The role of p38 MAPK activation in preconditioning mediated protection against ischaemia/reperfusion injury

[摘要] ENGLISH ABSTRACT:The ultimate consequence of the interruption of blood flow to the myocardium isnecrosis. In view of the prevalence of coronary artery disease in the generalpopulation, and the deleterious effects of myocardial ischaemia on myocardialtissue, it is important to develop new strategies to protect the myocardium againstischaemia. Necrosis of myocardial tissue has for a long time been considered to bethe main component of the damage incurred by myocardial infarction. Recently theimportance of the contribution of apoptotic cell death in the context of myocardialischaemia/reperfusion injury has become apparent.There is a general agreement that early reperfusion is necessary to salvagemyocardial tissue from cell death. Preconditioning is the phenomenon wherebybrief episodes of ischaemia and reperfusion protect the heart against a subsequentlonger period of ischaemia. This endogenous mechanism is the strongest form ofprotection against myocardial infarction that has yet been described. Apart fromischaemie preconditioning (IPC), protection can also be elicited with pharmacologicagents, such as activation of the beta-adrenergic receptor with isoproterenol.Ischaemie preconditioning protects the myocardium against necrosis, arrhythmiasand apoptosis, and increases functional recovery upon reperfusion. Betaadrenergicreceptor stimulated preconditioning (PPC) has been shown to improvepost-ischaemie functional recovery, but it is not known whether it also protectsagainst myocardial infarction and apoptosis.The signaling pathways involved in preconditioning have been extensively studied.A distinction is usually made between factors that act as triggers, or as mediatorsof protection. Triggers activate cellular responses before the onset of sustainedischaemia, and its involvement is demonstrated by showing that inhibitors of thetrigger bracketing the preconditioning protocol can block its protective effect, or thattransient administration with washout before sustained ischaemia can activate aprotective effect. A mediator operates during sustained ischaemia, and itsinvolvement is demonstrated by showing that infusion of an inhibitor of its actionimmediately prior to sustained ischaemia (without washout) can block its protectiveeffect. Another approach to demonstrate a mediator role is to attempt to activatesignal transduction pathways during sustained ischaemia. As it is not possible toinfuse substances during ischaemia, activators are infused immediately prior toischaemia without washout of the agent and subsequently its effect on protection isobserved.It is clear that the evolutionary conserved stress activated pathways are involved inpreconditioning. There are three pathways i.e., the extracellular receptor activatedpathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activatedprotein kinases (MAPK). The precise role of the p38 MAPK pathway has not beenelucidated. Experimental evidence has suggested a role for the activation of p38MAPK as a trigger, as well as a mediator of the protective effect of preconditioning.There is however also strong evidence that the attenuation of p38 MAPK activationduring sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only beeninvestigated in relation to its protection against necrosis, but not apoptosis.AIMS:The aim of this study was to:(I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture.The reason was that such a model could potentially enable one to rapidlyelucidate the signal transduction pathways in an environment without theinfluence of non-cardiac cells.(II) Investigate whether IPC and ~PC protect against necrosis and apoptosis.(III) Elucidate the role of the stress-activated kinase, p38 MAPK, inpreconditioning.METHODS:1. Neonatal rat cardiomyocyte cell culture modelA viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) was first developed using different concentrations - a concentration of0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cellcultures were subjected to sustained simulated ischaemia by using either 5 mMKCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min.Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCNfor 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation beforebeing exposed to sustained simulated ischaemia.2. Isolated adult rat cardiomyocyte modelIsolated cardiac myocytes were exposed to 2 hours of hypoxia, which was inducedby pelletting the cells by centrifugation, and covering them with a thin layer ofmineral oil. Some groups were preconditioned with either hypoxia for 10 min at37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes.The trypan blue exclusion method and MTT method developed in the neonatalcardiomyocytes were used to assess viability.3. Isolated perfused rat heart model3.1 Infarct size was determined in a model of regional ischaemia by usingtetrazolium staining and determining the area of necrosis (exclusion oftetrazolium) as a percentage of area at risk. These hearts were subjected to35 min global ischaemia and 30 min reperfusion. Some groups werepreconditioned by three cycles of 5 min global ischaemia or addition ofisoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before theonset of sustained regional ischaemia.3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation wasdetermined using antibodies against dual phosphorylated p38 MAPK (i.e.activated p38 MAPK). Apoptosis was measured by using antibodies againstactivated caspase-3, and against a fragment of PARP (PARP cleavage). Forthese experiments isolated rat hearts were exposed to global ischaemia for25 min followed by 30 min reperfusion. Some groups were preconditionedwith three cycles of 5 min global ischaemia. A global ischaemia model wasused in order to have sufficient tissue available for the Western blotdeterminations. This necessitated a shorter period of sustained ischaemia,as the globally ischaemie heart does not recover sufficiently after a longerperiod of ischaemia such as is necessary in regional ischaemiaexperiments.3.3 The role of p38 MAPK in ischaemie preconditioning was investigated byadministration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, eitherbracketing the preconditioning (i.e. to determine its role as a trigger) or for10 min immediately prior to sustained ischaemia (i.e. to determine its role asa mediator). The second approach was to use anisomycin, an activator ofp38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as amediator (10 min immediately prior to sustained ischaemia) in the samemodel as used for determination of p38 MAPK activity. The infusion ofanisomycin for 10 min has been shown to elicit activation of p38 MAPK to asimilar extent as has been observed with an ischaemie preconditioningprotocol. The endpoints used were infarct size and markers of apoptosis.RESULTS:1. Neonatal rat cardiomyocyte cell culture modelIt was not possible to establish a model of preconditioning of neonatalcardiomyocytes that was consistently successful. It was therefore decided toabandon the attempts and to use a different cell model.2. Isolated adult rat cardiomyocyte modelIsolated adult cardiomyocytes were preconditioned successfully, but produced toolittle material to perform simultaneous determinations of cell viability and Westernblots (p38 MAPK activation and markers of apoptosis). It was therefore decided touse the isolated perfused adult rat heart.3. Isolated perfused adult rat heart model3.1 Both IPC and PPCprotect against infarction and apoptosis:Using two models of preconditioning i.e., IPC and PPC, the protective effects ofpreconditioning were demonstrated convincingly against infarction (necrosis). IPCand PPC both caused a significant reduction in infarct size (12.2±1.4 and15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms ofpreconditioning also protected against apoptosis, by significantly reducing themarkers of apoptosis, caspase-3 activation and PARP cleavage. The protectionafforded by both forms of preconditioning was accompanied by a marked decreasein activation of p38 MAPK upon reperfusion. The relationship between p38 MAPKand the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. Toinvestigate the role of p38 MAPK as a mediator or a trigger in preconditioning, usewas made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii)a known activator of p38 MAPK i.e., anisomycin.3.2 p38 MAPK as a trigger of protection:Administration of SB 203580 during the IPC protocol and washed out beforesustained ischaemia did not abolish the protective effect of ischaemiepreconditioning, and resulted in a small, but significant increase in caspase-3activation and PARP cleavage. On the other hand, activation of p38 MAPK withanisomycin for 10 min followed by washout also resulted in a significant reductionin necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001)and both markers of apoptosis. The latter results suggested that p38 MAPK was atrigger of preconditioning. If this was the case, why didn't SB 203580 abolish theprotection of IPC? The most likely explanation was that multiple protectivemechanisms were activated during a multi-cycle protocol of ischaemicpreconditioning, of which activation of p38 MAPK was only one. Inhibition of p38MAPK with SB 203580 would therefore not be expected to block the activation ofthose mechanisms that were independent of p38 MAPK, but were still capable ofprotecting against necrosis or apoptosis. It is very interesting that a small increasein apoptosis was observed when SB 203580 was used in this situation, as it mayindicate that the protection against apoptosis was more dependent on theactivation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was notsensitive enough to detect such small effects.3.3 p38 MAPK as a mediator of protection:Inhibition of p38 MAPK activation with SB 203580 administered 10 min beforesustained ischaemia caused a significant decrease in infarct size compared toNon-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of heartspreconditioned with ischaemia. This was accompanied by a similar pattern ofprotection against apoptosis, with significantly reduced activation of caspase-3activation and PARP cleavage.These results strongly supported a role for the attenuation of p38 MAPK activationas a mediator of preconditioning against ischaemia/reperfusion-mediated necrosisand apoptosis. However, the results of the experiments with anisomycin were atfirst glance not compatible with such a conclusion. The administration of theactivator of p38 MAPK, anisomycin, for 10 min immediately prior to sustainedischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4%vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation andPARP cleavage indicating less apoptosis. The reason for these findings wereprobably that this method of administration of anisomycin did in fact not activatep38 MAPK during sustained ischaemia, but actually served as a trigger to protectagainst ischaemia - similarly as if it had been infused with washout of the drug.Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem ofnot being able to infuse a drug into the myocardium during ischaemia could not beovercome by immediate prior infusion, and that the administration of anisomycin inthis way had activated downstream effectors of the p38 MAPK signal transductionpathway. An important contender for such an effector would be heat shock protein27 (HSP27), which has been shown to play an important role in protection againstapoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibilitywas that anisomycin had activated the JNK stress activated kinases. Theelucidation of a role of this signal transduction pathway would necessitate the useof anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK.Final conclusion:The work in this thesis showed that the stress activated kinase, p38 MAPK, wasinvolved in the protective effect of ischaemie preconditioning. The resultssuggested a role for the activation of p38 MAPK as a trigger of protection, and theattenuation of p38 MAPK as a mediator of protection, which was observed in thereduction of both necrosis (infarct size) and apoptosis as determined with caspase-3 activation and PARP cleavage.

[发布机构] Stellenbosch University