The Role Of Parkin Dependent Mitophagy Pathway In Cardiomyocytes Anoxia And Reoxygenation Period

bjective: Autophagy,a normal physiological activity of cells,can also be activated when cells are subjected to various stimuli,and play a role in protecting cells or aggravating cell damage.The purpose of this study was to investigate the changes of autophagy activity and the relationship between autophagy activity and Parkin dependent mitophagy pathway in cardiomyocytes during anoxia and reoxygenation period.Methods: In this study,H9c2 cells were used to construct anoxia(A)injury model and anoxia/reoxygenation(A/R)injury model.Cells were randomly divided into three groups: normal control group,anoxia group,anoxia / reoxygenation group.Biochemical automatic analyzer was used to detect the activity of LDH 、CK and m PTP.Flow cytometry was used to measure the production of reactive oxygen species(ROS)and the level of mitochondrial membrane potential(Δψm).Real-time PCR method was used to detect the expression of related genes at m RNA level.Western blot was used to detect the expression of its protein level and the formation of autophagy was detected by MDC staining.The location of Parkin in mitochondria was detected by confocal laser microscopy.Results: 1.After anoxia,the activities of LDH and CK increased and the production of ROS increased slightly,and Δψm decreased.After reoxygenation,LDH and CK were further increased,which were significantly higher than those in group C and group A,and Δψm collapsed and m PTP were open.2.The results of Real-time PCR and Western blot showed that both LC3 and Beclin1 significantly decreased in group A compared with group C,however,VDAC1,Parkin and PINK1 increased.The expression of LC3 and Beclin1 in group A/R was higher than that in group A,and the expression of PINK1,Parkin and VDAC1 were further increased.3.The results of MDC showed that the number of autophagy in group A was lower than that in group C,suggesting that the number of autophagy in group A decreased after anoxia,but the number of autophagy in group A/R was significantly higher than that in group C and group A,indicating that the activity of autophagy was significantly increased.4.The sublocalization of Parkin in the cells was detected by confocal laser microscopy.The results showed that the Parkin of group A cells mainly existed in the cytoplasm,while the Parkin of group A cells was translocated to mitochondria.Conclusion: After anoxia,the autophagy activity decreased and the cell damage increased,but after reoxygenation,VDAC1 was upregulated significantly,Parkin was activated and promoted translocating to mitochondria,and the autophagy activity increased significantly,and the degree of cell injury was further deteriorated.Therefore,we speculate that anoxic autophagy should be harmful to the cells,while excessive increase of autophagy during reoxygenation may cause irreversible damage to cardiomyocytes.