Effect Of Down-regulation Of VDAC1 On Autophagy In H9c2 Cardiomyocytes Subjected To Anoxia/Reoxygenation

Objective: Previous researches have confirmed that excessive autophagy was activated in H9c2 cardiomyocytes exposed to ischemia/reperfusion(I/R),which increased apoptosis and aggravated injury.This study further explored the mechanism underlying the regulation of VDAC1 on autophagy during I/R injury and its relationship with PINK1 /Parkin autophagy pathway.Methods:This study established an in vitro anoxia/reoxygenation(A/R)model to mimic in vivo myocardial I/R injury using H9c2 cardiomyocytes.Cardiomyocytes were randomly divided into 4 groups,including control group,A/R group,p Super+AR group and p Super-VDAC1+AR group.Western blot analysis was performed to detect the expression of VDAC1,PINK1,Parkin,LC3-Ⅱ/Ⅰand Beclin1 proteins.LDH activities of each group were measured by spectrophotometer.The mitochondrial membrane potential(ΔΨm),reactive oxygen species(ROS)and apoptosis were assessed using flow cytometry.Moreover,MDC staining was used to detect autophagosome synthesis.In addition,laser confocal was carried out to identify subcellular location of Parkin.Results: 1.After A/R treatment,LDH activity in the medium significantly increased.The expression level of VDAC1 was also elevated(p< 0.01),which promotes ΔΨm collapsed,ROS burst.Thus,m PTP opening,and eventually the number of apoptotic cells increased obviously(p<0.01).On the contrary,silencing VDAC1 expression with RNA interference effectively attenuated A/R injury in cardiomyocytes.2.After A/R injury,the expression levels of PINK1 and Parkin,as well as two typical autophagy markers,LC3-II/I and Beclin1,were significantly up-regulated(p<0.01).MDC staining showed that autophagosomes accumulated in A/R-exposed cardiomyocytes.Notably,VDAC1 silencing cells expressed less autophagy-related proteins and produced less autophagosomes after A/R injury(p<0.01).3.The results of laser confocal microscope indicated that Parkin translocated to mitochondria after A/R injury in H9c2 cardiomyocytes.However,down-regulation of VDAC1 inhibited Parkin’s translocation,the majority of Parkin still located in the cytoplasm.Conclusion: In H9c2 cardiomyocytes,VDAC1 protein was upregulated after A/R injury,which resulted in excessive autophagy through the PINK1/Parkin pathway,and ultimately aggravated apoptosis.