Cardiac KCNE2 (MIRP1) Regulation by Estrogen and Cardiac Stress

描述（由申请人提供）：KCNE2是一种单一的跨膜调节亚基，在异种系统中可以调节多种K通道成孔亚基，包括Kv4.2和Kv4.3，它们是心脏快速瞬态外向K+电流（Ito,f）的主要成分。强调其临床相关性，KCNE2遗传突变与人类心律失常和长QT综合征有关。最近，Abbott博士在个人通信中告诉我，KCNE2基因敲除（KCNE2-/-）小鼠的Ito，f电流显著减少，并且倾向于药物引起的心律失常。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，在一些物种中具有心脏保护特性。特别是，在小鼠中，E2治疗改善了经主动脉收缩（TAC）引起的压力过载的肥厚反应。因此，我决定研究E2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恢复相关。初步数据表明：在非应激动物中，高E2心脏KCNE2转录水平显著上调。引人注目的是，E2倾向于KCNE2在t管中的定位，这应该有助于其与t管Kv a亚基的关联；而在TAC动物中，E2给药对心力衰竭（HF）有显著的拯救作用，恢复射血分数和心脏兴奋性，同时KCNE2转录上调和蛋白质重新分布到t管膜。因此，一般假设假设：心脏E2水平可能上调KCNE2转录物和蛋白水平，其目标是t小管，有利于KCNE2与Kv4.3/Kv4.2通道相互作用，增强Ito，f电流。为了验证整个假设，将使用多学科方法，包括分子生物学，生物化学，电生理学和蛋白质组学，并结合高分辨率共聚焦显微镜。具体目的是：1)在正常动物中确定雌激素诱导的Ito、f和IK表达变化的机制，以及Kv4.3/4.2和Kv1.5及其辅助亚基（KCNE2和KChIP2）的缓慢和功能性影响；2)探讨雌激素对TAC诱导心力衰竭的抢救作用机制。这些研究将为KCNE2和E2在心脏保护和心脏兴奋性中的作用提供新的信息，并为理解室性心律失常倾向的性别相关差异提供分子机制。公共卫生相关性：与男性相比，育龄女性患心脏病的几率较低，这支持了女性激素（雌激素）可能具有保护作用的观点。然而，人们对雌激素的作用及其机制知之甚少。该项目的目标是深入了解雌激素是如何通过调节一种基因的活性来诱导心脏保护的，这种基因是由雌激素精心调节的，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.