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: 8584984
• 负责人: Leslie Anne Leinwand
• 金额: $ 43.05万
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8584984
• 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; 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; Solutions; Stroke; Sudden Death; Suggestion; System; Testing; Thermodynamics; Time; United States; Work; cell motility; clinical phenotype; disease-causing mutation; mortality; mutant; novel; public health relevance; single molecule; stroke recovery

DESCRIPTION (provided by applicant): Although more than 200 mutations located in the motor domain of the human ??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 ??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 a- and b-cardiac myosin and mutant ? -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(由申请人提供)：尽管位于人类心肌肌球蛋白重链(MyHC)运动域的200多个突变导致肥厚型或扩张型心肌病(HCM或DCM)，但它们对肌球蛋白分子的分子作用仍然难以捉摸。为了确定这些突变对肌球蛋白的功能影响，大多数研究使用了非人类肌球蛋白，大多数没有使用心肌肌球蛋白。Leinwand和他的同事最近开发了一种生产重组人心肌肌球蛋白马达的系统，我们将利用这一系统首次系统研究人心肌肌球蛋白中HCM和DCM突变的影响。由于大多数导致疾病的突变位于肌球蛋白马达中，我们将把研究重点放在那里。我们选择了10个突变(5个HCM和5个DCM)进行研究，这既是因为它们的临床表型非常严重，也是因为它们位于肌球蛋白运动域。我们建议使用体外动力性和激光捕获单分子分析来分析溶液中突变的生化特性和生物力学特性。我们假设，导致力量产生增加的突变会导致HCM，而导致力量产生减少的突变会导致DCM，这与最近的建议一致。肌球蛋白导致力量产生减少或增加的机制可以是不同的。例如，通过改变马达弹性元件的弹簧常数，可以增加或降低马达的内在制力能力。另一方面，通过改变肌球蛋白的占空比(头部与肌动蛋白强烈结合的ATPase循环的一部分)，可以在两个方向上改变力量产生的能力。例如，增加占空比将导致更多的头部在任何时候都处于武力产生状态，从而导致武力的整体增加。此外，占空比可以通过多种方式改变。例如，ADP释放速率(确定强结合状态时间)、ATP水解步骤(定义恢复行程时间)或PI释放(进入强结合状态的速率)可能受到影响并导致占空比的改变。因此，有必要开始表征不同的突变，假设这些突变会影响马达的不同机械方面。这些研究将开始将运动功能的基本变化与最终不同的临床结果联系起来的过程。因此，我们将测定野生型人a-和b-心肌肌球蛋白马达和突变的？-肌球蛋白马达的稳态和瞬时动力学特性、零负荷和负荷条件下的速度、它们的占空比、它们的行程大小以及它们与肌动蛋白相互作用时产生的最大力。