Objectives To investigate the effect of BH3 only protein Bim (Bcl-2 interacting the mediator of the cell death) on apoptosis induced by hypoxia in rat cardiomyocyte.
Methods The Rat cardiomyocytes, 1-3days after birth, were cultured primarily and identified by using antibody targeting α-actin of striated muscle. Bim-siRNA were transfected transiently into cardiomyocytes by lipofectamine 2000. Establish a model of hypoxia (The cells after 48 h transfection, were given a deal with hypoxia for 12 h). The experiments were divided into five groups: Blank Control Group, Hypoxia Group, Hypoxia + Negative Control siRNA Group, Hypoxia + Mock control Group, Hypoxia + Bim-siRNA Group. With the inverted microscopy, the Cardiomyocyte beat frequency and rhythm can be observed and recorded; the content of lactate dehydrogenase (LDH) in cell culture fluid was detected by automatic biochemical analyser; The cell survival rate was determined by MTT method; the cell apoptosis rate was determined by flow cytometry; the expression of Bim, Bax, Bcl-2 and p-p38MAPK was assessed by Western Blotting.
Results Immunohistochemical identification confirmed that rat cardiomyocytes were successful cultured. The beat frequency of cardiomyocyte was slowed down after hypoxia stimulation, the rhythm was disordered (P < 0.01) and the content of lactate dehydrogenase (LDH) in cell culture fluid increased obviously (P < 0.01). The MTT result showed that the survival rate was decreased (p< 0.05). And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P < 0.05 or P < 0.01).the results of Western blotting showed that the transfection of Bim-siRNA decreased the expression of Bim (p<0.01). It confirmed that Bim was silenced by Bim-siRNA effectively. the hypoxia increased the expression of Bax and p-p38 (p<0.05),and decreased the expression of Bcl-2(p<0.01),while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P <0.05). These were greatly related to the decreasing of apoptosis.
Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of rat cardiomyocyte induced by hypoxia. Its mechanism is associated with the down-regulation of p-p38MAPK, Bax expression and up-regulation of Bcl-2. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.