Functional analysis of novel MYH7 mutations with prevalence in early-onset patients

早发患者中流行的新型 MYH7 突变的功能分析

• 批准号: 9053948
• 负责人: Carlos D Vera
• 金额: $ 3.51万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9053948
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Accounting; Actins; Adult; Affect; Affinity; Age; Animal Model; Animals; Automobile Driving; Biochemical; Biological Assay; Calcium; Cardiac Myocytes; Cardiac Myosins; Cardiomyopathies; Cause of Death; Cessation of life; Characteristics; Child; Childhood; Clinical; Clinical Treatment; Clinical Trials; Congenital cardiomyopathy; Contractile Proteins; Depressed mood; Diagnosis; Dilated Cardiomyopathy; Disease; Exhibits; Familial Hypertrophic Cardiomyopathy; Generic Drugs; Genes; Genetic; Heart; Heart Diseases; Heart Transplantation; Heart failure; Human; Hypertrophic Cardiomyopathy; Infant; Intervention; Kinetics; Lead; Length; Link; Measurement; Measures; Motor; Muscle; Muscle Cells; Mutation; Myocardium; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Patients; Phenotype; Population; Prevalence; Property; Protein Isoforms; Proteins; Recombinants; Relaxation; Sarcomeres; Skeletal Muscle; Sorting - Cell Movement; Structural defect; Structure; Symptoms; System; Systolic heart failure; Techniques; Testing; Therapeutic; Time; United States; beta-Myosin; disease-causing mutation; early onset; familial dilated cardiomyopathy; insight; interest; mutant; novel; outcome forecast; pediatric patients; protein folding; public health relevance; small molecule; small molecule therapeutics; sudden cardiac death

 DESCRIPTION (provided by applicant): Myosin is the motor driving contraction in cardiac and skeletal muscle. For cardiac muscle, the primary isoform is β-MyHC. Mutations have been identified for several decades in β-MyHC and other sarcomeric myosins and there has been a lot of interest in studying their functionality and how they might lead diseases such as Familial Hypertrophic Cardiomyopathy or Familial Dilated Cardiomyopathy. Recently, there have been novel mutations identified in β-MyHC and shown to be prevalent in early-onset patients. The clinical prognosis for these patients is very poor and their only alternative is heart transplant, hence the need for less invasive interventions. Understanding how these mutations lead to disease is crucial for developing treatments, but past studies of the sort are limited because they lack the human context, because almost all of these studies were performed in animals. Expressing human myosin in differentiated muscle cells enables purifying human β-MyHC to homogeneity and was pioneered by the Leinwand Lab to overcome these obstacles. In this proposal I am hypothesizing that: (1) HCM causing mutations consistently lead to enhanced myosin activity and conversely DCM causing mutations lead to depressed activity; (2) Omecamtiv mecarbil, a small molecule myosin activator currently in clinical trials for treatment of heart failure, will induce stronger kinetic effects on β-MyHC by increasing ATP-hydrolysis rate, ADP-release rates and actin affinity; (3) HCM-causing mutations increase duty ratio, therefore exhibiting longer cross-bridge kinetics and longer relaxation times, while DCM- causing mutations will show the opposite effect. I will complete these projects by expressing human myosin with known disease causing mutations and perform conventional kinetic assays such as ATPase and Stopped- Flow Kinetics to analyze how the different rates are affected. I will also perform contractility studies in isolated cardiomyocytes that express the mutations here proposed and relate the effect of the mutation to the biochemical rates. These aims should result in a comprehensive understanding of cardiomyopathy causing mutations and their functional and contractile characteristics.

 描述（由申请人提供）： 肌球蛋白是心肌和骨骼肌收缩的马达，其主要亚型是 β-MyHC。几十年来，β-MyHC 和其他肌节肌球蛋白的突变已被发现。人们对研究它们的功能以及它们如何导致家族性肥厚型心肌病或家族性扩张型心肌病等疾病产生了浓厚的兴趣。最近，在这些疾病中发现了新的突变。 β-MyHC 在早发患者中普遍存在，这些患者的临床预后非常差，他们唯一的选择是心脏移植，因此了解这些突变如何导致疾病至关重要。但过去的此类研究受到限制，因为它们缺乏人类背景，因为几乎所有这些研究都是在动物中进行的，在分化的肌肉细胞中表达人类肌球蛋白能够将人类 β-MyHC 纯化至同质性，并且是由 Leinwand 实验室开创的。到在此建议中，我假设：(1) HCM 突变导致肌球蛋白活性持续增强，相反，DCM 突变导致肌球蛋白活性降低；(2) Omecamtiv mecarbil，一种目前正在进行临床试验的小分子肌球蛋白激活剂。用于治疗 心力衰竭，将通过增加 ATP 水解速率、ADP 释放速率和肌动蛋白亲和力诱导对 β-MyHC 更强的动力学效应；(3) 引起 HCM 的突变增加占空比，因此表现出更长的跨桥动力学和更长的弛豫时间，而引起 DCM 的突变将表现出相反的效果，我将通过表达具有已知疾病突变引起的人类肌球蛋白并进行常规动力学测定（例如 ATP 酶和停流动力学）来分析不同的速率。我还将在分离的心肌细胞中进行收缩性研究，这些心肌细胞表达此处提出的突变，并将突变的影响与生化率联系起来。这些目标应该有助于全面了解引起突变的心肌病及其功能和收缩特征。