Effects And Protective Mechanism Of Calcitonin Gene Related Peptide On Mitochondrial Membrane Potential Of Cardiomyocyte In Hypoxia/Reoxygenation Rats

Objective:To investigate the effects of CGRP on mitochondrial membrane potential of cardiomyocytes in hypoxia/reoxygenation SD rats.To investigate the protective mechanism of CGRP on hypoxia/reoxygenation injury of cardiomyocytes by various drug interventions in cardiomyocytes cultured in vitro.Methods:Part 1:Cardiomyocytes were cultured in vitro and identified.The cardiomyocytes were from 1-3 days old SD rats and cultured for 3 days.The cardiomyocytes were identified by immunocytochemical SABC method.Part 2:to observe the changes of mitochondrial membrane potential of cardiomyocytes under different drug interventions.Establishing hypoxic/reoxygenation（H/R）model of cardiomyocytes.Cardiomyocytes were randomly divided into 8 groups,3 wells each:blank control group（control group）;hypoxia/reoxygenation group（hypoxia/reoxygenation group,H/R group）;CGRP+H/R group（CGRP group）;CGRP receptor antagonist CGRP8-37+CGRP+H/R group（CGRP8-37 group）;mitochondrial K_ATP channel(mitoK_ATP)agonist diazoxide+H/R group（DZ group）;mitoK_ATP specific blocker 5-hydroxydecanoate+CGRP+H/R group（5-HD group）;protein kinase A（PKA）inhibitor H-89+CGRP+H/R group（H-89 group）;protein kinase C（PKC）inhibitor chelerythine+CGRP+H/R group（CHE group）.The mitochondrial membrane potential of each group of myocardial cells was detected by jc-1.Results:The rat cardiomyocytes were successfully cultured and the hypoxia/reoxygenation model was successfully established,too.Compared with the control group,the mitochondrial membrane potential was significantly decreased in the H/R group;CGRP pretreatment increased the mitochondrial membrane potential of H/R cardiomyocytes.Compared with the blank group,the red/green fluorescence ratio of the H/R group was significantly lower（P<0.01）;compared with the H/R group,the red/green fluorescence ratio of the CGRP group and the DZ group was significantly higher（P<0.01）;Compared with the CGRP group,the red/green fluorescence ratio of the CGRP8-37 group,the 5-HD group and the CHE group was all decreased（P<0.05）.Conclusion:H/R treatment significantly reduced mitochondrial membrane potential in cardiomyocytes;CGRP pretreatment increased mitochondrial membrane potential and thus protected H/R cardiomyocytes;CGRP pretreatment may activate mitoK_ATPTP channels;PKC may be involved in the process of CGRP pretreatment to increase mitochondrial membrane potential in H/R cardiomyocytes.