The Mechanisms Of Cardioprotection Of Human Recombinant Erythropoietin On The Isolated Rat Heart Suffering From Ischemia Reperfusion Injury

Background:Myocardial ischemia reperfusion injury significantly depressed myocardial contractile and diastolic function for myocardiocyte necrosis and apoptosis,which were resulted from breakdown of ultrastructural organization and disorder of energy metabolism.Ischemia preconditioning or pharmacological preconditioning was used extensively in order to palliating the myocardial injury suffering from ischemia and reperfusion injury in both clinical and experimental settings.The purpose of this study was to determined the cardioprotective effect of rhEPO preconditioning on the isolated rat heart,and to identify the possible molecular mechanisms underlying the cardioprotection of rhEPO preconditioning by detecting the phosphorylated PKCε,p38MAPK and ERK1/2. PartⅠThe Cardioproteetive Effect of Human Recombinant Erythropoietin on the Cardiac Function of Isolated Rat Heart Suffering from Ischemia Reperfusion InjuryObjective:To investigate the effect of rhEPO preconditioning on the post-ischemic contractile and diastolic function and the cell viability after myocardial ischemia reperfusion injury.Methods:Hearts from normoxic rat(n=8/group) were isolated and perfused in the Langendorff mode.The hearts were perfused with rhEPO(10.0IU/ml) for 15 minutes prior to 30 minutes ischemia and 120 minutes reperfusion.The experimental protocol used was shown in Fig.1 PartⅠ.For mechanism studies,the hearts were pretreated with PKC inhibitor(chelerythrine),p38MAPK inhibitor(SB203580),ERK1/2 inhibitor (PD98059),potassium channel blockers(glibenclamide) or nitric oxide synthase inhibitors(L-NAME) for 15 minutes alone followed by 15 minutes in combination with rhEPO prior to ischemia..The left ventricular contractile and diastolic function(LVDP,RPP,+dP/dt_max,-dP/dt_max) and the coronary flow(CF) were recorded under steady state conditions prior to global no-flow ischemia and at 30,60,120min of reperfusion,and the cell viability was detected by MTT assay.Results:1.RhEPO significantly increased the LVDP,RPP,+dP/dt_max,-dP/dt_max but no effect on the CF at 30 minutes of reperfusion,while the LVDP,RPP,+dP/dt_max,-dP/dt_max and CF were all increased significantly at 60 and 120 minutes of reperfusion compared with I/R group.The cell viability was also increased significantly compared with I/R group.2.Inhibiting the activation and traslocation of PKCεwith chelerythrine abolished the cardioprotective effects of rhEPO.The LVDP,RPP,-dP/dt_max at 30 minutes of reperfusion,the LVDP,RPP,+dP/dt_max at 60 and 120 minutes of reperfusion,and the cell viability were all decreased significantly compare with rhEPO group.At the same time,in group pretreated with SB203580 or PD98059 following by rhEPO,the LVDP,RPP,±dP/dt_max at 30 minutes,the LVDP,RPP,±dP/dt_max,CF at 60 and 120 minutes, and the cell viability were also decreased significantly compared with rhEPO group.3.The LVDP,RPP,±dP/dt_max at 30 minutes of reperfusion,the LVDP,RPP,±dP/dt_max,CF at 60 and 120 minutes of reperfusion,and the cell viability were all decreased in group pretreated with K_ATP channel inhibitor glibenclamide following by rhEPO perfusion compared with rhEPO group.4.The inhibitor of NOS(L-NAME) can also abolish the cardioprotection of rhEPO preconditioning.The LVDP,RPP,+dP/dt_max,-dP/dt_max and CF at 30,60,120 minutes of reperfusion and the cell viability were all decreased in group pretreated with L-NAME following with rhEPO perfusion compared with rhEPO group.Conclusions:Our study demonstrated that rhEPO preconditioning increased the left ventricular post-ischemia contractile and diastolic function and coronary flow,as well as the cell viability after I/R injury.The mechanisms underlying the cardioprotection possibly associated with the phosphorylation of PKCε,p38MAPK and ERK1/2,for inhibiting the activity of PKCε,p38MAPK or ERK1/2 could abolish the cardioprotective effect of rhEPO absolutely.Besides,the opening of K_ATP channel and the synthesizing of NO might also involve in the cardioprotection of rhEPO preconditioning,for inhibiting the K_ATP channel or the NOS could abolish the cardioprotective effects of rhEPO absolutely as well.PartⅡThe Possible Molecular Mechanisms Underlying the Cardioprotection of Human Recombinant Erythropoietin on the Isolated Rat Heart Suffering from Ischemia Reperfusion InjuryObjective:To identify the possible molecular mechanisms underlying the cardioprotection of rhEPO preconditioning by detecting the phosphorylated PKCε,p38MAPK and ERK1/2.Methods:Hearts from rats were isolated and aerobically perfused with K-H buffer for 30 minutes,then perfused with erythropoietin(10IU/ml) for 10 minutes.To investigating the molecular mechanism of cardioprotection of rhEPO,the hearts were pretreated with PKCεinhibitor(chelerythrine),p38MAPK inhibitor(SB203580),ERK1/2 inhibitor (PD98059),potassium channel blockers(glibenclamide) or nitric oxide synthase inhibitor(L-NAME) for 10 minutes alone followed by 10 minutes in combination with rhEPO.Then the free wall of left ventricle was excised and immediately stored in liquid nitrogen for western blot analysis.Frozen myocardial tissue samples were powdered in a precooled stainless steel mortar and pestle.The powdered tissue was then processed to obtain cell lysates or fractionated to obtain cytosolic and particulate fractions.Equal amounts of protein was analyzed by SDS-PAGE and Western blotting by using either isoform-specific antibodies for phospho-PKCεdetection both in cytosolic and particulate fraction or specific antibodies against phosphorylated and nonphsophorylated of p38 MAPK and ERK1/2 in cell lysates.The blots were developed by ECL.Results:1.The analysis of cytosolic and particulate fractions revealed that in the rhEPO preconditioning(10IU/ml) hearts,PKCεwas activated and translocated from cytosolic fraction to particulate fraction after perfused with rhEPO for 10 minutes.And the expression of phosphorylation of p38MAPK and ERK1/2 in total cell lysate was increased significantly compared with control group as well.2.Administration of chelerythrine before rhEPO inhibited the activation and translocation of PKCε,and PKCεlocated in the particular fraction was decreased significantly compared with rhEPO group.While the SB203580 or PD98059 had no effect on the activation and translocation of PKCε.SB203580 pretreating before rhEPO abolished the phosphorylation and activation of p38MAPK.Pretreatment with chelerythrine before rhEPO could partially inhibit the phosphorylation and activation of p38MAPK.While PD98059 had no effect on the phosphorylation and activation of p38MAPK.PD98059 inhibited the phosphorylation and activation of ERK1/2,and chelerythrine could also partially inhibit the phosphorylation and activation of ERK1/2. While SB203580 had no effect on the phosphorylation and activation of ERK1/2.3.Pretreatment with glibenclamide followed by rhEPO resulted in partially inhibiting the phosphorylation and activation of p38MAPK and ERK1/2,but had no effect on the activation and translocation of PKCε. 4.Pretreament with L-NAME following by rhEPO partially inhibited the phosphorylation and activation of p38MAPK and ERK1/2,and partially inhibited the activation and translocation of PKCεas well.Conclusion:The activation and translocation of PKCεwas induced soon after pretreated with rhEPO(10IU/ml).And treatment with rhEPO also phosphorylated p38MAPK and ERK1/2 rapidly.PKCεwas the upstream kinase both of p38MAPK and ERK1/2,and blocking the activation of PKCεpartially inhibited the phosphorylation of p38MAPK and ERK1/2.PKCεwas the upstream kinase of K_ATP channel,and inhibiting the activation and opening of K_ATP channel had no effect on the activation and translocation of PKCε.In the contrary,p38MAPK and ERK1/2 were the downstream kinases of K_ATP channel,and blocking the opening of K_ATP channel partially inhibited the phosphorylation of p38MAPK and ERK1/2.So we concluded that the activation and phosphorylation of p38MAPK and ERK1/2 were K_ATP channel-dependent and K_ATP channel-independent.Inhibition the production of NO partially abolished the activation and phosphorylation of PKCε,and partially inhibited the activation and phosphorylation of p38MAPK and ERK1/2.The activation of PKCε,p38MAPK and ERK1/2 were all NO-dependent and NO-independent.