Cardiac KCNE2 (MIRP1) Regulation by Estrogen and Cardiac Stress

描述(申请人提供)：KCNE2是一个单一的跨膜调节亚基，在异源系统中可以调节各种K通道成孔亚基，包括Kv4.2和Kv4.3，这是心脏快速瞬时外向钾电流(Ito，f)的主要成分。突显其临床相关性的是，KCNE2遗传突变与人类心律失常的发生和长QT综合征有关。最近，Abbott博士在一次私人交流中告诉我，KCNE2基因敲除(KCNE2-/-)小鼠表现出Ito，f电流的显著减少和药物诱导的心律失常的倾向。此外，我们最近发现，KCNE2通过直接的基因组机制被雌激素(E2)激活，雌激素是一种众所周知的类固醇激素，在几个物种中具有心脏保护特性。特别是，在小鼠中，E2治疗改善了对经主动脉缩窄(TAC)引起的压力超负荷的肥大反应。因此，我决定研究雌激素诱导的KCNE2重塑及其亚细胞定位是否与Ito，f的变化和心脏应激的挽救有关。初步数据表明：在非应激动物中，高E2水平的心脏KCNE2转录水平显著上调。值得注意的是，E_2有利于KCNE_2在T管中的定位，这有利于其与T管Kvα亚单位的结合；而在TAC动物中，E_2可显著挽救心力衰竭(HF)，恢复射血分数和心脏兴奋性，并使KCNE_2转录上调和蛋白重新分布到T管膜。因此，一般的假说假设：心脏E2水平可能上调KCNE2的转录和蛋白水平，靶向于有利于KCNE2与Kv4.3/Kv4.2通道相互作用的T小管，从而增强Ito，f电流。为了验证整个假设，将使用包括分子生物学、生物化学、电生理学和蛋白质组学在内的多学科方法，并结合高分辨率共聚焦显微镜。其具体目的是：1)在正常动物中确定雌激素诱导的Ito、f和Ik表达改变的机制(S)、缓慢和功能后果：Kv4.3/4.2和Kv1.5及其辅助亚基(KCNE_2和KChIP2)的作用；以及(2)确定雌激素诱导的挽救TAC所致心力衰竭的机制(S)。这些研究将提供有关KCNE2和E2在心脏保护和心脏兴奋性中作用的新信息，并为理解与性别有关的室性心律失常倾向差异提供分子机制。公共卫生相关性：与男性相比，女性在育龄期间心脏病的发病率较低，这支持女性激素(雌激素)可能具有保护作用的观点。然而，雌激素的作用和涉及的机制却知之甚少。这个项目的目标是深入了解雌激素如何通过调节一种基因的活性来诱导心脏保护，该基因受到雌激素的精确调节，并改变心脏的兴奋性。

DESCRIPTION (provided by applicant): KCNE2 is a single transmembrane modulatory ¿ subunit that in heterologous systems can modulate a variety of K channel pore-forming 1 subunits including Kv4.2 and Kv4.3, major components of fast transient outward K+ currents (Ito,f) in the heart. Highlighting its clinical relevance, KCNE2 inherited mutations are associated with human cardiac arrhythmogenesis and long QT syndrome. Recently Dr. Abbott in a personal communication informed me that KCNE2 knockout (KCNE2-/-) mice display a major reduction in Ito,f currents and a propensity to drug induced arrhythmogenesis. Further, we recently found that KCNE2, via a direct genomic mechanism, is activated by estrogen (E2), a well-known steroid hormone with cardioprotective properties in several species. In particular, in mouse, E2 treatment ameliorates the hypertrophic response to pressure overload by transaortic constriction (TAC). Thus, I decided to investigate whether E2- induced KCNE2 remodeling including its subcellular localization correlates with Ito,f changes and rescue from cardiac stress. Preliminary data indicates that: In non-stressed animals with high E2 cardiac KCNE2 transcript levels are dramatically upregulated. Strikingly, E2 favored KCNE2 localization in the T-tubules which should facilitate its association with T-tubular Kv a-subunits; while in TAC animals, E2 administration exerted a dramatic rescue from heart failure (HF) restoring ejection fraction and cardiac excitability together with KCNE2 transcript upregulation and protein redistribution to the T-tubular membrane. Thus, the general hypothesis postulates that: Heart E2 levels may upregulate KCNE2 transcript and protein levels, with targeting to the T-tubules favoring KCNE2 interaction with Kv4.3/Kv4.2 channels potentiating Ito,f currents. To test the overall hypothesis, a multidisciplinary approach will be used including molecular biology, biochemistry, electrophysiology and proteomics in conjunction with high-resolution confocal microscopy. The Specific Aims are 1) To determine in normal animals the mechanism(s) of estrogen-induced changes on the expression of Ito,f and IK,slow and functional consequences: role of Kv4.3/4.2 and Kv1.5 and auxiliary subunits (KCNE2 and KChIP2); and 2) To determine the mechanism(s) of estrogen-induced rescue of TAC induced heart failure. These studies will provide new information on KCNE2 and E2 roles in cardioprotection and cardiac excitability and provide molecular mechanisms to understand gender related differences in propensity to ventricular arrhythmias. PUBLIC HEALTH RELEVANCE: Females during the reproductive age have low incidence to heart disease when compared with males supporting the view that female hormones (estrogen) may have a protective action. However, the role of estrogen and the mechanisms involved are little understood. The goal of this project is to gain insight in how estrogen may induce cardioprotection by regulating the activity of a gene that is exquisitely modulated by estrogen and that modifies cardiac excitability.