The Role Of ER Stress In The Process Of BH3-only Protein Bim-mediated Cardiomyocytes Apoptosis Induced By Hypoxia

OBJECTIVES:To investigate the effect of Bim and its mechanism of endoplasmic reticulum in cardiomyocytes apoptosis induced by hypoxia by using a model of hypoxic injury of cardiomyocytes transfected by siRNA targeting BH3-only protein Bim.METHODS:1.Rat cardiomyocytes were dissociated and then primary cultured cardiomyocytes were got from the infant rat aged1-3days. The antibody targeting a-actin of striated muscle was used to identify the cardiomyocytes.2.Cardiomyocyets were exposed to hypoxia for different periods(0hour,3hours,6hours,12hours,18hours,24hours). The expression of Bim protein was detected in all periods by western blotting and the period of highest expression of Bim was chosen.3.siRNAs of Bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by using western blotting.4.Cardiomyocytes were divided into five groups:Blank Control Group, Hypoxia Group,Hypoxia+Liposome Group, Hypoxia+Negative Control siRNA Group, Hypoxia+Bim-siRNA Group.5.The frequency of cardiomyocytes beat and rhythm were observed and recorded with the inverted microscopy. The content of lactate dehydrogenase(LDH) in culture medium was assessed by automatic biochemical analyzer, The cell survival rate was determined by MTT assay, The cell apoptosis rate and the concentration of calcium were measured by flow cytometry, The expressions of protein Caspase-12,InSP3were detected by western blotting.RESULTS:1.In hypoxia group, the expression of BH3-only protein Bim began increasing after3hours, reached to peak12hours later and then attenuated gradually.2. The expression of Bim was inhibited obviously after transfected by Bim-siRNAs and the silencing efficiency of Bim-siRNA2was the highest and 86.73%.3.The frequency of cardiomyocyte beat was slowed down after hypoxia and the rhythm was disordered, while the frequency of beat was increased obviously after inhibiting the expression of Bim.4.Compared with the control group, the concentration of lactate dehydrogenase(LDH) of culture medium was increased(92.28±11.05VS1935±4.12, p<0.01), and the survival rate of cardiomyocytes was reduced in hypoxia group(67.38±4.02VS100,p<0.05). After transfection of Bim-siRNA, the release of lactate dehydrogenase(LDH) was decreased, however,compared with hypoxia+Negative Control group, the survival rate of cardiomyocytes was increased.5.Compared with the control group,the apoptosis rate was increased (42.21±3.43VS2.98±0.72,p<0.01),the concentration of calcium in cardiomyocytes was increased in hypoxia group. And were both decreased with silencing the expression of Bim.6.Compared with the control group,the expression of Caspase-12(0.84±0.0370.15±0.029,p<0.05)and InSP3(0.70±0.034VS0.14±0.029,p<0.05)which were both endoplasmic reticulum stress markers were up-regulated in Hypoxia group, and which were down-regulated after inhibiting the expression of Bim.CONCLUSIONS:1.Bim plays an important role in the process of cardiomyocytes apoptosis induced by hypoxia.2.The expression of Bim in the cardiomyocytes can be inhibited effectively by RNA interference.3.Cardiomyocytes apoptosis induced by hypoxia can be inhibited by silencing the expression of Bim gene.4.Caspase-12and InSP3which are the markers of endoplasmic reticulum stress may participate in the process of Bim-mediated cardiomyocytes apoptosis induced by hypoxia.