基于CreER大鼠构建心脏Kcnh2基因敲除的LQT模型及在体研究

LQT syndrome is the most common phenotype of the arrhythmia.The Potassium ion channel coded by Kcnh2 gene plays the key role in the repolarization of action potential in cardiomyocyte and the mutation of Kcnh2 can lead to LQT syndrome, but the involved mechanism still is poorly understood. The Kcnh2 knock-out mouse model either failed to show the phenotype of LQT syndrome or leaded to the embryo lethal, resulting in a bottleneck for understanding the molecular mechanism underlying arrhythmia. We recently knock out a gene in rat in successful (Nature，2010）, suggesting it is possible to perform genetic manipulation on rat. In the project, we will first generate a CreER rat for cardiomyocyte specific, and then a conditional Kcnh2-knockout rat by homologous recombination, respectively. We plan to knock-out Kcnh2 gene in the young, middle-age and elder rat model based on expression of CreER induced by tamoxifen, and then to observe the expression and distribution of Erg protein on cell membrane, and the organizational structure of the heart combined with molecular and cell biology、 biochemical and histological techniques as well. We will further analyze the features of both potassium current and action potential in primary myocardial cells and embryonic stem cells from the Kcnh-/- model by patch clamp. We also detect ECG and the heart function by electrophysiology and echocardiography, so that we try to supply experiment references for the molecular mechanism of LQT syndrome led by Kcnh2 mutation.

LQT综合征是常见的心律失常之一。Kcnh2基因编码的钾离子通道在心肌细胞动作电位复极中起重要作用，其突变导致的功能异常是诱发LQT综合征的分子基础，但相关致病机制仍不清楚。以往利用小鼠构建的Kcnh2敲除模型不能获得典型的LQT表型或胚胎致死。大鼠是探索人类心脏疾病的理想模型，我们前期工作（Nature,2010）为遗传操作大鼠基因打下了基础。本研究拟用同源重组技术分别构建心肌细胞特异CreER工具大鼠和条件打靶Kcnh2大鼠，通过诱导分别对青年、中年和老年大鼠Kcnh2基因进行心肌细胞特异敲除，进一步结合分子生物、细胞、生化和组织学技术观察模型大鼠心肌细胞Kcnh2蛋白表达和细胞膜分布以及心脏组织结构状况；利用电生理和超声技术分别检测分析原代心肌细胞与ES细胞分化心肌细胞的钾电流和动作电位特点以及心电变化规律和心脏功能状况，为阐明Kcnh2突变诱导的心律失常发病机制提供实验依据。

Kcnh2基因编码Kv11.1钾通道，对维持心肌细胞中快速延迟整流K+电流的通透是必须的。人类的KCNH2突变可导致长QT综合征。但是，迄今其基本机制仍然不清楚。在本项目中，我们使用基于大鼠胚胎干细胞的基因靶向技术制备了Kcnh2突变大鼠模型。首先，我们设计并构建系列相关载体，如Myh7-CreER载体等，并进一步制备了CAG-Flpe转基因工具大鼠和CAG-Cre转基因大鼠。此外，我们还构建了CAG-Flpe转基因工具大鼠作为验证模型。最后，我们完成了Myh7-CreER敲入大鼠和Kcnh2-cKO嵌合体大鼠的制备。基于Kcnh2基因敲除模型，我们分别建立了Kcnh2-/- 和Kcnh2+/-胚胎干细胞株，发现kcnh2纯合子缺失抑制定向心肌细胞的分化。进一步发现Kcnh2+/-大鼠的心电图出现延长的QTc，而新生大鼠中的心肌细胞全电流正常。由于Kcnh2-/-胚胎心脏发育缺陷，大鼠通常在胚胎第11.5天死亡，并在纯合子突变体胚胎中触发了细胞凋亡，这归因于未能形成KCNH2与ITGB1的复合物，而此复合体对于抑制FOXO3A而阻止凋亡过程至关重要。因此，KCNH2在维持心律和早期胚胎发育中起着双重作用。