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GW24-e1392 The Role of ER stress in the BH3-only protein Bim-mediated cardiomyocytes apoptpsis induced by hypoxia
  1. Xia Zhen,
  2. Li Ju-xiang
  1. The Second Affiliated Hospital of Nanchang University

Abstract

Objectives To investigate the role of endoplasmic reticulum stress in the injury induced by Bim of hypoxic cardiomyocytes.

Methods The Rat cardiomyocytes, 1-3 days 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. The cell survival rate was determined by MTT method; the cell apoptosis rate and the change of the calcium concentration in cells were detdemined by flow cytometry; the expression of Bim and the markers of endoplasmic reticulum stress Caspase-12 and InSP3 were assessed by Western Blotting.

Results Immunohistochemical identification confirmed that rat cardiomyocytes were successful cultured. Green fluorescence was observed in the cells transfected of negative control siRNA group though the fluorescence microscope. The results of Western blot showed that the transfection of Bim-siRNA decreased the expression of Bim effectively (p < 0.01). Compared with the blank control group, The MTT assay determined that the survival rate of cardiomyocytes was decreased (p < 0.05) after the injured by hypoxia. And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P <0.01) and the concentration of calcium (p < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P <0.05 or P <0.01). Compared with the hypoxia + Negative Control siRNA group, the transfection of Bim-siRNA increased the cell survival rate, decreased cell apoptosis rate and the concentration of calcium (p < 0.01). The results of Western blotting showed that the transfection of Bim-siRNA reduced the expression of endoplasmic reticulum stress markers Caspase-12 and InSP3 (p < 0.05 or p < 0.01), and reduced the effects that hypoxia increased the expression of Caspase-12 and InSP3 (p < 0.05 or p < 0.01).

Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of cardiomyocytes induced by hypoxia. Its mechanism is associated with the endoplasmic reticulum stress. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.

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