Preconditioning

During the past decade, it has been paradoxically demonstrated that brief periods of myocardial ischemia followed by reperfusion provide endogenous myocyte protection from subsequent ischemic insult. This concept, now known as "ischemic preconditioning," has been widely investigated, but the process by which such protection is conferred remains to be elucidated. Understandably, further investigation into this area is warranted as ischemic heart disease remains a prominent source of morbidity and mortality, and the discovery of the mechanism(s) at work has far reaching clinical implications. This paper will focus on current areas of investigation in ischemic preconditioning, including proposed mechanisms and anticipated clinical applications. Ischemic Preconditioning Preconditioning was originally defined as a rapid, adaptive response to a brief ischemic insult which slowed the rate of cell death during a subsequent, prolonged period of ischemia [1]. Some investigators have expanded the definition to include other adverse consequences of ischemic insult, such as dysrhythmias and contractile dysfunction. These alternative end points may not represent the same phenomenon as originally described. Studies using dysrhythmias as an end point must be very carefully construed to avoid changes in infarct size which then confound interpretation of data. Altered contractile function is difficult to interpret, as it may be due to dysfunction of viable cells (stunning) as well as lethal injury. Originally, protection was found to be induced solely by intermittent ischemia and reperfusion but subsequently has been demonstrated (to some extent) by trials of hypoxia, stress, and pharmacological substances [2]. This phenomenon was first discovered in 1981 by Reimer et al. [3] while measuring adenosine triphosphate (ATP) depletion after reversible myocardial ischemic injury in dogs. They demonstrated that ATP depletion during a second period of ischemia occurred at a slower rate than in the initial period. The investigators concluded that ATP hydrolysis decreased with subsequent ischemic challenges. In 1983, the notion that serial occlusions of the left anterior descending coronary artery produced consistent electrocardiographic changes was tested by Barber [4]. In this experiment performed on dogs, it was unexpectedly noted that if the serial occlusions were separated by 3 min, the electrocardiographic changes observed after the second occlusion were significantly diminished with respect to those seen after the first. Similarly, Reimer et al. [5] later noted that myocardial necrosis occurred in only one of seven dogs subjected to four serial 10-min ischemic challenges. Had the dogs been subjected to one sustained 40-min coronary insult, significant myocardial necrosis would have been expected in all of the animals. However, substrate recovery during the intermittent perfusion could have explained these results. The results of these studies prompted a study by Murry et al. [6] in 1986, which showed that four distinct 5-min intervals of coronary occlusion, each separated by a 5-min period of reperfusion, decreased the ultrastructural changes in the myocardium produced by a subsequent continuous 40-min period of coronary occlusion. The results of this study reaffirmed the notion that preconditioning can have a profound impact on myocardial survivability after a prolonged ischemic event. Studies have documented the effect of preconditioning with regard to infarct size in several species, including the rat [7], pig [8], rabbit [9], and dog [5]. Considerable interspecies variation is observed in the ischemic time required to initiate preconditioning as well as the maximum duration where myocardial protection is afforded. However, it is generally agreed upon that the initial ischemic challenge should be at least 1 min but limited to no more than 5 min in duration, as irreversible myocardial damage results with prolonged ischemia. Similarly, the reperfusion period should persist for at least 1 min and may continue for as long as 90 min depending on the species being studied. For instance, it has been shown in rats that protection from infarction is lost with a 1-h period of reperfusion [7], while in rabbits this may occur after a period of only 30 min [10]. Clearly, the protection afforded by ischemic preconditioning is transient in nature, suggesting the possibility that an exhaustible pool of some protective substrate may exist. In general, preconditioning appears to provide electrical stability and tissue resistance to ischemia via some underlying process whereby metabolic conservation occurs. Nonetheless, while these studies demonstrate the existence of preconditioning, they shed little, if any, light on the mechanism(s) responsible for this phenomenon. Proposed Mechanisms Collateral Vessels Originally, it was widely theorized that protection by preconditioning was a consequence of the opening of collateral coronary vessels. However, this theory was soon dismissed by the realization that cardioprotection occurred despite the experimental manipulation of collateral flow in dogs [6]. Additionally, species known to have limited collateral flow, i.e., rabbits and rats, clearly demonstrate the preconditioning phenomenon. Finally, the temporal aspects of preconditioning cannot be explained by this hypothesis. While collateralization is not a mechanism for preconditioning, it is an important protective mechanism against ischemia. Nitric Oxide Researchers have suggested that preconditioning may be attributed to nitric oxide production. Vegh et al. [11] reported that Nomega-nitro-L-arginine methyl ester, an inhibitor of nitric oxide production, can partially attenuate the protective actions of preconditioning on ischemia-induced arrythmias in dogs. However, these researchers have also reported that there is no significant effect on dysrhythmias when nonpreconditioned hearts are subjected to L-arginine, an amino acid that enhances nitric oxide synthesis, in an attempt to induce preconditioning [12]. As nitric oxide acts by stimulating soluble guanylyl cyclase, Vegh et al. [12] speculated that these protective effects may be mediated by an increase in cyclic guanosine 3 prime,5 prime-monophosphate. The effector mechanisms that might mediate the antidysrhythmic actions of an increased cyclic guanosine 3 prime,5 prime-monophosphate level are not clear, although possibilities include modulation of cytosolic calcium levels and sarcoplasmic potassium channels. Convincing evidence establishing nitric oxide as a mechanism in preconditioning is lacking. ATP Sensitive K+ Channels Ischemia and hypoxia are potent stimuli for the opening of ATP-sensitive potassium channels (KATP) Figure 1. Increased potassium conductance has the effect of shortening the cardiac action potential with a rapid loss of contractile function while possibly decreasing ATP depletion [3]. Considerable evidence suggests that opening these channels is cardioprotective, while blocking them is proischemic, and may actually delay the subsequent recovery of myocardial function or result in an increase in the ultimate extent of myocardial contractile dysfunction after ischemia and reperfusion [13-16]. While potent ATP sensitive K+ channel blockers such as glibenclamide (glyburide) have been demonstrated to inhibit preconditioning in canines by Gross and Auchampach [17] and in pigs by Rohmann et al. [18], equivocal evidence [19] exists in rabbits [20], and negative evidence has been established in rats [21]. In spite of the negative findings in preconditioned rat hearts, it is still likely that KATP channels play a role in ischemic preconditioning. Several K+ channel openers are cardioprotective in the isolated rat heart [22]. In addition, an increase in infarct size was demonstrated in the presence of glibenclamide [23]. Interspecies variation in KATP channel effects on preconditioning may imply the presence of a mediator which affects the KATP channel.Figure 1: Schematic drawing of ATP sensitive potassium channel (KATP) and the possible interrelationships affecting channel activity. ATP = adenosine triphosphate; ADP = adenosine diphosphate; NDP = nucleotide diphosphates; PKC = protein kinase C; AMP = adenosine monophosphate.This effect was recently demonstrated in humans as well. Tomai et al [24] assessed preconditioning by measuring ST-segment shifts and subjective cardiac pain in humans. Oral glibenclamide was given to half of the subjects 90 min prior to coronary angioplasty. After repeated balloon inflations, the researchers concluded that pretreatment with glibenclamide completely abolished the protective effect of preconditioning, suggesting that it is mainly mediated by KATP channels. A mediator, protein kinase C (PKC), has been demonstrated to activate KATP channels [25]. PKC may prolong or enhance KATP channel opening by phosphorylating a membrane protein involved in maintaining KATP channel opening. The role of PKC will be discussed in more detail later. Thus there is substantial evidence supporting the role of KATP channels as an end-effector in preconditioning. Free Radicals It has long been known that significant tissue injury can occur secondary to the reoxygenation-reperfusion process [26]. Oxygen-derived free radicals are believed to play a major role in the development of such injury by interacting with cellular components such as nucleic acids, lipids, and proteins [27]. The notion that preconditioning might result as an adaptive response to protect the tissue from damage during the reperfusion phase was popularized following a study by Murry et al.1 In their study, canines receiving either catalase or superoxide dismutase did not benefit from preconditioning when infarct size was used as a measure of efficacy. Additionally, Tajima et al [29] showed that in rats subjected to chronic hypoxia, increased myocardial resistance to ischemia was observed, and that acute ischemic preconditioning increased this tolerance even further. However, other researchers were unable to demonstrate an effect on preconditioning when free radical scavengers were administered to rabbits. Moreover, Walsh et al. [30] demonstrated that hypoxia preceding an ischemic challenge confers resistance to infarction despite the lack of an intervening period of reoxygenation. These observations lend credence to the notion that preconditioning may not result as an adaptive response to a free radical insult. 1 Murry CE, Richard VJ, Jennings RB, Reimer KA. Preconditioning with ischemia: is the protective effect mediated by free radical induced myocardial stunning? [abstract]. Circulation 1988;78:77. Bradykinin The role of bradykinin with respect to preconditioning is currently being investigated. Initial investigation into the antidysrhythmic properties of bradykinin was stimulated by research on angiotensin-converting enzyme (ACE) inhibitors. ACE catabolizes bradykinin to inactive metabolites, and several ACE inhibitors have demonstrated an antidysrhythmic effect [31]. In support of bradykinin's role in preconditioning, Wall et al. [32] subjected rabbits to either preconditioning, no preconditioning, preconditioning plus intravenous HOE 140 (a bradykinin receptor antagonist), HOE 140 alone, intraatrial bradykinin infusion, or intraatrial bradykinin infusion plus HOE 140. Preconditioning alone reduced infarct size, as did preconditioning plus bradykinin infusion. However, pretreatment with HOE 140, as well as bradykinin infusion plus HOE 140, negated the cardioprotective effect. While these results support the idea that exogenously administered bradykinin may be cardioprotective, research must be done to satisfactorily demonstrate that this substance is actually released during ischemic preconditioning before it can be accepted as playing a major role. Stress-Related Proteins In recent years, heat shock and other stress-related proteins have gained notoriety with respect to preconditioning. Stress proteins are induced in the heart by various physiological and pathological conditions. Prior studies have demonstrated that brief episodes of ischemia can induce the production of HSP 70, a heat shock protein that may play a role in preconditioning.2 HSP 70 limits cardiac susceptibility to ischemia in rabbits [34]. In support of this theory, a recent experiment revealed that repeated ischemia and reperfusion facilitated the induction of several stress-related and antioxidant genes including HSP 70, 27, and 89, catalase, and Mn-SOD [35]. However, other studies have contradicted such findings with respect to heat shock proteins. In one such study, Kucukoglu et al. [36] demonstrated that HSP 70 is in fact not cardioprotective, and that any protection afforded was actually inversely proportional to concentrations of HSP 70. 2 Knowlton AA, Brecher P, Ngoy S, Apstein CS. Brief cardiac ischemia induces expression of heat shock protein 70 [abstract]. Circulation 1989;80:237. The main argument against the involvement of stress proteins in preconditioning is the inability to correlate the proteins with the timing of ischemic preconditioning. The protein message would have to be produced within a few minutes after reperfusion and be terminated after 90-120 min of reperfusion. The known stress proteins have been observed to continue increasing in quantities after this period [37]. Although stress proteins may ultimately lead to an endogenous route to myocardial protection, they do not appear to play a role in ischemic preconditioning. Myocardial Stunning The period(s) of ischemia and reperfusion that stimulate ischemic preconditioning also produce reversible deficits in myocardial contractility. These deficits are termed "myocardial stunning," and several researchers argue for stunning as the mediator of the cardioprotective effects observed with preconditioning. However, researchers have demonstrated that the temporal effects of stunning do not coincide with those seen with ischemic preconditioning [38]. Additionally, Matsuda et al. [39] recently demonstrated that reduced contractile effort is not required to produce a cardioprotective effect via preconditioning, and Miura et al. [40] showed that the infarct size-limiting effect of preconditioning did not correlate with the degree of myocardial stunning induced in their experiment. Thus, it appears that myocardial stunning is not causally responsible for the beneficial effects of preconditioning. PKC Support has recently been mounting with respect to PKC and its potential role in preconditioning. Researchers believe that endogenous adenosine and norepinephrine act on G-protein linked receptors, ultimately activating PKC. It is theorized that once PKC is activated, it phosphorylates an effector protein which in turn confers cardioprotection [41]. Speechly-Dick et al. [42] investigated the role of PKC in preconditioning the rat heart. They found that nonpreconditioned hearts treated with 1,2-dioctanoyl-sn-glycerol, a PKC agonist, had infarcts correlating with those seen in ischemic preconditioned hearts (not treated with 1,2-dioctanoyl-sn-glycerol) after a prolonged ischemic challenge. Additionally, this observed protective effect was abolished when chelerythrine, a potent selective PKC antagonist, was administered prior to preconditioning the rat hearts. Similarly, Liu et al. [43] showed that translocation of cytosolic PKC may be a key event during preconditioning of the rabbit myocardium. These initial studies indicate that PKC may play an integral role in ischemic preconditioning with respect to infarct size. However, additional studies in other species are needed prior to generalization of these findings. Norepinephrine The concept that norepinephrine (NE) is responsible for the cardioprotective effects seen with preconditioning has recently become popularized. One theory is that NE may act to stimulate adenosine production during ischemic preconditioning, thereby exerting an infarct-limiting effect. Toombs et al. [44] recently tested the hypothesis that NE release is quintessential to the preconditioning process. Rabbits were pretreated with reserpine, and depletion of their NE stores was confirmed via a tyramine challenge. Nonreserpinized rabbits served as controls, and they underwent 5 min of coronary occlusion followed by 10 min of reperfusion prior to a 30-min ischemic challenge. Half of the reserpinized rabbits underwent preconditioning prior to the prolonged ischemic challenge, while the other half did not. The control group had a significantly reduced average size of infarction when compared to that of the reserpinized rabbits that had undergone preconditioning as well as to those which had not. In light of their findings, Toombs et al. concluded that NE must be released during the preconditioning phase or during the phase of prolonged ischemia for a cardioprotective effect to be observed. While these findings are of interest, they do not explain the mechanism by which NE may act to confer cardioprotection. Additional studies are needed to shed light on this matter. Adenosine Adenosine has received by far the most interest as a potential mediator of the cardioprotective effect observed with preconditioning. Its role with respect to preconditioning was initially suggested by Ely et al. [45] in 1985, and since then many other studies have been published. Adenosine, an endogenous nucleoside synthesized primarily by the degradation of ATP, is released from the heart in a continuous fashion via myocardial metabolism. It exerts its effects primarily via extracellular membrane purinergic Type I receptors which are subclassified as A1 or A2. The A1 receptors are located within the myocardium, and the A (2) receptors are found predominantly within the coronary vasculature. Once activated, both receptor types stimulate regulatory G proteins which in turn act to stimulate (A2 receptor) or inhibit (A1 receptor) adenylate cyclase, open potassium channels (A1 receptor), and possibly increase guanalate cyclase and phospholipase C activity (A1 receptor) [46]. Although both receptor types have demonstrated an antidysrhythmic effect [47], the A1 receptor appears to be the more important receptor type with respect to preconditioning. Downey et al. [48] found that pretreatment with the selective adenosine receptor antagonists SPT and PD 115,199 inhibits the protective effect of preconditioning. These same researchers demonstrated that an intracoronary infusion of adenosine in the rabbit confers the same degree of antiinfarct protection as ischemic preconditioning, arguing for the hypothesis that preconditioning is initiated by adenosine receptors. In a recent study by Yao et al. [49], the infarctlimiting effects of adenosine were examined in anesthetized dogs. They found that pretreatment of unconditioned hearts with adenosine before 60 min of left anterior descending coronary artery occlusion produced an infarct-limiting effect similar to that seen with preconditioned hearts. However, the adenosine-induced cardioprotection waned within 60 min, while the protection conferred by ischemic preconditioning persisted for at least 60 min. Additionally, treatment with the potassium channel antagonists glibenclamide or 5-hydroxydecanoate blocked the protective effects of adenosine. Yao et al. concluded that adenosine may act via KATP channels to exert its cardioprotective effect, and that the difference in time course between the cardioprotection of exogenous adenosine versus preconditioning indicates that other may be to the infarct-limiting effects of ischemic preconditioning. While support for adenosine as the mediator of preconditioning is it is this theory can explain all aspects of the preconditioning phenomenon. For instance, and have demonstrated that adenosine is not responsible for preconditioning in They found that of an adenosine antagonist, produced no cardioprotective effect. Similarly, pretreatment with intravenous adenosine did not produce a protective effect, that adenosine no role on preconditioning the rat myocardium. Thus, while adenosine may in ischemic preconditioning, its role appears to be species Preconditioning may very well be a using several of the mechanisms Figure 2 and of that may explain both the and infarct-limiting effects of ischemic preconditioning. include calcium as well as a of metabolic by protein These are by of a of receptors, including and receptors, receptors, and the A1 and receptors. These receptors control the of and nucleotide regulatory proteins Ischemic preconditioning may ischemic by on these interacting and regulatory of adenosine A receptors in protein kinase C and modulation of KATP channel activity. = cyclic adenosine ATP = adenosine 1: Mechanisms to Myocardial and Ischemic is evidence to that the myocardium can be In et al. reported evidence to support this to coronary artery were into either a control or experimental The experimental group was subjected to preconditioning via periods of with a period of reperfusion. they were subjected to a 10-min period of with electrical The control group was subjected solely to the 10-min period of Myocardial tissue were at intervals during the and ATP was the of the ATP was similar between the and as it was decreased in the experimental group after the brief periods of However, after the 10-min ischemic challenge, ATP was in the experimental suggesting that ATP conservation had occurred secondary to preconditioning. et al. a similar study in coronary and their findings also that it is to the myocardium. et al. investigated the of several after acute myocardial infarction not by Researchers have long that is a clinical of induced preconditioning. They found that with acute myocardial not by including cardiac and than did had These findings that intermittent prior to a myocardial infarction may cardioprotection. of preconditioning could possibly lead to In to coronary artery and coronary both and as well as during cardiac may provide in which the myocardium is a of the mechanisms it may be possible to pharmacological and at increasing the against sustained ischemia. adenosine and the A1 receptor appear to the most with respect to the development of a pharmacological substance of the mechanisms against ischemic injury. Although adenosine if administered in the quantities to confer the development of more selective A1 may not be with such The of such a could have important clinical for the currently used during as well as for treatment of acute myocardial Although the past has seen in of ischemic preconditioning, it is that more research is A more of this phenomenon could result in a significant in the and morbidity with coronary artery disease and the of such