应用诱导多能干细胞建立体外PKP2和DSG2突变细胞模型探讨ARVC致病机制

Arrhythmogenic right ventricular cardiomyopathy (ARVC) comprises a group of hereditary myocardial diseases mainly involving the right ventricle. Clinical manifestations include palpitation, syncope and sudden cardiac death. The pathological features are myocardial necrosis and fat and fibrous tissue replacement, accompanied by severe inflammation. ARVC is currently considered a "desmosomes" disease but the intimate pathogenic mechanisms are unclear. The desmosomes are structures responsible for maintaining cell-cell adhesion and communication. PKP2 and DSG2 are the two most common disease genes. We intend to use exfoliated cells in urine of ARVC patients with specific mutations (reported by us) as a source to establish induced pluripotent stem cells (iPSCs) using protocols also reported by us. Then, we will differentiate the iPSCs into functional cardiomyocytes to study the molecular mechanism of the abnormal connection between the cardiomyocytes, the apoptosis ,myocardial inflammation and changes of action potential. This in vitro disease model will be used to explain the different clinical manifestations in patients carrying mutations in these 2 genes, which will provide a theoretical basis for drug screening and future clinical treatment.

致心律失常性右室心肌病（ARVC）是一种遗传性心肌疾病，表现为心悸、晕厥甚至心脏性猝死。其病理特征为右心室心肌坏死和脂肪纤维组织增生，并伴随炎症反应。目前认为ARVC是一种"桥粒病"，以PKP2和DSG2突变最为常见，但其致病机制有待阐明。本课题拟利用尿液中的脱落细胞建立PKP2 N852fsX930和DSG2 R46G（均为本研究组发现）的ARVC诱导多能干细胞（iPSCs），体外定向分化为心肌细胞，研究PKP2和DSG2突变引起的心肌细胞紧密连接、细胞凋亡、炎症反应等异常的分子机制及细胞电生理的改变。从而解释携带这两种不同基因突变患者临床表现的差异，为药物筛选和临床治疗提供理论依据。

研究背景：致心律失常性右室心肌病（Arrhythmogenic Right Ventricular Cardiomyopathy，ARVC）是一种遗传性心肌疾病，表现为心悸、晕厥甚至心脏性猝死。其病理特征为右心室心肌坏死和脂肪纤维组织增生，并伴随炎症反应。目前认为ARVC是一种“桥粒病”，以PKP2和DSG2突变最为常见，但其致病机制有待阐明。研究方法及结果：本课题利用尿液中的脱落细胞建立了ARVC-PKP2-IPSCs和ARVC-SG2-IPSCs细胞模型，并定向分化为心肌细胞，研究了PKP2突变引起的心肌细胞连接、炎症反应和细胞电生理改变。应用QPCR和免疫荧光鉴定发现ARVC -PKP2-IPSCs-心肌细胞PKP2 、Plakoglobin表达异常；炎症因子 MMP-9、MMP-2、TNF-α 和IL-6 的表达水平增高；应用膜片钳技术发现ARVC -PKP2-IPSCs-心肌细胞动作电位的间期明显不规整，并可见晚期后除极引发新的动作电位。研究意义：研究结果解释了ARVC PKP2突变患者的发病机制。ARVC-IPSCs细胞模型的建立为药物筛选和发病机制的深入研究提供了研究工具。