Mechanochemistry of myosin mutations that cause cardiomyopathy

导致心肌病的肌球蛋白突变的机械化学

基本信息

批准号：
10624860
负责人：
YALE E GOLDMAN
金额：
$ 53.06万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10624860
关键词：
ATP phosphohydrolase Actins Affect Amino Acid Sequence Architecture Biochemical Biological Assay Buffers Calcium Cardiac Cardiac Myocytes Cardiac Myosins Cardiomyopathies Contractile Proteins Contractile System DNA DNA Sequence Alteration Dependence Dilated Cardiomyopathy Disease EFRAC Electrophysiology (science)Etiology Fibrosis Filament Gene Mutation Gene Proteins Generations Genetic Goals Heart Heart Diseases Human Hypertrophic Cardiomyopathy Impairment In Vitro Kinetics Lead Left Ventricular Hypertrophy Measures Mechanics Microfilaments Microfluidics Modeling Molecular Motor Motor Activity Muscle Muscle Cells Mutate Mutation Myocardium Myofibrils Myopathy Myosin ATPase Nonmuscle Myosin Type IIA Outcome Output Performance Persons Pharmaceutical Preparations Production Proteins Regulation Research Sampling Sarcomeres Signal Transduction Symptoms System Techniques Testing Thin Filament Thinness Time Tissues Ventricular arm beta-Myosin biophysical techniques cell motility experimental study gain of function interstitial loss of function loss of function mutation mutant nanomachine optic trap optical traps preservation prevent single molecule sudden cardiac death therapy design

项目摘要

Project Summary Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiac disease affecting 1 in every 300-500 people. The disease is characterized by left ventricular hypertrophy, cardiomyocyte disarray, and interstitial fibrosis resulting in impaired diastolic function often with preserved or enhances systolic function. Dilated cardiomyopathy (DCM) has a similar occurrence and is characterized by thinning of one or both ventricular walls producing insufficient systolic function and diminished ejection fraction, hallmarks of a failing heart. Genetic mutations in sarcomere proteins have been identified to be associated with HCM and DCM, with mutations in - cardiac myosin (MYH7) strongly implicated as drivers of both conditions. A widely cited model relating myosin function to disease proposes that HCM arises from myosin mutations that enhance activity yielding hypercontractile myocytes, whereas DCM arises from loss-of-function mutations that diminish activity yielding hypo-contractility. Contractile abnormalities are proposed to be due to MYH7 gene mutations that affect ATPase activity, velocity, force production, the number or availability of motor domains, and thin filament activation. These molecular changes ultimately affect power output in a manner that impacts tissue architecture, electrophysiological signaling, and cardiac performance. Emerging research has provided examples that do not fit clearly into the prevailing model. For example, HCM mutations with decreased activity have been described in molecular assays and at the level of isolated myofibrils. To delineate the mechanisms by which myosin mutations lead to HCM and DCM, it is important to determine how changes in protein sequence lead to changes in activity at both the molecular and ensemble levels. We selected specific HCM and DCM mutations that are predicted to affect the mechanochemistry of the myosin motor. Our goal is to determine the effect of these mutations on myosin activity, and to test whether the HCM gain-of-function and DCM loss-of-function paradigm holds. We will utilize biochemical and biophysical approaches to assess the effect of mutations on the activity of single motors and regulated filament assemblies. Aim 1 will determine the biochemical and mechanical effects of key HCM and DCM mutations in human β-cardiac myosin. We will measure the ensemble kinetics and motility using biochemical and gliding assays. Changes in unitary forces, step-size, and force dependent kinetic steps will be measured using single molecule optical-trapping. Aim 2 will determine the effect of HCM/DCM mutations on heterogenous myosin assembles and thin filament activation. We will examine the effect the regulation of single molecules within a regulated system, and we will construct a myosin nanomachine using DNA origami that mimics cardiac muscle. We will use regulated thin filaments and myofilaments containing defined ratios of WT and mutant myosin. We expect that our approach will result in an unprecedented understanding of the underlying etiology of HCM and DCM.

项目概要肥厚型心肌病 (HCM) 是一种流行的遗传性心脏病，每 300-500 人中就有 1 人受影响。该疾病的特点是左心室肥厚、心肌细胞紊乱和间质纤维化导致舒张功能受损，但收缩功能得以保留或增强。心肌病 (DCM) 也有类似的情况，其特征是一侧或两侧心室壁变薄产生收缩功能不足和射血分数降低，这是心脏衰竭的标志。肌节蛋白突变已被确定与 HCM 和 DCM 相关，其中 - 心肌肌球蛋白 (MYH7) 与这两种疾病密切相关，是一种被广泛引用的与肌球蛋白相关的模型。疾病功能表明 HCM 源于肌球蛋白突变，增强了活性产生过度收缩的肌细胞，而 DCM 是由功能丧失突变引起的，该突变会减少活性产生收缩力异常被认为是由于影响 ATP 酶的 MYH7 基因突变所致。活动、速度、力的产生、运动域的数量或可用性以及细丝激活。分子变化最终会以影响组织结构的方式影响功率输出，电生理信号和心脏性能的新兴研究提供了一些例子。例如，已经描述了活性降低的 HCM 突变。在分子测定和分离的肌原纤维水平上描述肌球蛋白的机制。突变导致HCM和DCM，确定蛋白质序列的变化如何导致变化很重要我们选择了特定的 HCM 和 DCM 突变。预计会影响肌球蛋白运动的机械化学。我们的目标是确定这些的影响。肌球蛋白活性突变，并测试 HCM 功能获得和 DCM 功能丧失范式是否成立我们将利用生物化学和生物物理方法来评估突变对活性的影响。目标 1 将确定单电机和受控灯丝组件的生化和机械效应。我们将测量人类 β-心肌肌球蛋白中关键 HCM 和 DCM 突变的整体动力学和运动性。使用生化和滑动分析来改变单一力、步长和力依赖性动力学步骤。将使用单分子光学捕获进行测量，目标 2 将确定 HCM/DCM 突变的影响。我们将检查异质肌球蛋白组装和细丝激活的调节作用。调节系统内的单分子，我们将使用 DNA 折纸构建肌球蛋白纳米机器我们将使用含有规定比例的调节细丝和肌丝。我们期望我们的方法将对 WT 和突变肌球蛋白产生前所未有的理解。 HCM 和 DCM 的根本病因学。