GBF1调控hERG通道表达的机制及其在心肌细胞动作电位时程延长中的作用研究

Sudden cardiac death (SCD) are the leading causes of mortality worldwide, and prolongation of QT interval is an independent predictor of SCD. Genome-wide association studies (GWAS) have yielded associations between common gene variants at GBF1 with QT intervall, but the underlying mechanism is not clear yet. GBF1 mediates the cargo transport via vesicular trafficking. Dysfunction of the cardiac potassium channel, hERG, which is vital in cardiac repolarization, leads to cardiomyocytes action potential duration (APD) and electrocardiographic QT interval prolongation. In our previous studies, up-regulation of GBF1 increased the expression and function of hERG potassium channels. Thus we hypothesized that the malfunctioned GBF1 down-regulated the function and expression of hERG protein by affecting its trafficking, and consequently prolongs action potential duration. We will use the GBF1 knocked up or knocked down human pluripotent stem cell induced cardiomyocytes and hERG-HEK cells to record the action potential duration, together with the expression and function of hERG protein by molecular biology technologies , patch clamp and computational model of cardiomyocyte action potential, to investigate the effects of GBF1 on hERG channels and its corresponding molecular mechanisms. These findings will not only extend our understanding of the mechanisms of GBF1 gene variation associated QT interval prolongation, but also shed some light into new targets for SCD prevention.

心源性猝死（SCD）是全球首要致死原因，QT间期延长是其独立危险因素。GWAS研究发现GBF1基因变异与QT间期延长相关，但机制尚不明确。GBF1促进囊泡运输介导蛋白质转运。hERG通道介导的IKr是心肌细胞的主要复极电流，其功能降低是心肌细胞动作电位时程（APD）、心电图QT间期延长的重要原因。我们发现上调GBF1可增加hERG蛋白表达及功能，因此假设GBF1功能异常可影响hERG蛋白转运降低其表达及功能，延长APD。本研究采用人多能干细胞诱导分化的心肌细胞及稳定表达hERG的HEK细胞，利用病毒转染及GRISPR/Cas9技术上调或下调GBF1表达，分子生物学及膜片钳技术，检测hERG通道的表达及功能，记录APD，并建立计算机仿真模型，阐明GBF1功能异常影响hERG通道延长APD的分子机制。预期结果有助于揭示GBF1基因变异与QT间期延长的关系及机制，为预防SCD提供新的干预靶点。

心源性猝死（SCD）是全球首要致死原因，QT间期延长是其独立危险因素。GWAS研究发现GBF1基因变异与QT间期延长相关，但其机制尚不明确。GBF1通过促进COPI囊泡运输途径而介导蛋白质在细胞内的转运。hERG通道介导的IKr电流是心肌细胞的主要复极电流，其功能降低是心肌细胞动作电位时程（APD）、心电图QT间期延长的重要原因。本项目拟探索GBF1通过影响hERG蛋白表达及功能而延长动作电位时程，并进一步探索GBF1调控hERG功能的分子机制。我们发现利用CRISPR/Cas9技术下调GBF1表达可以延长心肌细胞动作电位时程，过表达GBF1可增加hERG蛋白表达及电流，而GBF1负显性突变体可以降低hERG蛋白表达及电流；免疫荧光激光共聚焦检测发现，GBF1能增加hERG在细胞膜及高尔基体的分布，实时定量PCR检测显示GBF1不影响hERG的mRNA水平，定点突变技术构建hERG转运缺陷突变体N470D和I573R发现，GBF1可增加hERG转运缺陷突变体水平，提示GBF1通过促进hERG蛋白转运影响其功能。另外采用RNA干扰技术敲低COPI囊泡形成蛋白β-COP及Arf1后，GBF1对hERG蛋白水平无影响。以上结果显示，GBF1通过COPI囊泡形成途径促进hERG蛋白向细胞膜转运。这可能是GBF1基因多态性与心电图QT间期延长的分子机制，是将来防治SCD新的干预靶点。