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）：尽管超过200个突变位于人类的运动域？？心肌肌球蛋白重链（MyHC）引起肥厚性或扩张性心肌病（HCM或DCM），其对肌球蛋白分子的影响尚不明确。大多数确定这些突变对肌凝蛋白功能影响的研究都使用了非人肌凝蛋白，大多数没有使用心脏肌凝蛋白。Leinwand和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，我们将利用该系统对人类HCM和DCM突变的影响进行首次系统研究。心脏肌凝蛋白。由于大多数导致疾病的突变都位于肌凝蛋白运动区，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，因为它们的临床表型很大程度上很严重，而且它们位于肌球蛋白运动结构域。我们建议使用体外运动和激光陷阱单分子分析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导致HCM，而导致力量产生减少的突变导致DCM，这与最近的建议一致。导致肌凝蛋白产生力的减少或增加的机制是多种多样的。例如，电机固有的产力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，肌凝蛋白（头部与肌动蛋白紧密结合的atp酶循环的一部分）的占空比的变化可以改变力的产生能力。例如，增加占空比会导致更多的头部在任何时刻都处于产生力的状态，从而导致力的整体增加。此外，占空比可以以不止一种方式改变。例如，ADP释放速率（决定强结合状态时间）、ATP水解步骤（决定恢复行程时间）或Pi释放（进入强结合状态的速率）都会受到影响，并导致占空比的变化。因此，有必要开始描述不同的突变，假设影响运动的不同机械方面。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。因此，我们将测定野生型人a型和b型心肌肌球蛋白及其突变体的生化和生物力学参数。-肌凝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、冲程大小以及它们与肌动蛋白相互作用时产生的最大力。