Role Of Mitochondrial ATP-sensitive Potassium Channel In The Protection Against Myocardial Ischemia/Reperfusion Injury In Isolated Hearts Of CGRP-preconditioned Rats

Objective:we established the current study to determine:1.To evaluate whether CGRP is involved in the cardioprotection of ischemic preconditioning and its effect on cardiac function and myocardial infarct size after reperfusion.2.Through drug intervention of cultured myocardial cells,To further explore the protective effect of CGRP on myocardial cells.Methods:Chapter 1: the effect of CGRP on the cardiac function and myocardial infarct size in isolated rat hearts after ischemia reperfusion Healthy male SD rats with body weight of 260-300 g.To establish an experimental model of myocardial ischemia reperfusion in isolated hearts of Langendorff.The experimental study is divided into two parts.Part 1: Before the isolated rat heart ischemia reperfusion,they were given CGRP,CGRP combined with CGRP receptor antagonist CGRP8-37(10-6mol/L),through the observation index of cardiac function(left ventricular systolic pressure(LVSP),left ventricular end diastolic pressure(LVEDP),left ventricular systolic blood pressure(LVDP = LVSP,LVEDP),heart rate(HR),myocardial contraction when the maximum change rate(+dp/dtmax),diastolic myocardial maximum change rate(-dp/dtmax))to evaluate the protective effect of CGRP on myocardium.Part 2: The first part of the isolated heart was collected,and then TTC.According to the observation of the results of staining,analysis the effect of exogenous CGRP on myocardial infarct size after ischemic preconditioning.Chapter 2: the effect of CGRP on the changes of cyt C in cultured neonatal rat cardiomyocytes after hypoxia / reoxygenation Part 1: Culture and identification of cardiomyocytes.The cardiomyocytes obtained from 1~3 day old SD rats were cultured for 72 hours for experimental study;The cultured myocardial cells were identified by immunocytochemical SABC method.Part 2: Effects of CGRP on the concentration of cytochrome C in neonatal rat cardiomyocytes.24 wells of neonatal rat cardiomyocytes were randomly divided into8 groups(3 wells in each group),control group,A/R group,CGRP+A/R group(10-8mol/L),CGRP+CGRP8-37+A/Rgroup(1umol/L),5-HD+CGRP+A/R group(500umol/L);H-89+CGRP+A/R group(1umol/L);chelerythine+CGRP+A/R group(2umol/L);DZ+A/R group(100umol/L).The concentration of cytochrome C in the cytoplasm of the cells was detected by ELISA after reoxygenation.Result:1.the effect of CGRP on the cardiac function and myocardial infarct size in isolated rat hearts after ischemia reperfusion:1.1 Cardiac function:There was no significant difference in cardiac function between the three groups when cardiac perfusion was stable.Compared with the basic values of the steady state,The HR,LVSP,LVDP,±d P/dtmax of the three groups decreased gradually(P<0.05),and LVEDP increased significantly(P<0.05).Compared with group I/R,LVSP,HR,LVDP,±d P/dtmax increased during reperfusion in CGRP group(P<0.05),while LVEDP decreased significantly(P<0.05).There was no significant difference between the CGRP+Gli+I/R group and the I/R group.1.2 The effection of CGRP to myocardial infarct size after reperfusion :Compared with group IR,the myocardial infarct size(IS/AAR%)of group CGRP decreased significantly(P<0.01),compared with group CGRP,the myocardial infarct size of group CGRP+CGRP8-37+I/R increased significantly.2.the effect of CGRP on the changes of cyt C in cultured neonatal rat cardiomyocytes after hypoxia / reoxygenation2.1 The model of primary cultured cardiomyocytes of SD rats was successfully established,presenting more than 90% of the cultured cells were cardiomyocytes and they were alive at 3 days of incubation.2.2 The hypoxia / reoxygenation model of myocardial cells was successfully established,and the content of cytochrome C in the cytoplasm of myocardial cells was significantly increased after hypoxia / reoxygenation.2.3 Compared with control group,The release of cyt C in CGRP+A/R group was no significant difference(P<0.05),but the release of cyt C was significantly lower than that of A/R group(P<0.05).Compared with group CGRP+A/R,the release of cyt C was significantly increased in group CGRP+CGRP8-37+A/R,group5-HD+CGRP+A/R and group chelerythine+CGRP+A/R(P<0.05).Compared with group H-89+CGRP+A/R,group chelerythine+CGRP+A/R significantly increased the release of cyt C(P<0.05).Compared with group A/R,the release of cyt C was significantly lower in group DZ+A/R(P<0.05),there was statistical difference.Conclusion:1.Pretreatment with exogenous CGRP before myocardial ischemia could stabilize hemodynamics and decrease infarct size.Its antagonist can antagonize the above action.Suggesting that CGRP acts on its receptor,through some way to regulate cardiac function,and thus have a protective effect on the heart.2.CGRP may play a role in myocardial protection through activation of protein kinase C and mito KATP channel opening,inhibition of mitochondrial pathway of apoptosis.