Myosin ELC, a novel therapeutic target for FHC

描述(申请人提供)：肌球蛋白基本轻链(ELC)是肌球蛋白交叉桥的结构成分，但对其功能或心脏特异性N末端ELC延伸对肌球蛋白与肌动蛋白相互作用、力量发展和心脏性能的意义知之甚少。最近发现的几种错义突变可导致家族性肥厚性心肌病(FHC)，这是一种遗传性疾病，表现为心室增大、肌丝紊乱和心脏性猝死(SCD)，突显了ELC的功能重要性。这一建议将阐明ELC介导的FHC的机制以及N端ELC-肌动蛋白直接相互作用在正常和FHC心脏中的作用。特异性目的1：确定ELC突变诱导FHC发生的机制。我们推测，ELC突变可能通过影响ELC与MHC形成稳定结构的能力而导致FHC，从而改变肌球蛋白在收缩周期和力的发展过程中的力学(刚性、力)和/或动力学性质。这项研究的重点是两个FHC突变，位于两个不同的ELC结构域，并表现出不同的临床表型。N端A57G突变导致典型的不对称性肥厚和SCD，C端E143K突变表现为中腔肥厚、收缩受阻和生理受限。我们建议确定：(1a)FHC突变对ELC与2-MHC结合、肌球蛋白杠杆臂硬度和肌球蛋白跨桥动力学的影响。(1B)去皮和完整乳头肌纤维中突变心肌的收缩特性。(1)通过组织病理学、超声心动图、有创血流动力学和MRI观察转基因突变小鼠心脏的年龄相关性形态和功能。特异性目的2：探讨ELC的N末端调控肌球蛋白力量生成能力和心肌收缩的机制。我们假设ELC的N-末端与肌动蛋白-原肌球蛋白-肌钙蛋白细丝相互作用，促进强烈的跨桥形成，从而作为一种机制来调节肌动蛋白-肌球蛋白相互作用和肌丝协作性，从而调节收缩力量。利用表达N末端截短的ELC的TG-43小鼠的心肌标本和表达全长ELC野生型的TG-WT，我们将研究：(2A)ELC的N末端对于肌球蛋白力量生成能力的重要性，这是在分子和肌肉纤维水平上确定的。(2B)N端ELC延伸对肌球蛋白跨桥动力学的影响。(2C)N端ELC依赖于心肌肥厚发展的机制。这些研究将有助于确定ELC和心脏N端特异性ELC延伸在调节健康和FHC疾病心脏的收缩力量中的作用(S)。 公共卫生相关性：这项建议的目标是了解心肌肌球蛋白基本轻链(ELC)调节肌球蛋白与肌动蛋白的相互作用、力量产生和心肌收缩的机制。此外，该提案还将揭示肌球蛋白ELC基因突变导致家族性肥厚型心肌病(FHC)的机制。我们开发的转基因动物模型以及将体外方法与体内心脏功能的生理评估相结合，将使我们能够解决有关ELC介导的心脏生理学和FHC连锁ELC突变的影响的基本问题。这一提议的成功实施可能为开发旨在改善ELC引起的心脏病的新的药理策略奠定基础。

DESCRIPTION (provided by applicant): The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but little is known about its function or the significance of the cardiac specific N-terminal ELC extension on the interaction of myosin with actin, development of force and heart performance. The functional importance of ELC is highlighted by the recent identification of several missense mutations shown to cause Familial Hypertrophic Cardiomyopathy (FHC), a genetic disorder manifested by ventricular enlargement, myofilament disarray and sudden cardiac death (SCD). This proposal will elucidate the mechanisms of ELC-mediated FHC and the role of the direct N-terminal ELC-actin interaction in the normal and FHC heart. SPECIFIC AIM 1: DETERMINE THE MECHANISM FOR THE ELC-MUTATION INDUCED DEVELOPMENT OF FHC. We hypothesize that ELC mutations may lead to FHC by affecting the ability of the ELC to form stable structures with the MHC, thus altering the mechanical (stiffness, force) and/or kinetic properties of the myosin cross-bridge during the contractile cycle and development of force. This study is focused on two FHC- mutations localized in two different ELC domains and demonstrating different clinical phenotypes. The N- terminal A57G mutation was shown to cause a classic asymmetric hypertrophy and SCD, and the C-terminal E143K mutation, which presented with mid-cavity hypertrophy, obstruction in systole and restrictive physiology. We propose to determine: (1A) The effect of FHC mutations on the binding of ELC to the 2-MHC, myosin lever arm stiffness and myosin cross-bridge kinetics. (1B) The contractile properties of the mutated myocardium in skinned and intact papillary muscle fibers. (1C) The age-dependent morphology and function of mutated transgenic mouse hearts by histopathology, echocardiography, invasive hemodynamics and MRI. SPECIFIC AIM 2: EXPLORE THE MECHANISMS BY WHICH THE N-TERMINUS OF ELC REGULATES MYOSIN FORCE GENERATING CAPABILITY AND CARDIAC MUSCLE CONTRACTION. We hypothesize that the N- terminus of ELC interacts with the actin-tropomyosin-troponin filaments to promote strong cross-bridge formation~ thus it serves as a mechanism to modulate the actin-myosin interaction and myofilament cooperativity, thereby regulating contractile force. Using cardiac muscle preparations from Tg- 43 mice expressing the N-terminal truncated ELC and Tg-WT expressing the full length ELC wild-type, we will study: (2A) the importance of the N-terminus of ELC for myosin force generating capability determined at the molecular and muscle fiber levels. (2B) the effect of the N-terminal ELC extension on myosin cross-bridge kinetics.(2C) The mechanism of the N-terminus ELC-dependent development of cardiac hypertrophy. These studies will help to determine the role(s) of the ELC and the N-terminal cardiac specific ELC extension in the regulation of contractile force in healthy and FHC-diseased hearts. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand the mechanism by which the cardiac myosin essential light chains (ELC) regulate the interaction of myosin with actin, force generation and cardiac muscle contraction. In addition, the proposal will unravel the mechanisms, by which genetic mutations in myosin ELC lead to Familial Hypertrophic Cardiomyopathy (FHC). Our developed transgenic animal models and the integration of in vitro approaches with a physiological assessment of cardiac function in vivo will enable us to address the fundamental questions regarding ELC-mediated physiology of the heart and the impact of FHC-linked ELC mutations. Successful execution of this proposal may lay a foundation for the development of novel pharmacological strategies aimed at improving ELC - induced cardiac disease.