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: 8843945
• 负责人: Leslie Anne Leinwand
• 金额: $ 42.76万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8843945
• 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; Solutions; Stroke; Sudden Death; Suggestion; System; Testing; Thermodynamics; Time; United States; Work; cell motility; clinical phenotype; disease-causing mutation; mortality; mutant; novel; 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）：尽管位于人心肌肌球蛋白重链（MyHC）运动结构域的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。大多数确定这些突变对肌球蛋白的功能影响的研究都使用非人类肌球蛋白，并且大多数没有使用心肌肌球蛋白。 Leinwand 及其同事最近开发了一种用于生产重组人心肌肌球蛋白马达的系统，我们将利用该系统首次系统研究 HCM 和 DCM 突变对人心肌肌球蛋白的影响。由于导致疾病的大多数突变都位于肌球蛋白运动中，因此我们将重点研究那里。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，也因为它们位于肌球蛋白运动域内。我们建议使用体外运动和激光陷阱单分子测定来分析溶液中突变的生化特性及其生物力学特性。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。导致肌球蛋白产生的力减少或增加的机制可以是多种多样的。例如，电机固有的产生力的能力可以通过改变电机弹性元件的弹簧常数的突变来增加或减少。另一方面，通过改变肌球蛋白的占空比（头部与肌动蛋白紧密结合的 ATP 酶循环部分），可以在任一方向上改变产生力的能力。例如，占空比的增加将导致更多的头在任何时刻处于产力状态，与肌动蛋白结合，从而导致力量的总体增加。此外，可以以不止一种方式改变占空比。例如，ADP 释放速率（决定强结合态时间）、ATP 水解步骤（定义恢复冲程时间）或 Pi 释放（进入强结合态的速率）可能会受到影响并导致在占空比的变化中。因此，有必要开始表征不同的突变，假设这些突变会影响电机的不同机械方面。这些研究将开始将运动功能的根本变化与最终不同的临床结果联系起来。因此，我们将测定野生型人a-和b-心肌肌球蛋白以及突变体?的生化和生物力学参数。 -肌球蛋白马达的稳态和瞬态动力学特性、零负载和负载条件下的速度、占空比、行程大小以及与肌动蛋白相互作用时产生的最大力。