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Cardiovascular Research 1992;26:734-739
Short review Ischaemic preconditioning: from mechanisms to exploitation David M Walker and Derek M Yellon
The discovery of ischaemic preconditioning In recent years significant advances have been made in our knowledge of the mechanisms underlying myocardial cell injury and death during ischaemia. It is now well known that the extent of any myocardial infarction depends on the duration and severity of the ischaemic insult, the presence and degree of any collateral circulation, the rate of oxygen consumption at the time of coronary occlusion, and the area of myocardium at risk.’* However, the exact biochemical mechanism of cell death remains unclear. At present most theories relate to either the loss of high energy metabolites such as ATP and pho~phocreatine~or the accumulation of catabolites such as lactate, H’ ions, and inorganic pho~phate.~ To clarify the situation, Reimer et a15 attempted to separate the effects of catabolite accumulation from those of high energy phosphate depletion by experiments in dogs in which repeated brief periods of ischaemia (interspersed with reperfusion to allow catabolite washout) were compared with a single ischaemic period of comparable length. Rather than a progressive fall in high energy phosphates with each ischaemic episode, they observed that intermittent reperfusion appeared to maintain ATP at a level similar to that seen after only one occlu~ion.~ In addition they also noted that no infarction occurred in six of the seven dogs evaluated. T i h was contrary to the previously accepted view that repetitive ischaemia could cumulatively cause infarction.‘ Reimer and colleagues then found that alterations in the heart induced by such brief periods of ischaemia and reperfusion were able to protect through a longer, sustained ischaemic insult, reducing infarct size to 25% of that seen in
a control group.7 Induction of ischaemic tolerance in this way has been called “ischaemic preconditioning”. General features of ischaemic preconditioning Since 1986, many research groups have found that ischaemic preconditioning can be induced by a variety of protocols in several different species. Initially in the dog, four five minute coronary occlusions were used.’ Subsequently a single 15 minute or one, six, or 12 five minute occlusive e isodes have been shown to be equally effective.’ In rabbits Po or rats,” I’ one five minute occlusion was sufficient whereas in pigs, two 10 minute occlusions were required.I3 In all these models. considerable limitation of infarct size has been achieved. Although ischaemic preconditioning seems to be an “all or nothing” response, with similar infarct size reduction occurring in different animal models, its decay as the intervening reperfusion is extended appears to be gradual. Murry et a1’ found that 120 minutes of reperfusion between the preconditioning and the sustained ischaemic insults resulted in considerable attenuation of the effect, with infarct size reduction falling from 92% (with only five minutes between the periods of ischaemia) to 54% in their canine model. In rabbits, however, the decay of ischaemic tolerance may be more rapid, with infarct size reduction falling from 84% to 45% when the intervening reperfusion is extended from 10 to 60 minutes and disappearing completely by 120 minutes.14 In pigs, protection disappears between 30 and 60 minutes of reperfusion, but no other time points have been assessed.” Differences in preconditioning protocols and/or animal species may therefore affect the time course for loss of protection. If instead of varying the length of the reperfusion period, the length of the final ischaemic insult is extended, the infarct size increases until it eventually matches that in controls. For example, no protection remains in dogs with an ischaemic insult of 90 minutes whereas at 60 minutes there is still marked protection.“ It therefore appears that the protection conferred by preconditioning is not absolute, delaying cell death but not preventing it. Various other consequences of ischaemidreperfusion have been reported to be attenuated by preconditioning. The incidence of reperfusion arrhythmias was initially studied by Shiki and Hearse in 1987.17 They used a five minute
Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, UCMSM, University College Hospital, Cower Street, London WC 1E 6 AU, United Kingdom. Correspondence to Dr Yellon.
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I
n recent years, there have been numerous attempts to design therapy aimed at limiting infarct size following acute myocardial infarction. Apart from the success of early restoration of blood flow to the ischaemic area by thrombolysis, results have largely been disappointing. As such, investigators have sought and are continually seeking new approaches to myocardial protection. The discovery of the phenomenon of ischaemic preconditioning has focused attention on the ability of the myocardium to protect itself and has raised the possibility of exploiting this endogenous form of protection by pharmacological means.
lschaernic preconditioning
’‘
Possible mechanisms of ischaemic preconditioning There have been many suggestions as to the possible mechanism of ischaemic preconditioning. We discuss the more popular theories below. Reduced energy demand ATP is depleted at a slower rate in preconditioned
myocardium, such that although some ATP is lost during preconditioning itself, levels are higher after 10 minutes of the sustained ischaemia.” In addition, glycogenolysis and anaerobic glycolysis both occur at a reduced rate and therefore Murry rt af have formed the hypothesis that there is a reduction in energy demand in preconditioned ischaemic myocardium.” As discussed earlier, improved cell viability could then be explained either by preservation of ATP stores or by reduced catabolite accumulation, or both. If a reduction in energy demand is an essential component of the protection afforded by preconditioning, this implies that either the metabolic work required to sustain viability has been reduced or that ATP utilisation has been redirected from nonessential to more critical pathways, eg, from ineffectual contractile activity to the maintenance of ionic gradients. As brief ischaemia causes some mechanical dysfunction, it has been suggested that the presence of this decreased contraction may be responsible for the reduced energy demand seen when the heart enters a second prolonged ischaemic episode. Attempts have therefore been made to link preconditioning with the presence of postischaemic mechanical dysfunction or “stunning”. So far these have been unrewarding. The time course of stunning is much more prolonged,’ stunned myocardium has an equal or increased oxygen consumption when compared with normal myocardium,” the degree of stunning does not correlate with the limitation in infarct size,” and the decrease in energy demand seen in preconditioned myocardium compared with control is not abolished in vitro by cardioplegic arrest.30 In addition, in swine myocardial preconditioning appears to be possible in the absence of stunning3’ Mitochondria1 ATPase inhibitor protein An alternative explanation for the reduction in energy demand seen in preconditioning involves the action of the mitochondrial ATPase inhibitor protein. Normally during ischaemia, the mitochondrial ATPase functions in reverse, potentially wasting ATP. In animals with slow heart rates (including rabbit, dog, pig, sheep, and human), there is a reversible inhibitor of this ATPase which binds during ischaemia, triggered by cytosolic acido~is.~’ It has been suggested, therefore, that preconditioning might in part be mediated by this inhibitor protein, either by binding more rapidly during a second ischaemic episode or by persistent binding during reperfusion.33 However, since its activity in the rat is minimal and yet preconditioning is still possible,’’ I‘ this hypothesis seems unlikely. Free radicals The role of free radicals in myocardial ischaemia and reperfusion is still unclear,’4 although these may be important in the pathogenesis of myocardial ~tunning.~’ With respect to their role in ischaemic preconditioning, preliminary studies have shown that free radical scavengers given to dogs prior to preconditioning cause attenuation of the response in 50% of cases.3h This suggests that free radicals might be beneficially involved in some way. However, Iwamoto et a13’ were unable to repeat this in rabbits, and Osada et allyfound that reperfusion arrhythmias were reduced by free radical scavengers in the isolated rat heart with no additional protective effect of preconditioning. Further work is required in this area to clarify these conflicting findings. Stress proteins Another area of interest with regard to the mechanism of preconditioning and “endogenous” myocardial protection in
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ischaemic insult to precondition rats and found that reperfusion arrhythmias following a second five minute coronary occlusion were markedly reduced. However arrhythmias, some requiring defibrillation, occurred frequently after the preconditioning ischaemia which led the investigators to suggest that the animals might simply be having their quota of rhythm disturbances earlier. Hagar et a1” resolved this issue by using three shorter coronary occlusions as their preconditioning protocol, which produced few arrhythmias by themselves and yet significantly reduced subsequent reperfusion arrhythmias. Similar protective effects have been noted in the isolated rat heart preconditioned by global ischaemia.” In the dog, preconditioning has been noted to reduce arrhythmias during the prolonged ischaemic phase as well as on reperfusion.*” Another group has looked at changes in wall motion resulting from prolonged ischaemia and has shown that in rabbits, preconditioning, allows much greater recovery of segmental shortening. Although it is likely that the mechanism underlying these observations is the same as that for infarct size reduction, at present some caution is required in extrapolating results between models. In particular, the incidence of reperfusion arrhythmias is known to be closely related to the length of the ischaemic insult, with a narrow time window for maximum arrhythmias.” It is possible that ischaemic preconditioning, by delaying cell necrosis, might delay this time window, such that more reperfusion arrhythmias would occur experimentally if a longer ischaemic insult was used. It is possible to induce ischaemic preconditioning by techniques other than coronary artery occlusion. In vitro experiments with the isolated perfused rat heart suggest that global preconditioning may result from a short period of zero flow followed by reperfusion - with either reperfusion arrhythmias” or recovery of function’2 being the end points used. However, this model has not been investigated using a subsequent regional ischaemic insult with infarct size as the end point. Also it is interesting to speculate that cardiac surgeons may have been preconditioning the heart globally for years with intermittent cross clamp fibrillation during bypass surgery. Another recent study has shown that rapid pacing can induce a preconditioned response in anaesthetised open chest dogs,23presumably via global ischaemia. This study looked at arrhythmias during ischaemia and reperfusion, and at ST elevation during ischaemia, to compare the paced and control groups. However, a similar study in rabbits with rapid atrial pacing failed to show a reduction in infarct size in spite of ECG and monophasic action potential evidence of ischaemia during the pacing.24This suggests that the severity of the brief ischaemic insult is crucial for preconditioning to occur. Recently it has been shown that it is possible to “precondition” myocardium with regional or global hypoxia, rather than i~chaemia.‘~ Although the mechanism is not necessarily the same, it does suggest that a phase of metabolite accumulation prior to washout by reperfusion is not essential for preconditioning.
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that the time to the onset of ischaemic contracture during global normothermic ischaemia is extended by pretreatment with adenosine or the A1 agonist, R-phenyl-isopropyladenosine (R-PIA). It appears likely therefore that adenosine released locally from ischaemic myocytes plays a role in attenuating ischaemic damage. This evidence has been linked to ischaemic preconditioning by Liu et a1.” They have shown that ischaemic preconditioning can be blocked by pretreatment with A1 adenosine receptor antagonists in an in vivo rabbit model and also that adenosine or R-PIA infused down the coronary artery of an isolated perfused rabbit heart causes infarct size limitation comparable to that achieved by preconditioning. Rabbit experiments with intravenous R-PIA infusion in vivo have demonstrated some limitation of infarct size, although this is much less marked when the rabbits are paced to prevent R-PIA induced brady~ardia.~”’~ A 1 receptors are coupled to Gi proteins and blockade of these proteins with pertussis toxin has also been shown to attenuate the protection afforded by pre~onditioning.~~ 6o If preconditioning is mediated by adenosine which accumulates during ischaemia and acts on A 1 receptors, then brief A1 stimulation must trigger biochemical changes in the heart that confer lasting protection against a subsequent ischaemic insult. It is not known at this stage what these changes might be, although the two most likely possibilities are stimulation of glycolysis and the opening of ATP regulated K’ channels.
Prostacyclin Other groups have proposed that preconditioning is dependent on the release of one or more endo enous protective substances. For example, Vegh et al-TF have implicated prostanoids. They found that sodium meclofenamate, an inhibitor of cyclo-oxygenase, attenuated the protective effect of preconditioning on arrhythmias in dogs. They suggest that prostacyclin release may be important as it is known to be released from ischaemic myocardium and it reduces arrhythmias during ischaemia and reperfusion when infused locally.” However, Li and Kloner” were unable to show any reduction of the effects of preconditioning on infarct size or arrhythmias by inhibiting cyclo-oxygenase with aspirin in the rat. Further work is required in this area.
Glycolytic flux A brief period of ischaemia increases glycolytic flux,” as does hypoxic perfusion or the infusion of adenosine.” Evidence supporting a role for glycolysis in maintaining cell viability during ischaemia was reviewed by Opie.62 Glycolytically derived ATP may be of particular importance in the preservation of cell membrane function and cell integrity while ATP from oxidative phosphorylation preferential12 supports contractile function.63 In addition, Fralix et a1 have shown that inhibition of glycolysis (by perfusing an isolated heart with pyruvate rather than glucose) appears to inhibit preconditioning. However preconditioning reduces glycogen stores and, as discussed earlier, during the subsequent ischaemic insult glycolysis is decreased2’ and so any theory based on improved glycolytic efficiency must explain this discrepancy. It is possible that enhanced glycolysis prior to the onset of the longer ischaemic insult leads to the accumulation of a compartmentalised store of ATP which subsequently protects the myocytes, even though total ATP at this stage is known to be decreased. This theory is, however, only conjecture.
Nitric oxide Alternatively, the release of nitric oxide during preconditioning may lead to a subsequent beneficial effect. Evidence for this comes from the attenuation of preconditioning seen with L-NAME (nitro L-arginine methyl ester), an inhibitor of nitric oxide synthesis, in an arrhythmia model.” However in contrast, Pate1 et al” failed to inhibit ischaemic preconditioning with a similar dose of L-NAME in the rabbit heart using infarct size as the endpoint. At present no firm conclusion can be drawn about the role of nitric oxide. Adenosine Perhaps the most likely candidate as an endogenous mediator of preconditioning is adenosine. It is released from many cells under stress (including ischaemic myocytes) and is thought to act as a local regulator modulating the function of the cell via a “feedback” mechanism.53 Studies in isolated, perfused rat hearts have shown that the increased glycolytic flux which occurs with hypoxia can be inhibited by 8-sulphophenyl-theophylline, an A 1 adenosine receptor antagonist. 54 Also in this model, the same group has shown
ATP regulated k? channels More is known about the role of ATP regulated (or sensitive) K’ channels (KATP).Noma‘’ found that the previously undefined outward current which increases significantly when cardiac cells are subjected to hypoxia, is due to a K’ channel the conductance of which increases at lower intracellular ATP concentrations. He also postulated that this channel is responsible for the shortening of the action potential which is seen during ischaemia and that this could reduce ATP depletion by reducing Ca2+influx and decreasing myocardial contractility. Since then, evidence has been accumulating to suggest that activation of KATPchannels plays an important protective role during ischaemia. For example, KATPchannel agonists such as cromakalim and pinacidil have been shown to improve postischaemic recovery of function in the isolated rat h e a d 6 and the
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general is stress (or heat stress) protein synthesis.38 This family of proteins, synthesised in response to a variety of stimuli, was originally identified in cells exposed to raised temperature^.'^ Recent studies have detected the 70 kDa stress protein in heart tissue of a variety of species, including the dog, rat, and rabbit, and increased expression can be induced by ischaemia, hypoxia, or pressure overload.3x Myocardial protection from heat stress has been shown in functional and biochemical terms in isolated hearts of rat“’ 4 1 and rabbit,4’ and also with infarct size reduction in the rabbit isolated heart Limitation of infarct size by prior heat stress has also been achieved in vivo in a rat model of infarctionu; however in vivo experiments in dogs4’ and rabbits46 have shown no protective effect. Evidence against the involvement of stress proteins in preconditioning comes from Thornton et a14’ who showed that protein synthesis inhibitors (actinomycin D and cycloheximide) did not attenuate preconditioning in rabbits, although specific stress protein levels were not determined. The 70 kDa stress protein has been measured in a similar study4’ and although its synthesis did not appear to be inhibited by these drugs, no rise was discernible in the protein until two hours after ischaemia. It therefore seems unlikely to be involved in preconditioning. This does not exclude the possibility of involvement of one of the other stress proteins and indeed recent studies have examined the role of the 65 kDa stress protein.49
Ischaemic preconditioning
’’
Evidence for ischaemic preconditioning in humans Considerable care must be taken in extrapolating results from animal models to humans. However. in view of the number of mammalian species in which ischaemic preconditioning occurs and the remarkable infarct size reduction achieved, it is likely to have significant clinical implications. Patients with unstable angina in the hours preceding a myocardial infarction might have a longer time window for reperfusion with thrombolysts, allowing greater limitation of infarct size. It is interesting that patients with angina prior to myocardial infarction have a less complicated in-hospital course than other groups, even though they have a higher risk profile for coronary artery disease (including hypertension, hyperlipidaemia, and diabetes).” Furthermore, it might be expected that during percutaneous transluminal coronary angioplasty (PTCA) the first balloon inflation would
x
protect the m ocardium against subsequent inflations. Deutsch et a f R have compared the first two 90 second balloon inflations in 12 patients undergoing elective PTCA and have found that subjective anginal discomfort. ST segmental changes, myocardial lactate production and coronary vein flow were all attenuated during the second in fl at ion. Conclusion In conclusion, ischaemic preconditioning is unique in the degree of resistance to ischaemic damage that it provides. As our knowledge increases. it may be possible to take advantage of the mechanisms underlying this endogenous form of myocardial protection such that in the future. patients may benefit from our ability to exploit the heart’s own adaptive responses to myocardial ischaemia. Received 5 February 1992. accepted I6 March I992 We are grateful to the Hatter Foundation for their continued support
Key terms. protection
ischaemic preconditioning.
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reperfusion.
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I Maxwell MP, Hearse 111. Yellon I>M Species vanation in the coronary collateral circulation dunng regional myocardial ischaemia a cntical determinant of the rate of evolution a d extent of myocardial infarction Cardiovosc Res 1987.21: 137-46 2 Schaper J. Schaper W Time course of myocardidl necrosis C~rdiovoscDrugs l h e r 1988.2: 17-25 3 Jennings RB. Hawkins HK, Lowe Jk. Hill ML. Klotman S. Reimer KA Relation between high energy phosphate and lethal injury i n myocardial ischemia in the dog Am I furhol 1978. 92:203 16 4 Seely JR, Grotyohann LV Role of glycolytic products in damage to the ischemic myocardium dissociation of adenosine InDhosDhate levels and recoverv of rewrfused ischemic hearts CA R;S 1984.ss:ni6- 24. 5 Reimer KA. .Mum/ CE. Yamasawa I, Hill MI.. Jenninrs RB Four bnef penods of Oischemid cause no cumularive AYP loss or necrosis Am I fh,siol 1986.251(11e~rr Circ f h v w d LO): f11306 I5 6 Geft I L . Fishhein MC. Sinomiya K. er a1 Intermittent hnef pcnodg of ischemia have a cumulative effect and may cause myocardial necrosis Crrculuorion 1982.66: I I50 3 7 Murry CI:. Jennings RB. Reimer KA Preconditioning with ischemia a delay of lethal cell injury in ischemic myocardium Ctrculurton 1986.74: I I24 36 8 M u q CE. Richard VJ. Jennings RB. Reimer KA Myocardial protection is lost hefore contractile function recovers from ischemic preconditioning Am J Phystol 1991.MO(Heorl Circ Ph vstol29): H796-804 9 1.1 K, Vasquer BS. Gallagher KP. Lucchesi BR Myocardial protection with preconditioning Ctnulorion 1990.82 09-19 10 Cohen .MV. 1.iu GS. Downcy JM Preconditioning c a u w improved wall motion as well ds smaller infarcts after transient coronary occlusion i n rabbits Circulo/ton 1W I .84:34 I 9 I I 1.1 Y. Kloner R A Cardioprotective effects of ischaermc “preconditioning” .ire not mediated by prostanoids Curdtovusc Res 1992.26:226-31 12 Alkhulaifi AM. Browne E, Yellon DM Iwhaemic preconditioning Iimiis infdrct sire i n the 1.11 heart (dhsuact) I Mol Cell Curdid 1992;24(suppl I):S93. 13 Schon RJ. Rohmann S. Braun ER. Schawr W Ischemic preconditioning rcducc\ infarct size i n swine myocardium Qrt Res 1990.66: 1 I33 42 14 Van Winkle I)M, Thornton J. Ilowney J M Cardioprotection from ischemic preconditioning is lost following prolonged reperfusion i n rabbits (abstract) Circularion 199 1.84(suppl I1):432 15 Schwarr ER. .Mohn M. Sack S. Arrds M Durdiion of infarct size limiting effect of irchermc preconditioning i n the pig (dhsrract) Circulation 199 I .84(suppl I1):432 16 Sao BS. McClanahan TB. Groh M A . Schott RJ. Gallagher KP The time limit of effective ixhemic preconditioning i n dogs (dhsuact) Ctnu/u/ton 1990.82(suppl 111):27 I
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arterially perfused guinea pig right ventricular w&’ at dosages which did not effect preischaemic contractility. Also in the isolated rat heart, a KATPagonist (nicorandil) causes a more rapid decrease in contractility during early ischaemia with delayed onset of contracture. whereas an antagonist (glibenclamide) delays the fall in developed pressure but contracture occurs earlier,” Grover and colleagues6’ have also been able to limit infarct size in open chest dogs by giving intracoronary cromakalim or pinacidil prior to ischaemia. There is still some debate as to whether KATPchannels are activated during the first few minutes of ischaemia, for experimentally they are inhibited at A’rP levels much lower than the intracellular ATP concentration seen at that stage.’” It ha$ k e n calculated that only minor increases in channel conductance ( < I % ) could have major effects on action potential d ~ r a t i o n . 7’~ !However in spite of this. quantitative calculations suggest that the fall in A’TP in early ischaemia is not sufficient to explain the observed action potential shortening.” Clearly for the KATPchannel to be responsible for preconditioning, channel activity would have to be increased by a five minute ischaemic episode or alternatively the channel would have to be modified in such a way that activity could increase more rapidly at the beginning of the sustained ischaernia. One possible explanation is that KATP channels, although belonging to the “ligand gated’ group of K’ channels, are modulated by adenosine via interaction with a G , protein.” If adenosine released during ischaemia decreases the channel sensitivity to ATP. this may therefore be a route by which channel activity could be increased during brief ischaemia and could explain the data supporting the adenosine hypothesis of preconditioning. The relevance of KATPchannels to preconditioning has been further tested using the antagonists sodium 5-hydroxydecanoate and glibenclamide, in an attempt to block preconditioning. This has been successful in dogs,” ’’ but not in rabbits,76 although in the latter study. glibenclamide did have a deleterious effect on infarct size. Further conflicting evidence comes from arrhythmia studies. KATPchannel agonists appear to be proarrhythmic during regional ischaemia in a conscious canine model of sudden death” and also during low flow global ischaernia in the isolated rat heart.” Reperfusion arrhythmias are, however, reduced by pinacidil in a global ischaemia modeLh7 By comparison. ischaemic preconditioning is antiarrhythmic during ischaemia and on reperfusion in the open chest dog,*’ as well as on reperfusion following global ischaemia.:’ N o firm conclusions can be drawn at present.
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buffer uerfused rabbit hearts (abstract). J Mol Cell Cardiol 1992;2&uppl I):S118. Donnellv TJ, Sievers RE, Vissern FW. Welch WJ. Wolfe CL. Heat shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion? Circulation 1992;85:769-78. Schott RJ, Nao B, Strieter R, et al. Heat shock does not precondition canine myocardium (abstract). Circulation 1990; 82(suppl III):464. Yellon DM, Pasini E, Cargnoni A, Marber MS, Latchman DS, Ferrari R. The protective role of heat stress in the ischaemic and reperfused rabbit myocardium. J Mol Cell Cardiol 1992 (in press). Thornton J, Striplin S, Liu GS, et a/. Inhibition of protein synthesis does not block myocardial protection afforded by Preconditioning. Am J Physiol 1990;259(Heart Circ Physiol 28): HI 822-5. Iliodromitis E, Kucukoglu S, Marber MS, Walker JM, Yellon DM. Preconditioning protects rabbit heart independent of stress protein synthesis (abstract). Eur Heart J 1991;12S:P720. Heads RJ, Patel VC, Latchman DS, Yellon DM. Ischaemic preconditioning causes rapid elevation of 65kDa stress protein in rabbit heart (abstract). J Mol Cell Cardiol 1992;24(suppl I):S94. Parratt JR, Coker SJ, Wainwright CL. Eicosanoids and susceptability to ventricular arrhythmias during myocardial ischaemia and reperfusion. J Mot Cell Cardiol 1987;19:55-6. Vegh A, Szekeres L, Parratt JR. Does nitric oxide play ;L role in ischaemic Dreconditioning? - .(abstract) J Mol Cell Cardiol 199 1: SUPPI PI vj:s72. Patel V, Woolfson RG. Sineh KJ. Neild GH. Yellon DM. Ischaemic preconditioning is- not prevented by inhibition of endothelium-derived nitric oxide. J Mol Cell Cardiol 1992;24(suppl 1):s 152. Stiles GL, Adenosine receptors: physiological regulation and biochemical mechanisms. News Physiol Sci 1991;6:1614. Wyatt DA, Edmunds MC, Rubio R, Berne RM, Lasley RD, Mentzer RM. Adenosine stimulates glycolytic flux in isolated perfused rat hearts by A I-adenosine receptors. Am J Physiol 1989;257(Henrt Circ Physiol 26):H 1952-7. Liu GS, Thornton 1, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A l adenosine receptors in rabbit heart. Circulation 1991;84:350-6. Thornton J, Liu GS, Olsson RA, Downey JM. Intravenous pretreatment with Al-selective adenosine analogues protects the heart against infarction. Circularion 1992;85:659-65. Van Winkle DM, Davis RE Ischemic preconditioning of myocardium: Effect of the adenosine agonist phenylisopropyl adenosine (PIA) (abstract). Circulation 1991;84(suppl II):305. Tsuchida A, Miura T, Iimura 0. Role of adenosine receptor activation in infarct size limitation by preconditioning in the heart (abstract). Circularion 1991;84(suppl 11): 191. Thornton J, Downey JM. G , proteins are involved in preconditioning's protective effect (abstract). Circularion 199 I ; 84(suppl 11): 192. Lasley RD, Barakat 0, Van Wylen DGL, Mentzer RM. Pertussis toxin inhibits adenosine mediated preservation of post-ischaemic myocardial function (abstract). Circularion 1991;84(suppl 11): 307. Jennings RB, Muny CE, Reimer KA Myocardial effects of brief periods of ischemia followed by reperfusion. In: Kelleremann, JJ Braunwald E, eds. Silent myocardial ischaemia: a critical appraisal (Advances in Cardiology, vol 37). Basel: Karger, 1990:7-31. Opie LH. Hypothesis: glycolytic rates control cell viability in ischaemia. J Appl Cardiol 1988;3:407-14. Weiss J, Hiltbrand B. Functional compartmentation of glycolytic versus oxidative metabolism in isolated rabbit heart. J CIin Invest 1985;75:436-47. Fralix TA, Murphy E, Steenbergen C, London RE. Role of glycolysis in preconditioning (abstract). Circulation 1991;84(suppl 11): 192. Noma A. ATP-regulated K' channels in cardiac muscle. Nature 1983;305:147-8. Grover GJ, McCullough JR, Henry DE, Conder ML, Sleph PG. The anti-ischemic effects of the potassium channel activators pinacidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide. J Phannacol Exp Ther 1989;251:98-110. Cole WC. McPherson CD. Sontag D. ATP-regulated K' channels protect the myocardium against ischemidrgperfusion damage. Circ Res 1991;69:571-81. Mitani A. Kinoshita K. Fukamachi K, et al. Effects of glibenclamide and nicorandil on cardiac function during ischemia and reperfusion in isolated perfused rat hearts. Am J Physiol
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17 Shiki K, Hearse DJ. Preconditionine of ischemic myocardium: reperfusion induced arrhythmias. Am J Physiol 198f253(Heart Circ Physiol 22kH1470-6. 18 Hagar JM- Hale SL, Kloner RA. Effect of preconditioning ischemia on reperfusion arrhythmias after coronary artery occlusion and reperfusion in the rat. Circ Res 1991;68:61-8. 19 Osada M, Sato T, Komori S, Tamura K. Protective effect of preconditioning on reperfusion induced ventricular arrhythmias of isolated rat hearts. Cardiovasc Res 1991;25:4414. 20 Vegh A, Szekeres L, Parratt JR. Protective effects of preconditioning of the ischaemic myocardium involve cyclooxygenase products. Cardiovasc Res 1990;24: 102Cb3. 21 Manning AS, Hearse DJ. Reperfusion induced arrhythmias: mechanisms and prevention. J Mol Cell Cardiol 1984;16: 497-5 18. 22 Barakat 0, Van Wylen DGL, Mentzer RM, Lasley RD. Ischemic preconditioning improves postischemic recovery of function but shortens time to onset of ischemic contracture in isolated rat hearts (abstract). Circularion 1991;84(suppI II):433. 23 Vegh A. Szekeres L. Parratt JR. Transient ischaemia induced by rapid cardiac pacing results in myocardial preconditioning. Cardiovasc Res 1991;25:105 1-3. 24 Marber MS, Walker DM, Yellon DM, Walker JM. Rapid atrial Dacine fails to orecondition the rabbit heart (abstract). J Mol Cell 'CardGI 1992;i4(suppl I):S92. 25 Shizukuda Y, Mallet RT, Lee S-C, Downey HF. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc Res 1992:26:534-42. 26 Tani M, Asakura Y, Ebihara Y, Nakamura Y. Effect of preconditioning with anoxic perfusion on sarcoplasmic reticulum calcium uptak; in reperfusid rat hearts (absiract). Circulation I991 ;84(suppl II):433. 27 M u m CE. Richard VJ. Reimer KA. Jennings RB. Ischemic precdnditioning slows energy metabolis; and delays ultrastructural damage during a sustained ischemic episode. Cirr Res 1990;66:9 13-3 I . 28 Stahl LD, Weiss HR, Becker LC. Myocardial oxygen consumption, oxygen supply/demand heterogeneity and microvascular patency in regionally stunned myocardium. Circulation 1988;77:865-72. 29 Miura T, Goto M, Urabe K, Endoh A, Shimamoto K, Iimura 0. Does myocardial stunning contribute to infarct size limitation by ischemic preconditioning? Circulation 1991;84:2504-12. 30 Jennings RB, Muny CE, Reimer KA. Energy metabolism in preconditioned and control myocardium: effect of total ischaemia. J Mol Cell Cardiol 1991;23:1449-58. 31 Rohmann S. Schott RJ, Harting J, Schaper W. Ischemic preconditioning is not a function of stunned myocardium (abstract). J Mol Cell Cardiol 1991;23(suppl V):S71. 32 Rouslin W, Pullman ME. Protonic inhibition of the mitochondrial adenosine 5'-triphosphatase in ischemic cardiac muscle. Reversible binding of the ATPase inhibitor protein to the mitochondrial ATPase during ischemia. J Mol Cell Cardiol 1987;19:661-8. 33 Jennings RB, Reimer KA, Steenbergen C. Effect of inhibition of the mitochondrial ATPase on net myocardial ATP in total ischemia. J Mol Cell Cardiol 1991;23:1383-95. 34 Yellon DM, Downey JM. Current research views on myocardial reperfusion and reperfusion injury. Cardioscience 1990;1:89-98. 35 Bolli R. Mechanism of myocardial stunning. Circulation 1990;82:723-38. 36 Muny CE, Richard VJ, Jennings RB, Reimer KA. Preconditioning with ischaemia: is the protective effect mediated by free radical induced myocardial stunning? (abstract) Circulation 1988; 78(suppl II):77. 37 Iwamoto T, Miura T, Adachi T, et a / . Myocardial infarct sizelimiting effect of ischemic meconditioning was not attenuated bv oxygei free radical scaveigers in the &bit. Circulation 199f; 83: 1015-22. 38 Yellon DM, Latchman DS. Stress proteins and myocardial protection. J Mol Cell Cardiol 1992;24: 113-24. 39 Lindouist S. Craig EA. The heat shock oroteins Annu Rev Genet 1986:22:631-7. 40 Cume RW. Ross BM. Davis TA. Heat shock remonse is associated with enhanced post ischaemic ventricular iecovery. Circ Res 1988;63:543-9. 41 Pasini E, Cargoni A, Ferrari R, Marber MS, Latchman DS, Yellon DM. Heat stress and oxidative damage following ischaemia and rewrfusion in the isolated rat heart (abstract). Eur Heart J 19'91;12S:PI 568. 42 Yellon DM. Pasini E. Ferrari R. Downev JM. Latchman DS. Whole body heat stress protects the isolateh perfused rabbit heart (abstract). Circularion 1990;82(suppl III):463. 43 Walker DM. Kucukoglu S, Pasini E, Marber MS, Latchman DS, Yellon DM. The effect of heat stress on infarct size in blood versus
Ischaemic preconditioning
75 Gross CJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res 1992;70:223-33. 76 Thornton J, Downey JM. Blockade of ATP-sensitive K' channels does not prevent preconditioning in rabbit heart (abstract). Circulation 1991;84(suppl IV):432. 77 Chi L, Uprichard ACG, Lucchesi BR. Profibrillatory actions of pinacidil in a conscious canine model of sudden coronary death. J Cnrdiovusc Pharmacol 1990;15:45244. 78 Wolleben CD, Sanguinetti MC, Siegl PKS. Influence of ATPsensitive potassium chanel modulators on ischaemia induced fibrillation in isolated rat hearts. J Mol Cell Cardiol 1989; 2 1~783-8. 79 Muller DWM, Topol EJ, Califf RM, e f a/ and the T.A.M.I. Study Group. Relationship between antecedent angina pectoris and short-term prognosis after thrombolytic therapy for acute myocardial infarction. Am Heart J I990;119:224-3 1. 80 Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW, Hemnann HC, Laskey WK. Adaptation to ischemia during percutaneous transluminal coronary angioplasty: clinical hemodynamic and metabolic features. Circuhtion 1990;82:2044-5 I .
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1991;261(Heart Circ Phvsiol 30):H 1864-7 I . 69 Grover GI, Dwonczyk S, Parham CS, Sleph PG. The protective effects of cromakalim and pinacidil on reperfusion function and infarct size in isolated rat hearts and anesthetized dogs. Cardiovasc Drugs Ther I990;15:465-74. 70 Nichols CG, Lederer WJ. Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am J Physiol 1991;261(Heart Circ Physiol 30):H1675-86. 71 Nichols CG, Lederer WJ. The regulation of ATP-sensitive K' channel activity in intact and permeabilized rat ventricular myocytes. J Physiol (Lund) 1990;423:91-110. 72 Nichols, CG, Ripoll C, Lederer WJ. KATPchannel modulation of the guinea-pig ventricular action potential and contraction. Circ Res 199I ;68:280-7. 73 Kirsch GE, Codina J, Birnbaumer L, Brown AM. Coupling of ATP-sensitive K' channels to A1 receptors by G proteins in rat ventricular myocytes Am J Phyiol 1990;259(Heart Circ Physiol 28):H820-6. 74 Auchampach JA, Grover GJ, Gross GJ. The ischemia selective ATP-sensitive K' channel antagonist, sodium 5-hydroxydecanoate, blocks myocardial preconditioning in dogs (abstract). Circulation I99 I ;84(suppl II):432.
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Cardiovascular Research 1992;26:734-739
Short review Ischaemic preconditioning: from mechanisms to exploitation David M Walker and Derek M Yellon
The discovery of ischaemic preconditioning In recent years significant advances have been made in our knowledge of the mechanisms underlying myocardial cell injury and death during ischaemia. It is now well known that the extent of any myocardial infarction depends on the duration and severity of the ischaemic insult, the presence and degree of any collateral circulation, the rate of oxygen consumption at the time of coronary occlusion, and the area of myocardium at risk.’* However, the exact biochemical mechanism of cell death remains unclear. At present most theories relate to either the loss of high energy metabolites such as ATP and pho~phocreatine~or the accumulation of catabolites such as lactate, H’ ions, and inorganic pho~phate.~ To clarify the situation, Reimer et a15 attempted to separate the effects of catabolite accumulation from those of high energy phosphate depletion by experiments in dogs in which repeated brief periods of ischaemia (interspersed with reperfusion to allow catabolite washout) were compared with a single ischaemic period of comparable length. Rather than a progressive fall in high energy phosphates with each ischaemic episode, they observed that intermittent reperfusion appeared to maintain ATP at a level similar to that seen after only one occlu~ion.~ In addition they also noted that no infarction occurred in six of the seven dogs evaluated. T i h was contrary to the previously accepted view that repetitive ischaemia could cumulatively cause infarction.‘ Reimer and colleagues then found that alterations in the heart induced by such brief periods of ischaemia and reperfusion were able to protect through a longer, sustained ischaemic insult, reducing infarct size to 25% of that seen in
a control group.7 Induction of ischaemic tolerance in this way has been called “ischaemic preconditioning”. General features of ischaemic preconditioning Since 1986, many research groups have found that ischaemic preconditioning can be induced by a variety of protocols in several different species. Initially in the dog, four five minute coronary occlusions were used.’ Subsequently a single 15 minute or one, six, or 12 five minute occlusive e isodes have been shown to be equally effective.’ In rabbits Po or rats,” I’ one five minute occlusion was sufficient whereas in pigs, two 10 minute occlusions were required.I3 In all these models. considerable limitation of infarct size has been achieved. Although ischaemic preconditioning seems to be an “all or nothing” response, with similar infarct size reduction occurring in different animal models, its decay as the intervening reperfusion is extended appears to be gradual. Murry et a1’ found that 120 minutes of reperfusion between the preconditioning and the sustained ischaemic insults resulted in considerable attenuation of the effect, with infarct size reduction falling from 92% (with only five minutes between the periods of ischaemia) to 54% in their canine model. In rabbits, however, the decay of ischaemic tolerance may be more rapid, with infarct size reduction falling from 84% to 45% when the intervening reperfusion is extended from 10 to 60 minutes and disappearing completely by 120 minutes.14 In pigs, protection disappears between 30 and 60 minutes of reperfusion, but no other time points have been assessed.” Differences in preconditioning protocols and/or animal species may therefore affect the time course for loss of protection. If instead of varying the length of the reperfusion period, the length of the final ischaemic insult is extended, the infarct size increases until it eventually matches that in controls. For example, no protection remains in dogs with an ischaemic insult of 90 minutes whereas at 60 minutes there is still marked protection.“ It therefore appears that the protection conferred by preconditioning is not absolute, delaying cell death but not preventing it. Various other consequences of ischaemidreperfusion have been reported to be attenuated by preconditioning. The incidence of reperfusion arrhythmias was initially studied by Shiki and Hearse in 1987.17 They used a five minute
Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, UCMSM, University College Hospital, Cower Street, London WC 1E 6 AU, United Kingdom. Correspondence to Dr Yellon.
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I
n recent years, there have been numerous attempts to design therapy aimed at limiting infarct size following acute myocardial infarction. Apart from the success of early restoration of blood flow to the ischaemic area by thrombolysis, results have largely been disappointing. As such, investigators have sought and are continually seeking new approaches to myocardial protection. The discovery of the phenomenon of ischaemic preconditioning has focused attention on the ability of the myocardium to protect itself and has raised the possibility of exploiting this endogenous form of protection by pharmacological means.
lschaernic preconditioning
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Possible mechanisms of ischaemic preconditioning There have been many suggestions as to the possible mechanism of ischaemic preconditioning. We discuss the more popular theories below. Reduced energy demand ATP is depleted at a slower rate in preconditioned
myocardium, such that although some ATP is lost during preconditioning itself, levels are higher after 10 minutes of the sustained ischaemia.” In addition, glycogenolysis and anaerobic glycolysis both occur at a reduced rate and therefore Murry rt af have formed the hypothesis that there is a reduction in energy demand in preconditioned ischaemic myocardium.” As discussed earlier, improved cell viability could then be explained either by preservation of ATP stores or by reduced catabolite accumulation, or both. If a reduction in energy demand is an essential component of the protection afforded by preconditioning, this implies that either the metabolic work required to sustain viability has been reduced or that ATP utilisation has been redirected from nonessential to more critical pathways, eg, from ineffectual contractile activity to the maintenance of ionic gradients. As brief ischaemia causes some mechanical dysfunction, it has been suggested that the presence of this decreased contraction may be responsible for the reduced energy demand seen when the heart enters a second prolonged ischaemic episode. Attempts have therefore been made to link preconditioning with the presence of postischaemic mechanical dysfunction or “stunning”. So far these have been unrewarding. The time course of stunning is much more prolonged,’ stunned myocardium has an equal or increased oxygen consumption when compared with normal myocardium,” the degree of stunning does not correlate with the limitation in infarct size,” and the decrease in energy demand seen in preconditioned myocardium compared with control is not abolished in vitro by cardioplegic arrest.30 In addition, in swine myocardial preconditioning appears to be possible in the absence of stunning3’ Mitochondria1 ATPase inhibitor protein An alternative explanation for the reduction in energy demand seen in preconditioning involves the action of the mitochondrial ATPase inhibitor protein. Normally during ischaemia, the mitochondrial ATPase functions in reverse, potentially wasting ATP. In animals with slow heart rates (including rabbit, dog, pig, sheep, and human), there is a reversible inhibitor of this ATPase which binds during ischaemia, triggered by cytosolic acido~is.~’ It has been suggested, therefore, that preconditioning might in part be mediated by this inhibitor protein, either by binding more rapidly during a second ischaemic episode or by persistent binding during reperfusion.33 However, since its activity in the rat is minimal and yet preconditioning is still possible,’’ I‘ this hypothesis seems unlikely. Free radicals The role of free radicals in myocardial ischaemia and reperfusion is still unclear,’4 although these may be important in the pathogenesis of myocardial ~tunning.~’ With respect to their role in ischaemic preconditioning, preliminary studies have shown that free radical scavengers given to dogs prior to preconditioning cause attenuation of the response in 50% of cases.3h This suggests that free radicals might be beneficially involved in some way. However, Iwamoto et a13’ were unable to repeat this in rabbits, and Osada et allyfound that reperfusion arrhythmias were reduced by free radical scavengers in the isolated rat heart with no additional protective effect of preconditioning. Further work is required in this area to clarify these conflicting findings. Stress proteins Another area of interest with regard to the mechanism of preconditioning and “endogenous” myocardial protection in
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ischaemic insult to precondition rats and found that reperfusion arrhythmias following a second five minute coronary occlusion were markedly reduced. However arrhythmias, some requiring defibrillation, occurred frequently after the preconditioning ischaemia which led the investigators to suggest that the animals might simply be having their quota of rhythm disturbances earlier. Hagar et a1” resolved this issue by using three shorter coronary occlusions as their preconditioning protocol, which produced few arrhythmias by themselves and yet significantly reduced subsequent reperfusion arrhythmias. Similar protective effects have been noted in the isolated rat heart preconditioned by global ischaemia.” In the dog, preconditioning has been noted to reduce arrhythmias during the prolonged ischaemic phase as well as on reperfusion.*” Another group has looked at changes in wall motion resulting from prolonged ischaemia and has shown that in rabbits, preconditioning, allows much greater recovery of segmental shortening. Although it is likely that the mechanism underlying these observations is the same as that for infarct size reduction, at present some caution is required in extrapolating results between models. In particular, the incidence of reperfusion arrhythmias is known to be closely related to the length of the ischaemic insult, with a narrow time window for maximum arrhythmias.” It is possible that ischaemic preconditioning, by delaying cell necrosis, might delay this time window, such that more reperfusion arrhythmias would occur experimentally if a longer ischaemic insult was used. It is possible to induce ischaemic preconditioning by techniques other than coronary artery occlusion. In vitro experiments with the isolated perfused rat heart suggest that global preconditioning may result from a short period of zero flow followed by reperfusion - with either reperfusion arrhythmias” or recovery of function’2 being the end points used. However, this model has not been investigated using a subsequent regional ischaemic insult with infarct size as the end point. Also it is interesting to speculate that cardiac surgeons may have been preconditioning the heart globally for years with intermittent cross clamp fibrillation during bypass surgery. Another recent study has shown that rapid pacing can induce a preconditioned response in anaesthetised open chest dogs,23presumably via global ischaemia. This study looked at arrhythmias during ischaemia and reperfusion, and at ST elevation during ischaemia, to compare the paced and control groups. However, a similar study in rabbits with rapid atrial pacing failed to show a reduction in infarct size in spite of ECG and monophasic action potential evidence of ischaemia during the pacing.24This suggests that the severity of the brief ischaemic insult is crucial for preconditioning to occur. Recently it has been shown that it is possible to “precondition” myocardium with regional or global hypoxia, rather than i~chaemia.‘~ Although the mechanism is not necessarily the same, it does suggest that a phase of metabolite accumulation prior to washout by reperfusion is not essential for preconditioning.
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that the time to the onset of ischaemic contracture during global normothermic ischaemia is extended by pretreatment with adenosine or the A1 agonist, R-phenyl-isopropyladenosine (R-PIA). It appears likely therefore that adenosine released locally from ischaemic myocytes plays a role in attenuating ischaemic damage. This evidence has been linked to ischaemic preconditioning by Liu et a1.” They have shown that ischaemic preconditioning can be blocked by pretreatment with A1 adenosine receptor antagonists in an in vivo rabbit model and also that adenosine or R-PIA infused down the coronary artery of an isolated perfused rabbit heart causes infarct size limitation comparable to that achieved by preconditioning. Rabbit experiments with intravenous R-PIA infusion in vivo have demonstrated some limitation of infarct size, although this is much less marked when the rabbits are paced to prevent R-PIA induced brady~ardia.~”’~ A 1 receptors are coupled to Gi proteins and blockade of these proteins with pertussis toxin has also been shown to attenuate the protection afforded by pre~onditioning.~~ 6o If preconditioning is mediated by adenosine which accumulates during ischaemia and acts on A 1 receptors, then brief A1 stimulation must trigger biochemical changes in the heart that confer lasting protection against a subsequent ischaemic insult. It is not known at this stage what these changes might be, although the two most likely possibilities are stimulation of glycolysis and the opening of ATP regulated K’ channels.
Prostacyclin Other groups have proposed that preconditioning is dependent on the release of one or more endo enous protective substances. For example, Vegh et al-TF have implicated prostanoids. They found that sodium meclofenamate, an inhibitor of cyclo-oxygenase, attenuated the protective effect of preconditioning on arrhythmias in dogs. They suggest that prostacyclin release may be important as it is known to be released from ischaemic myocardium and it reduces arrhythmias during ischaemia and reperfusion when infused locally.” However, Li and Kloner” were unable to show any reduction of the effects of preconditioning on infarct size or arrhythmias by inhibiting cyclo-oxygenase with aspirin in the rat. Further work is required in this area.
Glycolytic flux A brief period of ischaemia increases glycolytic flux,” as does hypoxic perfusion or the infusion of adenosine.” Evidence supporting a role for glycolysis in maintaining cell viability during ischaemia was reviewed by Opie.62 Glycolytically derived ATP may be of particular importance in the preservation of cell membrane function and cell integrity while ATP from oxidative phosphorylation preferential12 supports contractile function.63 In addition, Fralix et a1 have shown that inhibition of glycolysis (by perfusing an isolated heart with pyruvate rather than glucose) appears to inhibit preconditioning. However preconditioning reduces glycogen stores and, as discussed earlier, during the subsequent ischaemic insult glycolysis is decreased2’ and so any theory based on improved glycolytic efficiency must explain this discrepancy. It is possible that enhanced glycolysis prior to the onset of the longer ischaemic insult leads to the accumulation of a compartmentalised store of ATP which subsequently protects the myocytes, even though total ATP at this stage is known to be decreased. This theory is, however, only conjecture.
Nitric oxide Alternatively, the release of nitric oxide during preconditioning may lead to a subsequent beneficial effect. Evidence for this comes from the attenuation of preconditioning seen with L-NAME (nitro L-arginine methyl ester), an inhibitor of nitric oxide synthesis, in an arrhythmia model.” However in contrast, Pate1 et al” failed to inhibit ischaemic preconditioning with a similar dose of L-NAME in the rabbit heart using infarct size as the endpoint. At present no firm conclusion can be drawn about the role of nitric oxide. Adenosine Perhaps the most likely candidate as an endogenous mediator of preconditioning is adenosine. It is released from many cells under stress (including ischaemic myocytes) and is thought to act as a local regulator modulating the function of the cell via a “feedback” mechanism.53 Studies in isolated, perfused rat hearts have shown that the increased glycolytic flux which occurs with hypoxia can be inhibited by 8-sulphophenyl-theophylline, an A 1 adenosine receptor antagonist. 54 Also in this model, the same group has shown
ATP regulated k? channels More is known about the role of ATP regulated (or sensitive) K’ channels (KATP).Noma‘’ found that the previously undefined outward current which increases significantly when cardiac cells are subjected to hypoxia, is due to a K’ channel the conductance of which increases at lower intracellular ATP concentrations. He also postulated that this channel is responsible for the shortening of the action potential which is seen during ischaemia and that this could reduce ATP depletion by reducing Ca2+influx and decreasing myocardial contractility. Since then, evidence has been accumulating to suggest that activation of KATPchannels plays an important protective role during ischaemia. For example, KATPchannel agonists such as cromakalim and pinacidil have been shown to improve postischaemic recovery of function in the isolated rat h e a d 6 and the
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general is stress (or heat stress) protein synthesis.38 This family of proteins, synthesised in response to a variety of stimuli, was originally identified in cells exposed to raised temperature^.'^ Recent studies have detected the 70 kDa stress protein in heart tissue of a variety of species, including the dog, rat, and rabbit, and increased expression can be induced by ischaemia, hypoxia, or pressure overload.3x Myocardial protection from heat stress has been shown in functional and biochemical terms in isolated hearts of rat“’ 4 1 and rabbit,4’ and also with infarct size reduction in the rabbit isolated heart Limitation of infarct size by prior heat stress has also been achieved in vivo in a rat model of infarctionu; however in vivo experiments in dogs4’ and rabbits46 have shown no protective effect. Evidence against the involvement of stress proteins in preconditioning comes from Thornton et a14’ who showed that protein synthesis inhibitors (actinomycin D and cycloheximide) did not attenuate preconditioning in rabbits, although specific stress protein levels were not determined. The 70 kDa stress protein has been measured in a similar study4’ and although its synthesis did not appear to be inhibited by these drugs, no rise was discernible in the protein until two hours after ischaemia. It therefore seems unlikely to be involved in preconditioning. This does not exclude the possibility of involvement of one of the other stress proteins and indeed recent studies have examined the role of the 65 kDa stress protein.49
Ischaemic preconditioning
’’
Evidence for ischaemic preconditioning in humans Considerable care must be taken in extrapolating results from animal models to humans. However. in view of the number of mammalian species in which ischaemic preconditioning occurs and the remarkable infarct size reduction achieved, it is likely to have significant clinical implications. Patients with unstable angina in the hours preceding a myocardial infarction might have a longer time window for reperfusion with thrombolysts, allowing greater limitation of infarct size. It is interesting that patients with angina prior to myocardial infarction have a less complicated in-hospital course than other groups, even though they have a higher risk profile for coronary artery disease (including hypertension, hyperlipidaemia, and diabetes).” Furthermore, it might be expected that during percutaneous transluminal coronary angioplasty (PTCA) the first balloon inflation would
x
protect the m ocardium against subsequent inflations. Deutsch et a f R have compared the first two 90 second balloon inflations in 12 patients undergoing elective PTCA and have found that subjective anginal discomfort. ST segmental changes, myocardial lactate production and coronary vein flow were all attenuated during the second in fl at ion. Conclusion In conclusion, ischaemic preconditioning is unique in the degree of resistance to ischaemic damage that it provides. As our knowledge increases. it may be possible to take advantage of the mechanisms underlying this endogenous form of myocardial protection such that in the future. patients may benefit from our ability to exploit the heart’s own adaptive responses to myocardial ischaemia. Received 5 February 1992. accepted I6 March I992 We are grateful to the Hatter Foundation for their continued support
Key terms. protection
ischaemic preconditioning.
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reperfusion.
myocardial
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I Maxwell MP, Hearse 111. Yellon I>M Species vanation in the coronary collateral circulation dunng regional myocardial ischaemia a cntical determinant of the rate of evolution a d extent of myocardial infarction Cardiovosc Res 1987.21: 137-46 2 Schaper J. Schaper W Time course of myocardidl necrosis C~rdiovoscDrugs l h e r 1988.2: 17-25 3 Jennings RB. Hawkins HK, Lowe Jk. Hill ML. Klotman S. Reimer KA Relation between high energy phosphate and lethal injury i n myocardial ischemia in the dog Am I furhol 1978. 92:203 16 4 Seely JR, Grotyohann LV Role of glycolytic products in damage to the ischemic myocardium dissociation of adenosine InDhosDhate levels and recoverv of rewrfused ischemic hearts CA R;S 1984.ss:ni6- 24. 5 Reimer KA. .Mum/ CE. Yamasawa I, Hill MI.. Jenninrs RB Four bnef penods of Oischemid cause no cumularive AYP loss or necrosis Am I fh,siol 1986.251(11e~rr Circ f h v w d LO): f11306 I5 6 Geft I L . Fishhein MC. Sinomiya K. er a1 Intermittent hnef pcnodg of ischemia have a cumulative effect and may cause myocardial necrosis Crrculuorion 1982.66: I I50 3 7 Murry CI:. Jennings RB. Reimer KA Preconditioning with ischemia a delay of lethal cell injury in ischemic myocardium Ctrculurton 1986.74: I I24 36 8 M u q CE. Richard VJ. Jennings RB. Reimer KA Myocardial protection is lost hefore contractile function recovers from ischemic preconditioning Am J Phystol 1991.MO(Heorl Circ Ph vstol29): H796-804 9 1.1 K, Vasquer BS. Gallagher KP. Lucchesi BR Myocardial protection with preconditioning Ctnulorion 1990.82 09-19 10 Cohen .MV. 1.iu GS. Downcy JM Preconditioning c a u w improved wall motion as well ds smaller infarcts after transient coronary occlusion i n rabbits Circulo/ton 1W I .84:34 I 9 I I 1.1 Y. Kloner R A Cardioprotective effects of ischaermc “preconditioning” .ire not mediated by prostanoids Curdtovusc Res 1992.26:226-31 12 Alkhulaifi AM. Browne E, Yellon DM Iwhaemic preconditioning Iimiis infdrct sire i n the 1.11 heart (dhsuact) I Mol Cell Curdid 1992;24(suppl I):S93. 13 Schon RJ. Rohmann S. Braun ER. Schawr W Ischemic preconditioning rcducc\ infarct size i n swine myocardium Qrt Res 1990.66: 1 I33 42 14 Van Winkle I)M, Thornton J. Ilowney J M Cardioprotection from ischemic preconditioning is lost following prolonged reperfusion i n rabbits (abstract) Circularion 199 1.84(suppl I1):432 15 Schwarr ER. .Mohn M. Sack S. Arrds M Durdiion of infarct size limiting effect of irchermc preconditioning i n the pig (dhsrract) Circulation 199 I .84(suppl I1):432 16 Sao BS. McClanahan TB. Groh M A . Schott RJ. Gallagher KP The time limit of effective ixhemic preconditioning i n dogs (dhsuact) Ctnu/u/ton 1990.82(suppl 111):27 I
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arterially perfused guinea pig right ventricular w&’ at dosages which did not effect preischaemic contractility. Also in the isolated rat heart, a KATPagonist (nicorandil) causes a more rapid decrease in contractility during early ischaemia with delayed onset of contracture. whereas an antagonist (glibenclamide) delays the fall in developed pressure but contracture occurs earlier,” Grover and colleagues6’ have also been able to limit infarct size in open chest dogs by giving intracoronary cromakalim or pinacidil prior to ischaemia. There is still some debate as to whether KATPchannels are activated during the first few minutes of ischaemia, for experimentally they are inhibited at A’rP levels much lower than the intracellular ATP concentration seen at that stage.’” It ha$ k e n calculated that only minor increases in channel conductance ( < I % ) could have major effects on action potential d ~ r a t i o n . 7’~ !However in spite of this. quantitative calculations suggest that the fall in A’TP in early ischaemia is not sufficient to explain the observed action potential shortening.” Clearly for the KATPchannel to be responsible for preconditioning, channel activity would have to be increased by a five minute ischaemic episode or alternatively the channel would have to be modified in such a way that activity could increase more rapidly at the beginning of the sustained ischaernia. One possible explanation is that KATP channels, although belonging to the “ligand gated’ group of K’ channels, are modulated by adenosine via interaction with a G , protein.” If adenosine released during ischaemia decreases the channel sensitivity to ATP. this may therefore be a route by which channel activity could be increased during brief ischaemia and could explain the data supporting the adenosine hypothesis of preconditioning. The relevance of KATPchannels to preconditioning has been further tested using the antagonists sodium 5-hydroxydecanoate and glibenclamide, in an attempt to block preconditioning. This has been successful in dogs,” ’’ but not in rabbits,76 although in the latter study. glibenclamide did have a deleterious effect on infarct size. Further conflicting evidence comes from arrhythmia studies. KATPchannel agonists appear to be proarrhythmic during regional ischaemia in a conscious canine model of sudden death” and also during low flow global ischaernia in the isolated rat heart.” Reperfusion arrhythmias are, however, reduced by pinacidil in a global ischaemia modeLh7 By comparison. ischaemic preconditioning is antiarrhythmic during ischaemia and on reperfusion in the open chest dog,*’ as well as on reperfusion following global ischaemia.:’ N o firm conclusions can be drawn at present.
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