Mechanochemistry of myosin mutations that cause cardiomyopathy

导致心肌病的肌球蛋白突变的机械化学

• 批准号: 10230396
• 负责人: YALE E GOLDMAN
• 金额: $ 48.72万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/10230396
• 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; Pharmaceutical Preparations; Production; Proteins; Regulation; Research; Sampling; Sarcomeres; Signal Transduction; Symptoms; System; Techniques; Testing; Thin Filament; Thinness; Time; Tissues; Ventricular; arm; base; biophysical techniques; cell motility; experimental study; gain of function; interstitial; loss of function; loss of function mutation; mutant; nanomachine; optical traps; preservation; prevent; single molecule; sudden cardiac death; therapy design

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.

项目摘要 肥厚性心肌病（HCM）是一种普遍的遗传性心脏病，每300-500人中影响1人。 该疾病的特征是左心室肥大，心肌细胞混乱和间质纤维化的特征 经常保留或增强收缩功能，导致舒张功能受损。扩张 心肌病（DCM）的出现相似，其特征是一个或两个心室壁变薄 产生不足的收缩功能和减少的射血分数，心脏失败的标志。遗传 肉瘤蛋白中的突变已被鉴定为与HCM和DCM相关，与-的突变有关 心脏肌球蛋白（MYH7）强烈牵涉到两种情况的驱动因素。与肌球蛋白有关的广泛引用的模型 HCM源于肌球蛋白突变引起的疾病提案的功能，从而增强活性产生 高收缩肌细胞，而DCM是由功能丧失突变引起的，这些突变会减少活性产生 低收缩率。提议收缩异常是由于影响ATPase的MYH7基因突变 活动，速度，力产生，运动域的数量或可用性以及细丝激活。这些 分子变化最终以影响组织结构的方式影响功率输出 电生理信号传导和心脏性能。新兴研究提供了不 清楚地适合盛行的模型。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，重要的是确定蛋白质序列的变化如何导致变化 在分子和集合水平的活性。我们选择了特定的HCM和DCM突变 预计会影响肌球蛋白电动机的机械化学。我们的目标是确定这些效果 肌球蛋白活性的突变，并测试HCM功能获得和DCM功能丧失范式是否 持有。我们将利用生化和生物物理方法来评估突变对 单电机和受管制的细丝组件。 AIM 1将确定生化和机械效应 人β-心肌球蛋白中的关键HCM和DCM突变的。我们将测量合奏动力学和运动能力 使用生化和滑行测定。统一力，尺寸和依赖力动力学步骤的变化 将使用单分子光捕获来测量。 AIM 2将确定HCM/DCM突变的效果 在异质肌球蛋白组件和细丝激活上。我们将研究调节的影响 调节系统中的单分子，我们将使用DNA折纸构建肌球蛋白纳米机器 模仿心肌。我们将使用受调节的细丝和包含定义比率的肌膜 WT和突变肌球蛋白。我们期望我们的方法将使人们对 HCM和DCM的基础病因。