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目的探讨心肌细胞支链氨基酸(BCAA)代谢障碍在儿茶酚胺诱导的心肌细胞毒性中的作用。方法分离并培养新生大鼠心室肌细胞(NRVMs),给予异丙肾上腺素(ISO)20μmol/L处理48 h模拟儿茶酚胺毒性,ISO刺激同时给予支链α-酮酸脱氢酶激酶(BDK)特异性抑制剂3,6-二氯-2-苯并噻吩羧酸(BT2)处理(40μmol/L)。48 h后,蛋白质印迹法(Western blot)检测BCAA代谢相关酶及细胞凋亡相关酶蛋白的表达;分光光度法检测支链-α酮酸脱氢酶(BCKD)活性;给予外源性BCAA(5 mmol/L)后,分时间点检测培养基中BCAA浓度,直接反映心肌细胞BCAA降解速率;流式细胞术检测心肌细胞凋亡。结果 ISO处理激活心肌细胞线粒体凋亡通路并引起心肌细胞凋亡。与对照组比较,ISO组心肌细胞中BDK表达明显上调,引起BCKD活性下降和BCAA降解障碍。给予BDK抑制剂BT2处理后,能够逆转ISO刺激造成的心肌细胞BCAA降解障碍并明显抑制心肌细胞凋亡。结论抑制BDK能够减轻儿茶酚胺毒性诱导的心肌细胞凋亡,改善心肌BCAA降解障碍有可能成为减轻儿茶酚胺毒性的新策略。
Objective To investigate the role of cardiomyocyte branched chain amino acid (BCAA) metabolism in catecholamine-induced cardiomyocyte cytotoxicity. METHODS: Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured. Isoproterenol (ISO) 20 μmol / L for 48 h was used to simulate catecholamine toxicity. ISO stimulated with branched-chain α-keto acid dehydrogenase kinase (BDK) The sex inhibitor 3,6-dichloro-2-benzothiophene carboxylic acid (BT2) treatment (40μmol / L). After 48 h, Western blotting was used to detect the expression of BCAA metabolism-related enzymes and apoptosis-related enzyme proteins; Spectrophotometry was used to detect BCKD activity; BCAA 5 mmol / L), BCAA concentration in culture medium was measured at different time points, which directly reflected the rate of BCAA degradation in cardiomyocytes. Cardiomyocyte apoptosis was detected by flow cytometry. Results ISO treatment activated cardiomyocyte mitochondrial apoptotic pathway and caused cardiomyocyte apoptosis. Compared with the control group, BDK expression in cardiomyocytes of ISO group was significantly upregulated, which caused the decrease of BCKD activity and the degradation of BCAA. Administration of BDK inhibitor BT2 reversed the impairment of cardiomyocyte BCAA degradation induced by ISO stimulation and significantly inhibited cardiomyocyte apoptosis. Conclusion Inhibition of BDK can reduce catecholamine-induced cardiomyocyte apoptosis and improve myocardial BCAA degradation may be a new strategy to reduce catecholamine toxicity.