycin as a pharmacological tool (Bauer et al., 1999). Ischemic PC in the rabbit heart causes an increase in inositol 1,4,5-trisphosphate, which is prevented by neomycin treatment. However, infarct size was similar in both neomycin and control preconditioned hearts. In contrast, it was determined whether an agonist and an antagonist of the second messenger inositol 1,4,5trisphosphate signaling, D-myo-inositol-1,4,5-trisphosphate hexasodium salt and 2-aminoethoxydiphenyl borate, respectively, given that they mimic this biphasic profile, would mimic infarct size reduction with PC in isolated rabbit hearts (Gysembergh et al., 1999). Infarct size was reduced with PC and in all D-myo-inositol 1,4,5-trisphosphate hexasodium salt- and 2-aminoethoxydiphenyl borate-treated groups versus control. Thus, pharmacological manipulation of the second messenger inositol 1,4,5-trisphosphate signaling appeared to mimic the cardioprotective effects of PC in isolated rabbit heart (Gysembergh et al., 1999). Furthermore, it is noteworthy that the hypothesis has been forwarded that tumor necrosis factor (TNF)-a also has a role in ischemic PC. Using perfused rat heart, it has been shown that ischemia reperfusion induced increases in TNFa in the rat heart and impaired myocardial function, while sequestration of myocardial TNF-a improved postischemic

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myocardial function (Meldrum et al., 1998). TNF-a also establishes a complex network with the protease-activating receptor-2 (Cirino et al., 2000) during myocardial ischemic injury in the rat (Napoli et al., 2000). Finally, some studies have suggested that free radicals (oxygen radicals) can contribute to PC by directly stimulating PLs and/or transcription signaling (Baines et al., 1997; Tritto et al., 1997), and that hypercholesterolemia did not appear to limit the PC effect (Kremastinos et al., 2000) in the rabbit heart. 3. From preclinical studies to new clinical features in myocardial ischemic preconditioning Several clinical conditions may mimic ischemic PC (reviewed in Dana et al., 1998b; Schwarz et al., 1999; Yellon & Baxter, 2000). Repeated coronary artery occlusions during coronary angioplasty might simulate PC (Deutsch et al., 1990; Cribier et al., 1992). In this condition, adenosine can mimic the protective effect (Leesar et al., 1997) and, accordingly, the adenosine antagonists block it (Claeys et al., 1996). Preliminary data in humans also suggest that naloxone may reduce the effects of PC during angioplasty (Tomai et al., 1999b). Pharmacological PC has also been shown to be induced by nicorandil in patients undergoing coronary angioplasty (Matsubara et al., 2000). Finally, in a recent study (Leesar et al., 1999), bradykinin appears to precondition human myocardium against ischemia in vivo in the absence of systemic hemodynamic changes. Pretreatment with bradykinin appeared to be just as effective as ischemic PC, and could be used prophylactically to attenuate ischemia in patients undergoing coronary angioplasty. Thus, several compounds with PC-like effects could reduce myocardial ischemia during angioplasty. In the Acute Myocardial Infarction Study of Adenosine trial, the hypothesis was tested in humans that adenosine given as an adjunct to thrombolysis would reduce myocardial infarct size (Mahaffey et al., 1999). The Acute Myocardial Infarction Study of Adenosine trial was a prospective, open-label trial of thrombolysis, with randomization to adenosine or placebo in 236 patients within 6 hr of infarction onset. The primary endpoint was infarct size, as determined by 99Tc-Sestamibi single-photon emission-computed tomography imaging. Secondary endpoints were myocardial salvage index and in-hospital clinical outcome (death, reinfarction, shock, congestive heart failure, or stroke). There was a significant reduction in infarct size with adenosine (33%; P < 0.05) associated with a better clinical outcome, indicating a possible application of adenosine-mediated PC in larger multicenter clinical studies. Interestingly, nucleoside transport inhibitors, that is, dipyridamole, have been shown to enhance infarct size limitation by PC in the rabbit heart, most likely as a consequence of elevated interstitial adenosine levels (Suzuki et al., 1998). Since dipyridamole is widely used clinically, a possible role for modulating adenosine levels in PC of humans has been

debated (Seiler & Billinger, 1998). Previous studies showed that intracoronary application of dipyridamole in patients undergoing coronary angioplasty significantly improved myocardial function during the angioplasty procedure (Strauer et al., 1996). Similarly, intravenous low-dose dipyridamole increased peak adenosine plasma levels and mimicked PC with respect to electrocardiographic and echocardiographic findings (Pasini et al., 1996). Low-dose dipyridamole infusion increases exercise tolerance in patients with chronic stable angina, possibly by endogenous adenosine accumulation acting on high-affinity A1 myocardial receptors involved in PC or positively modulating coronary flow through collaterals (Tommasi et al., 2000). Finally, in the double-blind randomized Clinical European Studies in Angina and Revascularization-2 investigation, the addition of nicorandil to anti-anginal treatment was shown to reduce transient myocardial ischemia and arrhythmias in patients with unstable angina, an effect that can be attributed to chemical PC (Patel et al., 1999). PC is also observed in preinfarction angina and during coronary artery bypass surgery (see Section 3.4). The ``warm-up'' phenomenon may represent another clinical counterpart of myocardial ischemic PC (Okazaki et al., 1993; Maybaum et al., 1996; Baxter, 1997; Cohen & Downey, 1999). In fact, patients with coronary heart disease (usually with stable angina) are able to exercise longer before developing chest pain, and may develop less angina and signs of myocardial ischemia during a second exercise test compared with the first exercise when exercise tests are divided by a rest period. The pathophysiology and the mechanisms of the warm-up phenomenon are not well established (Cohen & Downey, 1999). In patients with exertional angina, the size of the warm-up response is related to the maximum intensity rather than the duration of first exercise (Kay et al., 2000). Patients with coronary heart disease were randomized to receive glibenclamide or placebo, and then stimulated to perform serial exercise tests (Tomai et al., 1999a). After placebo administration, ratepressure product at 1.5 mm ST-segment depression significantly increased during the second exercise test compared with the first, but it did not change after glibenclamide. Thus, glibenclamide, at a dose previously shown to abolish ischemic PC during coronary angioplasty, prevented the increase of ischemic threshold observed during the second of two sequential exercise tests. Mortality in diabetic patients in the Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction study with AMI is predicted by age, previous heart failure, and severity of the glycometabolic state at admission, but not by conventional risk factors or sex (Malmberg et al., 1999). Intensive insulin treatment reduced long-term mortality, despite high blood glucose at admission and glycosylated hemoglobin. Patients receiving oral hypoglycemic agents for diabetes mellitus have been indicated to be at an increased relative risk of cardiovascular mortality after direct angioplasty for myocardial infarction (Garratt et al.,

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1999). Since oral hypoglycemic agents are inhibitors of the KATP channel, it has been suggested that myocardium from patients taking long-term oral hypoglycemic agents would be resistant to protection by ischemic PC. Although the research is very active in understanding the mechanisms of PC and several large studies are actually in progress, we sought to analyze the ``state of the art'' clinical evidence for ischemic PC. In particular, we attempt to describe the newly developed hypothesis that has arisen from preclinical studies and, therefore, new possible clinical challenges. 3.1. Preinfarction angina and ``new-onset'' angina as models of myocardial ischemic preconditioning in humans Several studies (Iwasaka et al., 1994; Kloner et al., 1995; Nakagawa et al., 1995; Ottani et al., 1995; Napoli et al., 1998b) support the idea that preinfarction angina may act as a common PC stimulus. In this condition, two key points should be kept in mind. First, the analysis of the mechanism(s) by which previous episodes of angina exert a beneficial clinical effect on myocardial function after myocardial infarction. Second, the exact temporal definition of the term ``preinfarction angina.'' For instance, patients with angina prior to AMI have been shown to have better preserved left ventricular performance (Matsuda et al., 1984; Hirai et al., 1992; Iwasaka et al., 1994), and the Thrombolysis and Angioplasty in Myocardial Infarction Study Group (Muller et al., 1990) reported less short-term complications and fewer episodes of reocclusion after thrombolysis in patients with preinfarction angina within 1 week. Probably, the difference in residual contractile function was due in part to recruitment of coronary collateral flow. In fact, Cortina et al. (1985) reported better preservation of ventricular function in patients with preinfarction angina (ranging from 1 month to 8 years), but it was shown angiographically that visible collaterals played a significant protective role. More recently, Nakagawa et al. (1995) also showed the protective effect of previous angina in patients with reperfused anterior wall myocardial infarction. However, we have to consider that large studies showed that those with angina did worse in terms of prognosis (Barbash et al., 1992; Behar et al., 1992). Indeed, patients with antecedent stable angina often have multivessel coronary heart disease, and are prescribed several anti-ischemic drugs that are taken for several months or years. These considerations can additionally affect the clinical outcome following myocardial infarction. In order to minimize the impact of several variables, we analyzed the recovery of regional myocardial contractile function in patients after thrombolysis, in whom myocardial infarction occurred unheralded, in comparison with patients who had new-onset angina within 48 hr before their first myocardial infarction (Napoli et al., 1998b). We showed for the first time that recovery of myocardial contractile function after thrombolysis was more prompt and durable in patients in

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whom myocardial infarction was preceded by new-onset angina, as compared with patients in whom infarction occurred unheralded. Moreover, TIMI-9B prospectively determined the importance of the time of onset of preinfarction angina in relation to 30-day outcomes (Kloner et al., 1998c). Of the 3002 patients entered into the study, 425 reported angina before their myocardial infarction, and, more important, patients with angina onset within 24 hr of infarction had a lower 30-day cardiac event rate (4%) in terms of mortality, recurrent myocardial infarction, heart failure, or shock than those with onset of angina >24 hr (17%). Therefore, these temporal observations (Kloner et al., 1998c; Napoli et al., 1998b) suggest that human PC is induced by new-onset preinfarction angina. Interestingly, although a complete myocardial reperfusion is mandatory for PC (Ovize et al., 1992b), it has been shown that the presence of a critical coronary artery stenosis does not abolish the protective effect of PC (Kapadia et al., 1997). This phenomenon may be similar to the clinical scenario in which brief ischemic episodes and reperfusion, superimposed on a critical coronary stenosis, precede a prolonged occlusion determining AMI. Thus, new-onset angina may be a representative clinical condition in which the presence of a critical stenosis does not abolish the beneficial effect of ischemic PC. Moreover, further studies are necessary in order to better understand pathophysiological mechanisms underlying atherogenesis in humans (Napoli et al., 1999a; Ross, 1999), coronary vasomotion (Maseri et al., 1999), and myocardial microcirculation (Tritto & Ambrosio, 1999). Finally, it is well known that early administration of thrombolysis after AMI is crucial in both limiting infarct size and preserving left ventricular function (Lincoff & Topol, 1993). Andreotti et al. (1996) proposed that the benefit of preinfarction angina with respect to infarct size may partially depend on faster coronary thrombolysis, besides ischemic PC. New-onset angina may be associated with newly formed thrombi that are thrombolyzed faster than isolated and persistent growth thrombi, and a trend toward shorter reperfusion times was also seen in other studies (Ottani et al., 1995; Napoli et al., 1998b). 3.2. Is the development of myocardial tolerance to ischemia in humans due to ischemic preconditioning or to collateral recruitment? The time needed for developing new collaterals after acute coronary occlusion in humans is still unclear (Ambrose & Fuster, 1983; Sasayama & Fujita, 1992). Schwartz et al. (1984) showed that  2 weeks is required to develop new visible collateral vessels after myocardial infarction. Better preservation of ventricular function in patients with preinfarction angina may depend upon collaterals (Cortina et al., 1985). However, the retrospective analysis of patients enrolled in the Thrombolysis in Myocardial Infarction 4 Trial has shown that preinfarction angina resulted in a lower incidence of in-hospital death, severe

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heart failure, or shock, and a smaller infarct size, and this protection was not dependent on angiographically visible epicardial coronary collateral blood vessels (Kloner et al., 1995). Similarly, the absence of visible collaterals during the beneficial effects of PC was seen in other studies (Ottani et al., 1995; Napoli et al., 1998b). In a recent study, the contribution of ischemic, as well as adenosine-induced, PC and of collateral recruitment to the development of tolerance against repetitive myocardial ischemia has been elegantly investigated in patients with quantitatively determined, poorly developed coronary collaterals (Billinger et al., 1999). Myocardial adaptation to ischemia was measured using intracoronary electrocardiographic ST segment elevation changes during three subsequent 2-min balloon occlusions in 30 patients undergoing coronary angioplasty. Simultaneously, an intracoronary pressure-derived collateral flow index was determined as the ratio between distal occlusive minus central venous pressure, divided by the mean aortic minus central venous pressure. Results showed that collateral flow index at the first occlusion was not different between the groups, and it increased significantly during the third occlusion. Thus, even in patients with few coronary collaterals, the myocardial adaptation to repetitive ischemic episodes appeared to be closely related to collateral recruitment. When adenosine was used before coronary angioplasty, the phenomenon did not occur. Therefore, the variable responses of ECG indicators of ischemic adaptation to collateral opening suggest that ischemic PC is a relevant factor in the development of ischemic tolerance. 3.3. Myocardial stunning and ischemic preconditioning The postischemic myocardial dysfunction termed ``myocardial stunning'' should not be considered as a single entity, but rather, as a ``syndrome'' observed in several experimental settings, which include the following: (1) stunning after a single, completely reversible episode of regional ischemia in vivo; (2) stunning after multiple and reversible episodes of regional ischemia in vivo; (3) stunning after a partly reversible episode of regional ischemia in vivo (subendocardial infarction); (4) stunning after global ischemia in vitro and in vivo; and (5) stunning after exercise-induced ischemia (high-flow ischemia) (Bolli & Marban, 1999). Although the pathogenesis of myocardial stunning has not been definitively established, the two major hypotheses are that it is caused by the generation of oxygen radicals (radical hypothesis) and/or by a transient Ca2 + overload (Ca2 + hypothesis) during reperfusion (Bolli & Marban, 1999). However, these hypotheses are not mutually exclusive and are likely to represent different aspects of the same pathophysiological scenario. In fact, increased oxygen radical formation could also cause cellular Ca2 + overload, which would damage myocardial contractile function, and this could also directly alter filaments of myocytes in a manner that renders them less responsive to Ca2 +.

Murry et al. (1991) firstly studied the relationship between ischemic PC and stunning in an experimental model. If the reperfusion phase between the brief PC ischemia and the 40-min occlusion was extended to 120 min, the myocardium remained severely stunned after the brief ischemia, but the myocardial infarct size plotted against collateral flow returned toward nonpreconditioned values. Matsuda et al. (1993) showed that dobutamine could be used to reverse stunning induced by four 5-min coronary occlusions in the dog model, but that reversing stunning did not prevent PC. These studies suggested that although brief periods of ischemia induce both stunning and PC, the two phenomena could be dissociated and PC may be not due only to reduced contractile function of stunned myocardium. In other studies, the efficacy of ischemic PC in reducing stunning has not been as consistent as its ability to reduce necrosis. It is unclear, therefore, whether the protection afforded by PC against stunning is mediated by the same mechanism that mediates its protection against lethal cell injury (Asimakis et al., 1992; Ovize et al., 1992a). More recently, experimental studies have shown that ischemic PC significantly increased the maximal inotropic response and diminished the contractile dysfunction of early stunning (Mosca et al., 1998), and PC also reduced myocardial stunning, preserving high-energy phosphates after cardiac transplantation (Landymore et al., 1998). However, it is important to note that contractile dysfunction may persist for hours, or even for as long as several days, before function recovers (Bolli & Marban, 1999). In contrast, prolonged contractile impairment may stem from development of reversible myocyte damage caused by ischemic episodes, whereas evidence of contractile dysfunction in the presence of abnormalities of myocardial flow manifests persisting or recurrent ischemia (Bolli & Marban, 1999). Thus, the effects of late PC (second window of protection) may affect prolonged contractile impairment. In this respect, we showed that patients with new-onset angina before infarction had an endogenous protection of left ventricular ejection fraction and wall motion until 3 months of follow-up (Napoli et al., 1998b). Moreover, ischemic PC also improved cardiac function at 30 min and 12 hr after reperfusion in valve-replacement patients (Li et al., 1999). The possible protective mechanism was that ischemic PC decreased the production of oxygen radicals. Taken together, these studies (Napoli et al., 1998b; Li et al., 1999) strongly suggested that late PC may exert beneficial effects on postischemic contractile dysfunction, that is, stunning, in humans. Accordingly, it was also demonstrated recently in a single-center prospective study that the beneficial effect of preinfarction angina on left ventricular wall motion is independent of collateral flows (Noda et al., 1999). The greater protective effect of a longer time interval between angina pectoris and AMI also suggested that the protection was due to a delayed PC effect. MLA was shown to attenuate myocardial stunning in dogs, and a recent study investigated this drug in stunning

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and PC (Elliott et al., 1998). To induce stunning, anesthetized dogs were subjected to five cycles of 5 min of coronary occlusion with 10 min of reperfusion, and finally, followed by 2 hr of reperfusion. Single intravenous doses of MLA in the range of 10 ±35 mg/kg given 24 hr before ischemia resulted in an improvement in the number of hypokinetic segments over a 2-hr reperfusion period. Cardioprotection against stunning with MLA appeared to require activation of KATP channels during ischemia, because glibenclamide completely blocked the protective effect of MLA. Thus, MLA improved stunning by a KATPsensitive channel-dependent process during late ischemic PC. These studies suggest that MLA also may exert positive effects on stunning and PC in humans. 3.4. Preconditioning and coronary artery bypass heart surgery Mentzer et al. (1997) were the first to apply adenosinemediated PC to patients undergoing coronary artery bypass surgery. More recently, minimally invasive direct coronary artery bypass (MIDCAB), which utilizes alternative incisions and ``port-access'' technology, provides many anesthetic challenges, including intense monitoring, managing of myocardial ischemia, and pain control (Chitwood et al., 1999). Recent advances in videoscopic visualization and evolving mechanisms of myocardial protection may justify the expanding application of MIDCAB. A recent study has evaluated the monitoring requirements and the potential benefits of PC and intrathecal morphine sulfate in MIDCAB patients (Heres et al., 1998). Transesophageal echocardiography was used, and a pulmonary artery catheter was used in 43% of the patients. PC did not prevent increases in systemic or pulmonary artery pressures during coronary occlusion. MIDCAB may reduce the length of hospital stay for patients with single-vessel coronary artery lesions, when compared with classical median sternotomy. Early PC induced by a single 5-min test coronary occlusion did not protect against subsequent regional ischemic dysfunction in the subset of patients with normal baseline function. The effects of early ischemic PC during MIDCAB were also investigated in another recent study (Malkowski et al., 1998). Left ventricular wall motion score increased significantly from baseline to coronary occlusions 1 and occlusion 2, whereas no difference in wall motion was noted between coronary occlusions 1 and 2. Pulmonary artery systolic pressure increased significantly from baseline to coronary occlusion 1 and occlusion 2. Pulmonary artery diastolic pressure also increased significantly from baseline to coronary occlusion 1 and occlusion 2, whereas no significant differences in pulmonary artery pressures were noted between coronary occlusions 1 and 2. Thus, ischemic dysfunction was precipitated by the early ischemic PC induced by a 5-min coronary occlusion, as shown by the increase in left ventricular wall motion score and pulmonary artery pressure. This study indicated that early ischemic PC

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induced by a 5-min coronary occlusion and the resulting ischemia did not alter regional left ventricular systolic function during subsequent ischemia in humans. 3.5. Arrhythmias and myocardial ischemic preconditioning The efficacy of early ischemic PC in reducing arrhythmias in experimental models has not been as consistent as its ability to reduce necrosis (Sariahmetoglu et al., 1998; Przyklenk & Kloner, 1995; Hagar et al., 1991; Shiki & Hearse, 1987). More recently, it was demonstrated that KATP channels and opioid receptors may be partly involved in the suppression of reperfusion arrhythmias, although their roles may be compensated for by other antiarrhythmic mechanisms in repetitive PC (Kita et al., 1998). In contrast with the effects of early PC against infarction, PKC is unlikely to play a major role in protection afforded by PC against reperfusion arrhythmias in the rat (Kita et al., 1998). Interestingly, in a very recent study, it was shown that in rats neither maturation nor gender influence the antiarrhythmic effect of early ischemic PC; however, female rats exhibit a lower level of arrhythmic activity during sustained coronary artery occlusion than male rats, both in vivo and in vitro (Humphreys et al., 1999). There are also effects of PC that are species-related (reviewed in Wainwright, 1992; Sun et al., 1996;). For example, ischemic PC increases both the arrhythmic index and the incidence of ventricular fibrillation during the early phase of a subsequent ischemic period in the pig (Grund et al., 1997). The progressive electrocardiographic deterioration and increasing incidence of ventricular arrhythmias during repetitive 15-min occlusions in pigs suggested increasing metabolic derangement. However, the progressively faster normalization of the ST segment and the reduced incidence of ventricular arrhythmias during reperfusion suggested an increasingly faster restoration of the metabolic and ionic balance (Figueras et al., 1996). An important question on the possible mechanism exerted by PC in protecting the myocardium from arrhythmias in humans was postulated by Lawson and Hearse (1994). It is important to understand whether this beneficial effect is due to an antiarrhythmic action per se or to a general anti-ischemic phenomenon. At present, the exact nature of the protective mechanism(s) exerted by PC on arrhythmias is still not well established. It is well known that QT interval dispersion reflects regional variations in ventricular repolarization and cardiac electrical instability. The effect of ischemic PC on the manner of ventricular repolarization was investigated by assessing the change in QT dispersion during coronary angioplasty (Okishige et al., 1996). The gradual decrease in QT dispersion provoked by coronary artery occlusion and reperfusion during coronary angioplasty may be associated with the electrophysiologic effects of ischemic PC on human myocardium. Moreover, animals treated with cardiac pacing (Parratt et al., 1996) and patients with severe angina within 24 hr of onset of their first myocardial infarction, but

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not those without preceding angina (Tamura et al., 1997), had reduced occurrence of life-threatening ventricular tachyarrhythmias and late potentials mainly associated with reperfusion. Early PC also induced a significant protection against ischemia-induced complex ventricular arrhythmias (more than five premature ventricular beats per minute or repetitive ventricular arrhythmias) in patients with variant angina (Pasceri et al., 1996). This beneficial effect was not related to a reduction in either severity or duration of ischemia, suggesting that arrhythmic protection was a direct consequence of PC rather than an epiphenomenon of ischemic protection. 3.6. A loss of preconditioning in the aging heart? The geriatric population is considerably increased around the world. Thus, more elderly patients have coronary heart disease, which is often the cause of sudden death (Gersh, 1986; Toefler et al., 1988). Abete et al. (1996) first showed that ischemic tolerance induced by PC was reduced in the rat senescent heart. This finding appeared to be species-related. In fact, in the ovine

senescent heart, the cellular pathways involved with the PC response were well preserved (Burns et al., 1996). Later, Tani et al. (1997) confirmed that hearts became more vulnerable to ischemia with age, and that the beneficial effects of PC were reversed in middle-aged rat hearts. More recently, it was demonstrated that in the senescent rabbit myocardium, the cellular pathways involved ischemic PC, such as postischemic dysfunction, and that functional recovery was worse compared with that of the adult myocardium (Uematsu & Okada, 1998). Moreover, using adenosineenhanced ischemic PC, it was shown that this treatment provided similar protection to Mg2 + -supplemented K + cardioplegia, significantly enhancing postischemic functional recovery and decreasing infarct size in the rabbit senescent myocardium (McCully et al., 1998). PC also failed to lessen the increased [Na + ]i or to protect the aging hearts, probably due to the pre-existence of increased glycogen level (Tani et al., 1999). However, preliminary data indicate that exercise training may partially restore ischemic PC in the senescent rat heart (Abete et al., 2000). It is well known that there is a shorter survival time after AMI in elderly patients compared with younger patients

Fig. 1. Some of the inhibitors, agonists, and antagonists used in experimental procedures of PC to determine involvement of different triggers, transducers, and end-effectors, and possible signaling pathways activated. The continuous arrow indicates stimulation while the dotted arrow indicates inhibition. Substances quoted in the figure are reported with the corresponding reference in parentheses: RO31-8220, inhibitor of PKC (Tosaki et al., 1997); 5-HD, inhibitor of KATP mitochondrial channels (Baines et al., 1999); R-PIA, A1 receptor agonist (Smits et al., 1998); [D-Ala(2),D-Leu(5)]enkephalin (DADLE), d1 opioid receptor agonist (Kevelaitis et al., 1999); DOG, inhibitor of PKC (Yabe et al., 1998); icatibant, inhibitor of B2 receptor (Bouchard et al., 1998); TAN67, d1 opioid receptor agonist (Schultz et al., 1998b); PMA, PKC stimulator (Fryer et al., 1998); Lavendustin A, tyrosine kinase inhibitor (Galinanes et al., 1998); isoproterenol, b-adrenoreceptor agonist (Yoshida et al., 1997); fostriecin, PP inhibitor (Armstrong et al., 1997; Weinbrenner et al., 1998); captopril, ACE inhibitor (Armstrong et al., 1998; Yang, X. P., et al., 1997); sodium oleate, PLD agonist (Eskidilsen-Helmond et al., 1996); MLA, KATP channel and iNOS stimulator (Elliott, 1998; Kersten et al., 1998; Tosaki et al., 1998); 7-benzylidenenaltrexone (BNTX, d1 opioid receptor agonist (Schultz et al., 1997); IBMECA, A3 adenosine receptor agonist (Tracey et al., 1997); naloxone, opioid receptor antagonist (Schultz et al., 1995); cromakalim, KATP channel opener (Haruna et al., 1998); BWA1443, A1 receptor agonist (Cohen et al., 1998); IAA-94, chloride channel inhibitor (Diaz et al., 1999); hsp70 (Marber et al., 1995); nicorandil and pinacidil, KATP opener (Critz et al., 1997; Kita et al., 1998); MAPK and JNK (Ping et al., 1999b); TK (Fryer et al., 1998; Ping et al., 1999b; Valhaus et al., 1998); genistein, TK inhibitor (Fryer et al., 1998); glibenclamide, KATP inhibitor (Gross & Fryer 1999; Tomai et al., 1999b). aPKC, activated PKC; MEK, MAPK kinase, MEK2 resembles MEK1 in its substrate specificity; NFkB, nuclear factor kB.

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(Gersh, 1986; Toefler et al., 1988). Retrospective studies strongly suggest that ischemic PC (Abete et al., 1997) and the warm-up phenomenon (Napoli et al., 1998a, 1999b) are insufficient to protect the senescent, but not the adult, myocardium in humans. Another retrospective study, however, suggested that preinfarct angina was still beneficial in older patients (Kloner et al., 1998b). Since multiple mechanisms may be responsible for age-related effects of myocardial ischemic PC (Napoli & Ambrosio, 1998), the hypotheses presented here need to be tested in large prospective clinical studies. In particular, these studies will have to also evaluate the exact contribution of collaterals to myocardial function in elderly people with previous angina. Nevertheless, in a very recent study, it was confirmed that beneficial effects of prodromal angina are lost in elderly patients with AMI (Ishihara et al., 2000). Nine hundred ninety patients (722 < 70 years and 268 >70 years) who underwent coronary angiography within 12 hr after the onset of AMI were studied. Prodromal angina in the 24 hr before infarction was found in 190 of 722 nonelderly patients and in 66 of 268 elderly patients (26% vs. 25%, P = 0.61). In nonelderly patients, prodromal angina was associated with lower peak creatine kinase levels, lower in-hospital mortality rates (3.7% vs. 8.8%, P = 0.02), and better 5-year survival rates ( P = 0.007). On the contrary, in elderly patients, there was no significant difference in peak creatine kinase levels ( P = 0.51), in-hospital mortality rate (21.2% vs. 17.4%, P = 0.49), and 5-year survival rates ( P = 0.47). A multivariate analysis showed that prodromal angina in the 24 hr before infarction was associated with a 5-year survival rate in nonelderly patients (odds ratio 0.49, P = 0.009), but not in elderly patients (odds ratio l.12, P = 0.65). Thus, in nonelderly patients, but not in elderly patients, prodromal angina in the 24 hr before infarction was associated with a smaller infarct size and better shortand long-term survival, suggesting a relationship to ischemic PC. 4. Concluding remarks It is very difficult to understand which is the leading mechanism(s) in the PC phenomenon. Even though several triggers, transducers, end-effectors, agonists, antagonists, and inhibitors have been tested in experimental settings, the question whether it is possible to chemically simulate PC remains open. In an effort to give to the reader an overview of what has been discussed in this review, some of the drugs used in experimental models and some of the most acknowledged mechanisms are reported in Fig. 1. Obviously, this figure does not pretend to summarize all that is known, but attempts to give an outline on the complexity of this phenomenon. Delayed effects of PC (second window of protection) and their possible mediators are reported in Table 1. The same criteria used to create Fig. 1 have also been used for Table 1. However, at the present, the possi-

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bility to develop a safe drug that will mimic PC still seems to be far away, except for a preliminary clinical study with adenosine (Mahaffey et al., 1999). Obviously, results from clinical trials are more complicated than those obtained from models used in experimental studies. PC is more likely to occur in patients with ``newonset'' angina before myocardial infarction, as well as during MIDCAB or coronary angioplasty. The role of ischemic PC in preventing arrhythmias, stunning, and with increasing age is still poorly understood. Large prospective multicenter trial studies are needed to better understand the possible pathophysiological role of the endogenous protection induced by PC in these clinical conditions. Acknowledgments C. Napoli would like to dedicate this paper to Dr. G. Ambrosio (Perugia, Italy), his mentor in the pathophysiology of myocardial ischemia and oxygen radicals. The authors also gratefully acknowledge Drs. P. Abete, A. Liguori, F. Cacciatore, M. Chiariello, and L. Sorrentino (Naples, Italy) and Drs. V. Anania, F. Franconi, and M.P. DeMontis (Sassari, Italy) for valuable discussions in this field, and Dr. C.L. Wainwright (Glasgow, UK) for insightful suggestions regarding the organization of the paper. This work was supported by grants from Ministero della Universita' e Ricerca Scientifica e Tecnologica (MURST 97/60%, 96/40%) to the Federico II University of Naples (G. Cirino and C. Napoli), by grant ISNIH.99.56980 (C. Napoli), and by grant MURST 98/60% to the University of Salerno (A. Pinto). References Abete, P., Ferrara, N., Cioppa, A., Ferrara, P., Bianco, S., Calabrese, C., Cacciatore, F., Longobardi, G., & Rengo, F. (1996). Preconditioning does not prevent postischemic dysfunction in the aging heart. J Am Coll Cardiol 26, 1777 ± 1786. Abete, P., Ferrara, N., Cacciatore, F., Madrid, A., Bianco, S., Calabrese, C., Napoli, C., Scognamiglio, P., Bollella, O., Cioppa, A., Longobardi, G., & Rengo, F. (1997). Angina-induced protection against myocardial infarction in adult and elderly patients. A loss of preconditioning mechanism in the aging heart? J Am Coll Cardiol 30, 947 ± 954. Abete, P., Calabrese, C., Ferrara, N., Cioppa, A., Pisanelli, P., Cacciatore, F., Longobardi, G., Napoli, C., & Rengo, F. (2000). Exercise training restores ischemic preconditioning in the aging heart. J Am Coll Cardiol 36, 643 ± 650. Ambrose, J. A., & Fuster, V. (1983). Coronary collateral vessels and myocardial protection. Int J Cardiol 3, 417 ± 420. Andreotti, F., Pasceri, V., Hackett, D. R., Davies, G. J., Haider, A. W., & Maseri, A. (1996). Preinfarction angina as a predictor of more rapid coronary thrombolysis in patients with acute myocardial infarction. N Engl J Med334, 7 ± 12. Arai, M., Minatoguchi, S., Takemura, G., Uno, Y., Kariya, T., Takatsu, H., Fujiwara, T., Higashioka, M., Yoshikuni, Y., & Fujiwara, H. (1998). N-methyl-1-deoxynojirimycin (MOR-14), an a-glucosidase inhibitor, markedly reduced infarct size in rabbit hearts. Circulation 97, 1290 ± 1297.

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