MG53调节心脏辅助亚基KChIP2表达的分子机制及其在心脏电稳态调节中的作用

KChIP2 is the obligatory beta subunit of cardiac transient outward current (Ito), which is essential for regulating Ito function. KChIP2 protein levels increase with the development of heart, which determines the developmental increase of cardiac Ito. Furthermore, the transmural gradients of KChIP2 transcription across the free ventricular wall (epicardial myocytes > mid- cardial myocytes > endocardial myocytes) in many species contribute to the Ito transmural grandients which are critical determinants of myocardial function. In multiple heart diseases, such as hypertrophy, infarct cardiomyopathy, heart failure, etc, Ito amplitudes are consistently decreased, predisposing the heart to the risk of ventricular arrhythmia. A series of studies demonstrate that decrease in KChIP2 protein expression is an important mechanism for the downregulation of Ito in diseased hearts. Therefore, KChIP2 plays an important role in maintaining cardiac electrical stability. Unfortunately, the mechanisms underlying physiological and pathological regulation of KChIP2 expression and Ito function are largely unknown. MG53 is a member of TRIM family, which is specifically expressed in myocardium and skeletal muscle. Our preliminary data demonstrate that adenoviral overexpression of MG53 increased KChIP2 protein levels in cardiomyocytes, whereas MG53 knockdown by RNA interference decreased KChIP2 protein expression. Concomitantly, MG53 overexpression in cardiomyocytes increased the amplitude of Ito, while MG53 knockdown decreased cardiac Ito. These findings implicate that MG53 is an intrinsic regulator of KChIP2 expression in the heart. Based on the findings, we intend to address the following important questions in this project. First, what are the molecular mechanisms underlying MG53 regulation of KChIP2 expression in cardiomyocytes. Second, what is the role of MG53 in the developmental increase of KChIP2 expression and Ito function, and what are the underlying mechanisms. Third, whether and how MG53 regulates L-type Ca2+ current and IK,slow which have been suggested to be regulated by KChIP2 in cardiomyocytes. Furthermore, we will explore the alteration of MG53 in infarct cardiomyopathy and its role in the pathogenesis of electrical disturbances which are related to downregulation of Ito due to the decrease of KChIP2 expression. The potential therapeutic effect of modulating MG53 expression on the treatment of arrhythmia in diseased hearts will also be investigated in this project. Through these efforts, we hope to uncover novel mechanisms participating in maintaining cardiac stability and new therapeutic targets for the treatment of arrhythmia in heart diseases.

KChIP2是心脏瞬间外向钾电流Ito的必须b亚基，对其功能起不可或缺的调节作用。KChIP2还决定Ito的发育变化特性和在心室壁的跨壁梯度，对维持心脏电稳态至关重要。多种心脏疾病通过降低KChIP2蛋白表达而抑制Ito电流，引起心律失常。但KChIP2的生理和病理调节机制尚待查明。MG53是心肌和骨骼肌特异表达的TRIM家族成员，我们前期工作发现，过表达MG53增加心肌细胞KChIP2蛋白和Ito电流,降低MG53表达则起相反作用，表明MG53是心脏内在的KChIP2调节分子。据此，本项目拟开展以下研究：1.MG53调节KChIP2表达的机制是什么？2.MG53在KChIP2和Ito发育变化中的作用？3.MG53对也受KChIP2调节的L-型钙通道和IK,slow的调节作用？4.MG53在心肌梗死心电紊乱发生中的作用及治疗意义？以此阐明心脏电稳态调节的新机制，探寻心律失常治疗的新靶点。

KChIP2是心脏快瞬间外向钾电流（fast transient outward current, Ito,f）的必须beta亚基，对维持心脏电稳态至关重要。在心肌肥大和心衰中发现，KChIP2表达下调，导致Ito,f电流减小，引起室性心律失常的发生。但是无论是生理抑或是病理条件下KChIP2表达的调节机制目前尚不完全清楚。. MG53是心肌和骨骼肌特异性表达的TRIM家族成员，前期高通量测序分析发现，心肌细胞过表达MG53增加KChIP2的mRNA水平，而敲低MG53表达则降低KChIP2的mRNA水平。在此基础上，本研究进一步证实MG53调节KChIP2蛋白表达，并一致性地调节Ito,f电流幅度。同时，我们还引进了MG53敲基因小鼠，发现敲除MG53降低心肌细胞KChIP2表达和Ito,f电流，延长动作电位（AP），增加心脏发生室性心律失常的敏感性。. 通过检测NF-kB（p65）在细胞核的蛋白水平、免疫荧光核定位和启动子区转录活性发现，MG53负向调节NF-kB信号通路，而反向调节NF-kB活性能逆转MG53过/低表达对KChIP2和Ito,f的调节。通过染色质免疫沉淀（chromatin immunoprecipitation，ChIP）技术进一步证实MG53通过调节NF-kB在KChIP2启动子区的募集而调节KChIP2转录水平。通过免疫共沉淀（co-IP）还发现，MG53与NF-kB信号通路的TAK1和IkBa相互作用，从而调节NF-kB活性。. 在心肌肥大和梗死性心肌病时，KChIP2表达明显减少，我们同时还发现MG53表达也显著降低。在细胞水平，通过过表达MG53可以抑制肥大或缺氧引起的KChIP2表达下调和Ito,f电流减小。在主动脉狭窄引起的心肌肥大模型发现，敲除MG53基因加重KChIP2表达下调和Ito,f电流减小，进一步延长AP，并大大增加了室性心律失常的发生风险。. 因此，本项目率先提出MG53是KChIP2和Ito,f的内在调节分子，通过负向调节NF-kB转录活性，调节KChIP2表达和Ito,f电流，在心电稳定性维持中起重要作用。同时提出心肌肥大和心梗等病理条件下，MG53表达降低在KChIP2和Ito,f功能下调所致的室性心律失常中起关键作用，为临床心律失常治疗提供了新靶点。