Pathogenesis of hERG Mutations in Human Long QT Syndrome

描述（由申请方提供）：长QT综合征是一种与心脏复极延迟和心电图QT间期延长相关的疾病，可导致室性心律失常和猝死。7号染色体连锁遗传性长QT综合征（LQT 2）是由人类ether-a-go-go相关基因（hERG）突变引起的。hERG编码心脏中快速激活延迟整流钾通道的成孔亚基。在LQT 2患者中已发现超过200种hERG突变。这些LQT 2突变导致hERG通道功能降低，从而导致动作电位延长和心律失常。以往的研究大多集中在突变蛋白和通道功能的分析上。超过30%的LQT 2突变是无义、移码或剪接位点突变，可能影响hERG mRNA的剪接和稳定性。然而，LQT 2突变对hERG mRNA剪接和稳定性的影响在很大程度上尚未探索。此外，大多数以前的研究涉及在非洲爪蟾卵母细胞或哺乳动物细胞系中表达致病突变，但对LQT 2突变在心肌细胞中表达时如何发挥作用知之甚少。本申请的具体目的是：1）研究剪接位点突变引起的hERG mRNA异常剪接，2）研究携带提前终止密码子的LQT 2突变是否通过无义介导的mRNA衰变导致hERG mRNA转录水平下降，3）研究前体mRNA剪接、mRNA稳定性、蛋白质运输、亚细胞分布、和心肌细胞中表达的LQT 2突变体的药理学拯救。我们将使用分子生物学、生物化学和电生理学方法来研究HEK 293细胞以及新生和成年大鼠心肌细胞中表达的LQT 2突变。我们还将分析从携带LQT 2突变的患者淋巴细胞中分离的内源性表达hERG mRNA的剪接模式和稳定性。这些研究的结果将增加我们对LQT 2突变如何在mRNA和蛋白水平上导致hERG通道功能障碍的了解。阐明这些机制不仅将加强我们对人类长QT综合征中hERG突变的发病机制的理解，而且还将为开发长QT综合征的新治疗策略提供宝贵的信息。

DESCRIPTION (provided by applicant): Long QT syndrome is a disease associated with delayed cardiac repolarization and prolonged QT intervals on the electrocardiogram, which can lead to ventricular arrhythmias and sudden death. The chromosome 7- linked inherited long QT syndrome (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). hERG encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. More than 200 hERG mutations have been identified in patients with LQT2. These LQT2 mutations result in decreased hERG channel function, which leads to action potential prolongation and cardiac arrhythmias. Most of the previous studies were focused on the analysis of mutant proteins and channel function. More than 30% of LQT2 mutations are nonsense, frameshift, or splice site mutations, which may affect hERG mRNA splicing and stability. However, the effect of LQT2 mutations on hERG mRNA splicing and stability are largely unexplored. In addition, most of the previous studies involved expressing disease-causing mutations in Xenopus oocytes or mammalian cell lines, but little is known about how LQT2 mutations function when expressed in cardiac myocytes. The specific aims of this application are: 1) To study abnormal splicing of hERG mRNA caused by splice site mutations, 2) To study whether LQT2 mutations that carry premature termination codons cause a decrease in the level of hERG mRNA transcripts by nonsense mediated mRNA decay, and 3) To study pre-mRNA splicing, mRNA stability, protein trafficking, subcellular distribution, and pharmacological rescue of LQT2 mutants expressed in cardiac myocytes. We will use molecular biology, biochemical, and electrophysiological approaches to study LQT2 mutations expressed in HEK 293 cells and in neonatal and adult rat cardiac myocytes. We will also analyze the splicing patterns and stability of endogenously expressed hERG mRNA isolated from lymphocytes of patients carrying LQT2 mutations. The results from these studies will increase our knowledge of how LQT2 mutations lead to hERG channel dysfunction at both the mRNA and protein levels. Elucidating these mechanisms will not only strengthen our understanding of the pathogenesis of hERG mutations in human long QT syndrome, but will also provide invaluable information directed towards the development of new therapeutic strategies for long QT syndrome.