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 肥厚型心肌病(HCM)是一种常见的遗传性心脏病，每300-500人中就有1人发病。 本病以左室肥厚、心肌细胞紊乱和间质纤维化为特征。 导致舒张期功能受损，常伴有收缩功能的保留或增强。扩张的 心肌病(DCM)也有类似的发生，其特征是一侧或两侧室壁变薄 产生收缩功能不足和射血分数减少，这是心力衰竭的特征。遗传 肌节蛋白的突变已被证实与肥厚性心肌病和扩张性心肌病有关，-1基因的突变。 心肌肌球蛋白(MYH7)强烈地与这两种情况的驱动因素有关。一个被广泛引用的肌球蛋白模型 疾病功能提示，肥厚性肌球蛋白起源于肌球蛋白突变，这种突变增强了活性。 过度收缩的心肌细胞，而扩张性心肌病源于功能丧失的突变，这种突变降低了活性。 低收缩能力。收缩异常被认为是由于影响ATPase的MYH7基因突变所致 活动度、速度、作用力、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， 电生理信号和心脏功能。新兴的研究提供了一些例子，但并不是 显然符合流行的模式。例如，已经描述了活性降低的hcm突变。 在分子分析和分离的肌原纤维水平上。来描述肌球蛋白的作用机制 突变导致HCM和DCM，重要的是确定蛋白质序列的变化是如何导致变化的 在分子和系综水平上的活性。我们选择了特定的HCM和DCM突变 预计会影响肌球蛋白马达的机械力化学。我们的目标是确定这些措施的效果 突变对肌球蛋白活性的影响，并测试HCM功能获得和DCM功能丧失范例 等一下。我们将利用生化和生物物理方法来评估突变对酶活性的影响。 单电机和可调节灯丝组件。目标1将确定生化和机械效应 人类β-心肌肌球蛋白中关键的hcm和dcm突变。我们将测量系综的动力学和运动性 使用生化和滑行分析。单位力、步长和与力相关的动力学步数的变化 将使用单分子光学捕获法进行测量。目标2将确定HCM/DCM突变的影响 关于肌球蛋白的异质性组装和细丝激活。我们将考察监管的效果 在一个受调控的系统中的单分子，我们将使用DNA折纸构建肌球蛋白纳米机器 它模仿了心肌。我们将使用受监管的细丝和肌丝，其中包含 WT和突变型肌球蛋白。我们希望，我们的方法将导致对 肥厚型心肌病和扩张型心肌病的潜在病因。