Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM: To investigate the effect of BH3-only protein Bim( Bcl-2 interacting mediator of cell death)on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS: Rat cardiomyocytes were isolated from infant rats aged 1 ～ 3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome,and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group,hypoxia group,hypoxia + liposome group,hypoxia + negative control siRNA group and hypoxia + bim-siRNA group. The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase( LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay.The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim,Bax,Bcl-2,p-p38 MAPK and p38MAPK was detected by Western blotting. RESULTS: Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest( 86. 73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered,while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group,the LDH in the culture medium was increased( P 0. 01),and the viabilityof the cardiomyocytes was reduced in hypoxia group( P 0. 05). The apoptotic rate was increased( P 0. 01). After transfection with bim-siRNA,the release of LDH was decreased,and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and pp38 MAPK( P 0. 05),and decreased the expression of Bcl-2( P 0. 01),while transfection with bim-siRNA reduced the effects caused by hypoxia( P 0. 05). These were greatly related to the decrease of apoptosis. However,the expression of p38 MAPK was not changed. CONCLUSION: The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.