遗传性隐匿性1型长QT综合征分子机制研究

The inherited long QT syndrome (LQTS) is a serious heart disease, and the leading cause of cardiac death in children and adolescents. Among the LQTS patients, a large proportion are in a silent state without any clinical sympotoms, however, triggered frequently by sympathetic stimulation (such as physical or emotional stress). Yet, the underlying mechanisms remain unclear. LQTS type 1 (LQT1), caused by the mutations of KCNQ1 that is the α-subunit of cardiac IKs potassium channel, accounts for the majority of genotyped patients. Since most LQT1 patients have experienced cardiac events induced by sympathetic stimulation. We therefore speculate that the sympathetic stimulation, leading to the activation of protein kinase A (PKA), cannot normally cause the phosphorylation of the cardiac IKs potassium channel due to KCNQ1 mutations. Ulteriorly, the increase of outward IKs currents is attenuated during the plateau phase of cardiac action potential, which results in the excessive ventricular action potential duration and the QT interval prolongation on electrocardiogram. In the present study, the KCNQ1 mutation is constructed by methods such as heterologous transgene expression and PCR-based site-directed mutagensis, and transfected together with KCNE1 (the β-subunit of IKs) into mammalian cells. And then both functions and protein expressions of IKs channels are analyzed by using patch-clamp technique and immunocytochemistry. The aim of present study is to uncover the pathogenesis of silent LQT1 at the molecular level, so as to provide scientific evidence for both prevention and cure of the LQTS.

遗传性长QT综合征(LQTS)是一种严重危害人类健康的心脏病，为儿童和青少年心源性猝死的首要原因。LQTS中很大一部分处于无任何临床症状的"隐匿"状态，但交感神经兴奋时（如游泳、跑步运动或情绪激动）易发病，然而发生机制至今不明。占多数LQTS的1型LQT1是由心脏IKs钾离子通道的α亚基KCNQ1基因变异所致，患者大都经历过交感神经激动后发生的心脏意外。我们推测交感神经激动后，蛋白激酶A激活，由于KCNQ1基因变异，致使心脏IKs通道磷酸化异常，动作电位平台期外向IKs电流不能正常增加，从而导致动作电位时程和心电图QT间隔延长。本课题利用转基因和碱基定点变异等方法，构建KCNQ1变异后将其与IKs通道的β亚基KCNE1一起表达到哺乳动物细胞，再采用膜片钳和免疫细胞化学方法对IKs通道的功能和蛋白表达进行分析，以期从分子水平上揭示隐匿性LQT1的发病机理，为LQTS的防治提供提供科学依据。

中文摘要： .项目背景：遗传性长QT综合征(LQTS)是一种严重危害人类健康的心脏病。分子遗传学研究发现，遗传性LQTS是由编码心脏离子通道及其调控蛋白的基因突变所致。基因分析证实的LQT患者中，部分突变基因携带者处在隐匿状态，无任何临床异常症状，静态心电图ECG正常，但可因运动或体力劳作而突发晕厥、心搏停止，甚至是心脏猝死。然而，有关隐匿性LQTS的发病机制目前尚不清楚。..研究内容：利用膜片钳和免疫细胞化学等方法观察了KCNQ1基因突变R174C、V254M、R519H和T587M等对钾离子通道的影响；还观察了SCN5A-N1774D对钠离子通道的影响，以期从分子学机制上揭示隐匿性LQTS的发病机制。..重要结果：①发现杂合状态下四种KCNQ1突变（R174C、V254M、R519H和T587M）对IKs钾离子通道均产生单倍剂量（haploinsufficiency）不足式的影响，杂合状态下四种突变对离子通道门控动力学参数未产生显著影响。②发现V254M突变对KCNQ1通道蛋白的表达无明显影响，在细胞膜和细胞内均有大量表达。R174C、R519H和RT87M单纯突变通道蛋白在细胞膜上鲜有表达，主要分布在细胞内；但突变（R174C、R519H或T587M）与野生型的杂合通道蛋白WT+R174C、WT+R519H和WT+T587M在细胞膜和细胞内均有表达。提示，V254M突变不影响KCNQ1通道蛋白的转运，R174C、R519H和T587M突变虽然对通道蛋白的转运产生了抑制，但杂合状态下通道蛋白转运却无明显异常。③进一步观察R174C、R519H或T587M三种杂合突变通道对交感神经激动剂异丙肾上腺素的反应，发现KCNQ1野生（WT）与KCNQ1-R174C、KCNQ1-V254M和KCNQ1-R519H杂合突变通道对异丙肾上腺素的反应明显减弱。④发现KCNQ1与hERG之间存在相互作用。⑤发现由SCN5A-N1774D突变构成的INa钠离子通道存在较大晚Na电流，而交感神经激动剂异丙肾上腺素和PKA激动剂（Forskolin + IBMX）均可使晚Na电流继续增加。..关键数据及科学意义：KCNQ1突变造成IKs通道对交感神经的反应降低，致使IKs电流不能在交感神经激动时正常增加，为隐匿性LQT1的主要病发机制。心脏离子通道相互作用可能也是引发隐匿性LQT1的病发