LRP6通过去磷酸化的L-型钙通道和缝隙连接蛋白Cx43对大鼠室性心动过速调控的研究

The preliminary study in our research group identified that low density lipoprotein receptor related protein 6 (LRP6) was dominantly distributed to intercalated disc and directly bound to the gap junction protein connexin 43 (Cx43), and found that the knockout LRP6 impaired the intracellular trafficking of the core gap junction protein connexins, reduced the currents of L-type calcium channel, and played an important role in ventricular tachycardia (VT) of non-organic heart disease mice. Thus, we assume that LRP6 may regulate VT targeting intracellular gap junction protein and L-type calcium channel dephosphorylation? This study is designed to establish acute myocardial infarction (AMI) model of organic heart disease, to separate the LRP6 cardiac specific gene knockout of rat myocardial cells, to detect the relevant molecular changes of intracellular gap junction protein and L-type calcium channel dephosphorylation, the current changes of L-type calcium channel in single myocyte, and the potential spreading in multiple myocytes, etc. Then uncover the electrophysiological mechanisms of LRP6-mediated VT in AMI, and elucidate the possible molecular pathways mechanisms of LRP6-mediated VT in AMI, which may eventually provide new insights into the preventive and therapeutic strategy for VT in coronary heart disease.

我们前期研究揭示低密度脂蛋白受体相关蛋白6（Low-density lipoprotein Receptor related Protein 6，LRP6)优势分布在闰盘，与缝隙连接蛋白Cx43直接结合，且LRP6心肌特异性敲除不但导致心肌细胞间缝隙连接蛋白胞内运输缺陷，还引起L-型钙通道电流的明显降低，在无器质性心脏病室性心动过速（Ventricular Tachycardia, VT）中起到重要作用，因而我们提出LRP6是否通过去磷酸化的缝隙连接蛋白和L-型钙通道调控VT？本项目拟建立器质性心脏病急性心肌梗死模型，通过分离LRP6心肌特异性基因敲除的大鼠心肌细胞，检测缝隙连接蛋白和L-型钙通道去磷酸化后相关分子变化、单个心肌细胞膜L-型钙通道电流变化、多细胞电位扩布等，揭示LRP6调控急性心肌梗死后VT的电生理学机制和潜在分子通路机制，最终为冠心病VT防治提供新的基础理论和干预策略。

既往研究表明低密度脂蛋白受体相关蛋白6（Low-density lipoprotein Receptor related Protein 6，LRP6）在心血管健康和稳态中起到关键作用。课题组前期研究揭示LRP6心肌特异性敲除在室性心动过速（Ventricular Tachycardia,VT）中起到重要作用，因而我们提出LRP6是否通过去磷酸化的缝隙连接蛋白和L-型钙通道调控VT？本项目建立急性心肌梗死小鼠模型，通过生物信息学分析,构建circRNA-miRNA-LRP6调控网络，并筛选靶向LRP6的miR-152-3p及与其结合的circRNA1615，同时采用RNA干扰、荧光素酶和RIP实验等技术，验证了LRP6的上游基因circRNA1615通过海绵吸附miR-152-3p后，改善LRP6功能，并调控其下游靶点L型钙通道(L-VOCC)和缝隙连接蛋白Cx43的磷酸化状态，进而调节VT发生的分子机制。同时，我们亦通过使用Loss-and-Gain方法和Real-time PCR及Western blot等技术进一步探索了LRP6通过调控其下游靶点G蛋白亚单位（Gas），进而影响Cx43的磷酸化水平，提出并验证“LRP6-Gas-Cx43”信号轴参与室速（VT）发生的科学假说。本项目有望拓展心梗中VT发生新的调控机制，为寻找心肌梗死后VT新的治疗靶点提供实验依据。

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