Molecular Characterization of Cardiomyopathy Mutations in Human Cardiac Myosin

人心肌肌球蛋白心肌病突变的分子特征

• 批准号: 9058602
• 负责人: Leslie Anne Leinwand
• 金额: $ 43.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-20 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058602
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actinin; Actins; Affect; Binding; Biochemical; Biological Assay; Biomechanics; Cardiac Myosins; Cardiomyopathies; Clinical; Data; Defect; Dependence; Dilated Cardiomyopathy; Disease; Elements; Genetic; Head; Health; Heart; Heart Diseases; Human; Hypertrophic Cardiomyopathy; In Vitro; Kinetics; Lasers; Lead; Link; Location; Measurement; Measures; Molecular; Motor; Mutation; Myosin ATPase; Myosin Heavy Chains; Nucleotides; Outcome; Process; Production; Property; Proteins; Recombinants; Stroke; Sudden Death; Suggestion; System; Testing; Thermodynamics; Time; United States; Work; cell motility; clinical phenotype; disease-causing mutation; familial dilated cardiomyopathy; mortality; mutant; novel; single molecule; stroke recovery

DESCRIPTION (provided by applicant): Although more than 200 mutations located in the motor domain of the human ß-cardiac myosin heavy chain (MyHC) cause hypertrophic or dilated cardiomyopathy (HCM or DCM), their molecular effects on the myosin molecule remain elusive. Most studies to determine the functional impact of these mutations on myosin have used non-human myosins and most have not used cardiac myosins. Leinwand and colleagues have recently developed a system for producing recombinant human cardiac myosin motors and we will exploit this system for the first systematic study of the effects HCM and DCM mutations in human ß-cardiac myosin. Since the majority of mutations that cause disease are located in the myosin motor, we will focus our studies there. We selected for study 10 mutations (5 HCM and 5 DCM), both because of their largely severe clinical phenotypes and because of their locations within the myosin motor domain. We propose to analyze the biochemical properties of the mutations in solution and their biomechanical properties using in vitro motility and laser trap single molecule assays. We hypothesize that mutations that lead to increased force production lead to HCM while those that lead to decreased force production lead to DCM, consistent with recent suggestions. Mechanisms that lead to decreased or increased force production by myosin can be varied. The inherent force-producing capability of the motor, for example, could be increased or decreased by mutations that change the spring constant of the elastic element of the motor. On the other hand, the force- producing capability could be changed in either direction by changes in the duty ratio of the myosin (the fraction of the ATPase cycle that the head is strongly bound to actin). An increase in the duty ratio, for example, would result in more heads bound to actin in a force-producing state at any moment, leading to an overall increase in force. Furthermore, the duty ratio can be changed in more than one way. For example, the ADP release rate (which determines the strongly-bound state time), the ATP hydrolysis step (which defines the recovery stroke time) or the Pi release (the rate of entry into the strongly bound state) can be affected and result in a change in the duty ratio. Thus, it is essential to begin to characterize different mutations, hypothesized to affect different mechanistic aspects of the motor. These studies will begin the process of linking the fundamental changes in motor function to the eventual different clinical outcomes. Therefore, we will determine the biochemical and biomechanical parameters of wild type human α- and ß-cardiac myosin and mutant ß-myosin motors with respect to their steady-state and transient kinetic properties, velocities at ~zero load and under loaded conditions, their duty ratios, their stroke sizes, and the maximum force they produce upon interacting with actin.

描述（由适用提供）：尽管在人ß-肌球蛋白重链（MYHC）的运动结构域中有200多个突变引起肥厚或扩张的心肌病（HCM或DCM），但它们对肌球蛋白分子的分子作用仍然难以捉摸。确定这些突变对肌球蛋白的功能影响的大多数研究都使用了非人类肌球蛋白，并且大多数没有使用心脏肌球蛋白。 Leinwand及其同事最近开发了一种用于生产重组人心脏肌球蛋白电动机的系统，我们将利用该系统进行对人ß-心脏肌球蛋白中HCM和DCM突变的首次系统研究。由于大多数引起疾病的突变位于肌球蛋白运动中，因此我们将在那里集中研究。我们选择了研究10个突变（5 HCM和5 dCM），这都是由于它们在很大的临床表型中以及它们在肌球蛋白运动结构域内的位置。我们建议使用体外运动和激光陷阱单分子测定法分析溶液中突变及其生物力学特性的生化特性。我们假设导致力增加的突变导致HCM，而那些导致力产生降低的突变导致DCM，这与最近的建议一致。导致肌球蛋白产生降低或增加的力的机制可以变化。例如，通过改变电动机弹性元件的弹簧常数的突变，可以增加电动机的继承力产生能力。另一方面，产生力的能力可以通过肌球蛋白的占空比变化（ATPase循环的比例与肌动蛋白密切绑定的ATPase循环的比例）来改变任一方向。例如，占空比的增加将导致更多的头部抑制肌动蛋白在产生力量状态，从而导致总体增加的力量。占空比可以以多种方式更改。例如，ADP释放速率（决定了强大的状态时间），ATP水解步骤（定义恢复笔划时间）或PI释放（进入强限制状态的率）可能会受到影响，并会影响占空比比率的变化。这是必不可少的，必须开始表征不同的突变，假设会影响电动机的不同机械方面。这些研究将开始将运动功能的基本变化与事件不同的临床结果联系起来的过程。因此，我们将确定野生型人类α-和β-甲肌球蛋白和突变体的生物化学和生物力学参数相对于它们的稳态和短暂性动力学，〜零负载和载荷条件下的速度和载荷条件下的速度，在载荷条件下，其职责率，其效率，以及最大的力量，它们在互动中产生了互动。