儿茶酚胺释放对心肌BCKDK表达的影响及其机制研究

目的 探究在缺血、高血压、心衰等疾病条件下儿茶酚胺释放对心肌细胞中支链酮酸脱氢酶激酶（BCKDK）的表达影响及其作用机制。 方法 使用异丙肾上腺素（ISO）腹腔注射模拟小鼠心脏儿茶酚胺导致的心肌病变模型。小鼠分为对照（Vehicle）组和ISO注射（ISO）组。分别使用ISO、过氧化氢（H₂O₂）、鱼藤酮（rotenone）和抗霉素（antimycin ）A 模拟多种病理刺激处理心肌细胞。使用Western blot检测BCKDK、含有NHL重复序列蛋白（NHLRC）1 和热休克蛋白（Hsp）70蛋白水平表达;实时荧光定量PCR检测BCKDK mRNA水平表达;免疫荧光检测BCKDK、NHLRC1和Hsp70的细胞内定位;免疫共沉淀探究蛋白-蛋白相互作用和BCKDK泛素化水平。 结果 与Con组相比,ISO处理显著增加BCKDK蛋白水平（P<0.01）,而mRNA水平无显著差异,BCKDK与NHLRC1的结合降低且泛素化水平降低。而过表达NHLRC1上调BCKDK泛素化水平并降低其蛋白表达。与ISO组相比,Hsp70敲低能完全逆转ISO诱导的BCKDK升高和泛素化降低逆转ISO诱导的支链氨基酸（BCAA）代谢紊乱（P<0.01）。 结论 ISO暴露通过诱导Hsp70与BCKDK结合竞争性抑制NHLRC1介导的BCKDK泛素化,从而导致BCKDK的上调和心肌细胞中BCAA分解代谢紊乱。

Effects of catecholamine release on cardiac BCKDK expression and its underlying mechanism

AIM To explore the mechanism of BCKDK up-regulation in cardiomyocytes under pathological conditions such as ischemia, hypertension and heart failure. METHODS Isoproterenol (ISO) was injected intraperitoneally to simulate the cardiomyopathy model induced by catecholamine. Mice were divided into two groups: control group (Vehicle) and ISO injection group (ISO). Isoproterenol (ISO), hydrogen peroxide (H₂O₂), rotenone (rotenone) and antimycin A (antimycin A) were used to simulate various cardiac pathological stimuli. Western blot was used to detect the expression level of BCKDK, NHLRC1 and Hsp70 and real-time fluorescence quantitative PCR was used to detect the expression level of BCKDK mRNA. Immunofluorescence was used to detect the intracellular localization of BCKDK, NHLRC1 and Hsp70 and immunoprecipitation was used to explore the protein-protein interaction and the ubiquitination level of BCKDK. RESULTS Compared with those in Con group, BCKDK protein level in ISO group was significantly increased (P<0.01), but mRNA level remained unchanged. ISO treatment decreased the binding of BCKDK and NHLRC1 and significantly down-regulated the ubiquitination level of BCKDK. Overexpression of NHLRC1 upregulated the ubiquitination level of BCKDK and decreased its protein expression. Compared with ISO group, Hsp70 knockdown reversed the increase of BCKDK and the decrease of ubiquitination induced by ISO, and reversed the catabolism disorder of BCAA induced by ISO (P<0.01). CONCLUSION ISO exposure reduced the ubiquitination of BCKDK mediated by NHLRC1 by inducing the binding of HSP70 and BCKDK, thus leading to the upregulation of BCKDK and the disorder of BCAA catabolism in cardiomyocytes.