Activation Of AMPK Mediates Cardioprotection Induced By Brief Calcium Depletion Treatment In Isolated Heart

Background:Myocardial injury induced by ischemia and reperfusion （I/R） is verycommon in cardiovascular diseases, which is a severe threat to health in theworld, and the morbidity and mortality of cardiovascular diseases related tomyocardial injury have also increased in China recently. However, the presenttreatment for these diseases is hard to satisfy both doctors and patients, therefore,it’s imperative to come up with countermeasures against I/R-induced myocardialinjury. Ischemic preconditioning （IP） is administering several alternate processesof transitory ischemia and reperfusion, before long-term ischemia, whichprovides strong cardioprotection against I/R-induced myocardial injury and isone of the strongest mechanisms of endogenous myocardial preservation up tonow. In addition, administering transitory Ca²⁺-free Krebs-Henseleit buffersolution to isolated hearts, before long-term ischemia, also provides strong cardioprotection against I/R-induced myocardial injury, which is calledrepetitive Ca2+depletion and repletion for short duration （CP）. CP has been animportant method of studying Ca²⁺signal in many laboratories, and the study oncardioprotection induced by CP is another hot spot in cardiovascular studyingfield, however, the mechanisms of the cardioprotection have not been clarified.Apparently, elucidating the mechanisms of IP and CP, as well as utilizing thecardioprotection, is very important to the prevention and therapeutics ofischemic heart disease.AMP-activated protein kinase （AMPK） is a regulator of cellular energymetabolism and is activated during myocardial ischemia. Activated AMPKimproves glucose uptake, enhances fatty acid oxidation and inhibits apoptosisafter ischemia, which presents protective effects on hearts during I/R. AcetylCoA carboxylase （ACC） is a target molecule of AMPK, and phosphorylationlevel of ACC is an indication of activated state of AMPK, which phosphorylatesand blunts ACC, resulting in inhibition of fatty acid synthesis and promotion offatty acid oxidation, and elicits vital protective effects on hearts during ischemia.Previous studies showed that IP activated reverse mode sodium/calciumexchanger （NCX）, and led to a transitory increase in calcium level of cytoplasm,which provided cardioprotection against myocardial injury. Meanwhile, theincrease in intracellular calcium activated protein kinase C （PKC） to translocateto cytomembrane, leading to activation of AMPK and promotion of geneexpression of glucose transporter4（GLUT4）, which played an important role incardioprotection induced by NCX-calcium preconditioning.However, whether AMPK is involved in the protective effect of CP againstischemia/reperfusion injury has not been determined. If so, it is also necessary toinvestigate the role of NCX and PKC in the activation of AMPK by CP. The results help us understand the physiological mechanisms of CP more deeply, andpresent much theoretical significance and practical value in the prevention andtherapeutics of cardiovascular diseases.Aims:（1） To study the role of AMPK in cardioprotection induced by CP againstI/R-induced myocardial injury;（2） To study whether NCX and PKC are involved in cardioprotection of CPmediated by AMPK.Methods:Adult Sprague-Dawley male rats （220²80g） were anesthetized withpentobarbital and anti-coagulated with heparin, then hearts were quicklyremoved and underwent Langendorff perfusion. CP consisted of three cycles ofCa2+depletion （1minute each） and Ca2+repletion （5minutes each）, and IPconsisted of three cycles of ischemia （5minute each） and reperfusion （5minuteseach）. The hemodynamics was recorded during the whole experiment by usingAcqKnowledge3.8.1（BIOPAC Systems, Inc） software.2,3,5-triphenyltetrazolium chloride （TTC） staining was used to analyze the viabletissue in heart. Immunobloting assay was used to detect the total andphosphorylation level of AMPK and ACC.Results:（1） The role of AMPK in cardioprotection of CP and IP against myocardialinjury was expounded as follows. After I/R, the level of left ventricularend-diastolic pressure （LVEDP） of isolated hearts increased markedly, whilelevels of left ventricular developed pressure （LVDP）, maximum rates of increaseand decrease in left ventricular pressure （±dp/dtmax）, rate pressure product （RPP）and coronary artery flow （CF） decreased significantly. Compared to I/R group, the level of LVEDP of hearts in CP and IP group was decreased noticeably, andlevels of LVDP,±dp/dtmax, RPP and CF were improved significantly. The AMPKinhibitor, P5499, which was administered to the isolated hearts before ischemia,had no significant influence on heart functions and CF, but attenuated thecardioprotection of CP and IP remarkably.（2）10μmol/L P5499, which is the inhibitor of AMPK, attenuatedcardioprotection induced by CP and IP. After I/R, the ratio of myocardial injuryarea to total area was39.6%±1.49%. Compared to vehicle pretreatment （VP）group, the myocardial injury areas in CP and IP group were decreasedremarkably, and phosphorylation levels of AMPK and ACC were increasedsignificantly. P5499, which was administered to the isolated hearts beforeischemia, had no significant influence on myocardial injury area andphosphorylation levels of AMPK and ACC, but attenuated the cardioprotectionof CP and IP remarkably.（3）3μmol/L KB-R7943（KB-R）, which is the inhibitor of NCX, attenuatedcardioprotection induced by CP and IP. Compared to VP group, the myocardialinjury areas in CP and IP group were decreased remarkably, and phosphorylationlevels of AMPK and ACC were increased significantly. KB-R, which wasadministered to the isolated hearts before ischemia, had no significant influenceon myocardial injury area and phosphorylation levels of AMPK and ACC, butattenuated the cardioprotection of CP and IP remarkably.（4）3μmol/L chelerythrine chloride （CHE）, which is the inhibitor of PKC,attenuated cardioprotection induced by CP and IP. Compared to VP group, themyocardial injury areas in CP and IP group were decreased remarkably, andphosphorylation levels of AMPK and ACC were increased significantly. CHE,which was administered to the isolated hearts before ischemia, had no significant influence on myocardial injury area and phosphorylation levels ofAMPK and ACC, but attenuated the cardioprotection of CP and IP remarkably.Conclusions:In addition to verifying activation of AMPK, NCX and PKC in themechanisms of cardioprotection induced by IP, our studies also demonstrate thatactivation of AMPK mediates the cardioprotection induced by brief calciumdepletion treatment, and suggest that activation of NCX and PKC may beinvolved in the process of activation of AMPK induced by CP, which may belocated in the upper stream of AMPK, and play important roles in thecardioprotection induced by brief calcium depletion treatment.