通过iPSC细胞模型对KCNH2基因新突变导致LQTs的机制及干预研究

Although the mortality of long QT syndrome (LQTs) is high, the traditional therapies could not prevent syncope or sudden cardiac death. We found two LQT2 mutations from 15 LQTs patients, KCNH2-W927X and KCNH2-W410R. KCNH2-W927X mutation was proved to lead to Ikr current decreased, and the latter has not been reported so far. KCNH2-W410R was a missense mutation, located at S1 section of HERG potassium channel, with Tryptophan being replaced by Arginine. The W410R mutation was predicted to cause abnormal structure of HERG protein, which could lead to LQT2. As a kind of Chinese herb, Wenxin Keli (WXKL) has been proved to be able to influence the cardiac ion channels, but it has not been used to treat LQTs patients. Based on the successfully established cardiomyocyte model derived from induced pluripotent stem cells (iPSC-CM), this study is designed to investigate the mechanism of KCNH2-W410R mutation and KCNH2-W927X mutation leading to LQTs in CHO cell model and iPSC-CM model, and to examine the effect of WXKL on HERG potassium channel, then to test whether WXKL could treat LQTs.

长QT综合征（LQTs）是致死率极高的恶性室性心律失常，常用临床治疗方式不能完全预防晕厥或猝死。课题组在对15例LQT患者的基因筛查的基础上发现两个LQT2突变，KCNH2-W927X和KCNH2-W410R。预实验证明KCNH2-W927X突变可导致Ikr电流密度明显下降，从而导致LQT2；而KCNH2-W410R突变迄今为止尚未见报道，位于HERG钾通道的S1跨膜区。稳心颗粒为一被证明对离子通道有明显干预作用的中成药，已被广泛用于临床，然而上尚未应用于LQTs的治疗。本研究拟在已经成功构建正常人诱导多能分化干细胞分化的心肌细胞（iPSC-CM）模型的基础上，从经典细胞电生理途径（CHO细胞模型）和iPSC-CM途径对W410R和W927X突变致LQTs的机制进行研究；并分别用稳心颗粒进行干预，研究稳心颗粒对KCNH2所编码HERG钾通道的功能影响，从而探讨其对LQT2的治疗作用。

以往研究表明，hERG钾离子通道突变与2型长QT综合征（LQT2）有关。临床工作中，我们在一长QT综合征患者体内发现新的突变——KCNH2-W410R突变，此突变造成的电生理功能的改变有待验证。我们通过蛋白质印迹以及免疫荧光来检测hERG通道蛋白的表达；运用膜片钳技术及计算机模拟技术分别探究突变对IKr电流及心室肌细胞动作电位的影响。与野生型的hERG通道（hERGWT）相比，突变型的hERG通道（hERGW410R）可显著降低IKr电流，并将hERG通道的稳态激活曲线向去极化方向转变。此外，hERGW410R通道对hERGWT通道产生显性抑制作用。值得关注的是，我们发现hERG通道阻断剂E-4031可以部分挽救hERGW410R通道的功能。通过Luo-Rudy模型，我们发现hERGW410R通道明显延长了心室肌细胞动作电位的复极化。总体而言，W410R突变降低了hERG通道的电流，这归因于突变通道的膜表达减少。hERGW410R通道电流的减少导致心肌细胞复极化过程延长，这可能诱导了LQT2的发病。此外，E-4031可以部分挽救hERGW410R通道活性。我们的研究确定了一个新的功能缺失突变——KCNH2-W410R，该突变可能为LQT2的临床干预提供新的思路。