Reversal of Hypertrophic Cardiomyopathy via Conditional Ablation of Cardiac Myosin Light Chain Kinase

项目摘要 肥厚型心肌病（HCM）是年轻人心脏性猝死的主要原因， 大约500个个体中有1个，其特征在于舒张功能障碍、纤维化组织形成、受损 收缩特性，最明显的是左心室肥大和肌丝Ca 2+敏感性增加。 迄今为止，HCM尚无有效的非侵入性治疗方法。肌节蛋白的突变-包括 心肌肌钙蛋白C（cTnC）是心肌收缩过程中的Ca ~（2+）感受器，可能是心肌梗死的致病因子。的 然而，对疾病机制以及各种突变如何导致肌病的了解甚少。更 具体而言，已在HCM患者中鉴定出cTnC中的Ala 8Val（A8 V）突变， 独立的先证者，并导致我们开发了基于其致病性的基因敲入小鼠模型。A8V 人类和小鼠的突变导致左心室肥大和过度收缩，心房扩大， 增加肌丝Ca 2+敏感性（后者仅在小鼠中测试）。最近，心肌肌球蛋白轻链激酶 显示cMLCK（cMLCK）敲除小鼠表现出降低的射血分数。这是由于减少 调节性肌球蛋白轻链磷酸化，已知其降低肌丝Ca 2+敏感性。 因此，我们假设cTnC-A8 V（HCM表型）小鼠的条件性cMLCK敲除可 使心脏功能向野生型正常化。我们相信这将改善心脏形态，血液动力学， 和蛋白表达水平，通过ECHO、压力-容积环、组织病理学、心脏至胫骨测量 长度比、心肌细胞Ca 2+和颤搐瞬变以及Ca 2+处理蛋白的蛋白质印迹。此外，本发明还提供了一种方法， 我们将使用先进的生物信息学工具，如蛋白质组学和RNA测序，以量化整体变化 在这些小鼠模型中。迄今为止，心肌肌球蛋白轻链激酶被表征为专用激酶， 使其成为有希望的治疗靶点。因此，拟议的工作可以为新颖，有针对性的心脏奠定基础 HCM的疾病治疗。

Project Summary Hypertrophic cardiomyopathy (HCM), as the leading cause of sudden cardiac death in young adults, affects approximately 1 in 500 individuals and is characterized by diastolic dysfunction, fibrotic tissue formation, impaired contractile properties and, most evidently, left ventricle hypertrophy and increased myofilament Ca2+ sensitivity. To date, there are no effective non-invasive therapies for HCM. Mutations in sarcomeric proteins—including cardiac troponin C (cTnC), the Ca2+ sensor in contraction—seem to be the causative agents in the disease. The disease mechanisms and how the various mutations lead to myopathy, however, are poorly understood. More specifically, the Ala8Val (A8V) mutation in cTnC has been identified among HCM patients and within three independent probands and led us to develop the knock-in mouse model based on its pathogenicity. The A8V mutation in humans and mice causes left ventricular hypertrophy and hypercontractility, atrial enlargement, and increased myofilament Ca2+ sensitivity (the latter only tested in mice). Recently, cardiac myosin light chain kinase (cMLCK) knock-out mice were shown to exhibit reduced ejection fraction. This is attributed to the reduction in regulatory myosin light chain phosphorylation, which is known to decrease myofilament Ca2+ sensitivity. Consequently, we hypothesize that a conditional cMLCK knock-out of a cTnC-A8V (HCM phenotype) mouse can normalize cardiac function toward wild-type. We believe this will improve heart morphology, hemodynamics, and protein expression levels, as measured via ECHO, pressure-volume loops, histopathology, heart to tibia length ratios, cardiomyocyte Ca2+ and twitch transients, and western blots for Ca2+ handling proteins. In addition, we will use advanced bioinformatics tools such as proteomics and RNA sequencing to quantify overall changes in these mouse models. Cardiac myosin light chain kinase has thus far been characterized as a dedicated kinase, making it a promising therapeutic target. As such, the proposed work can set the stage for novel, targeted heart disease therapy for HCM.