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（适用提供）：肌球蛋白是心脏和骨骼肌的运动驾驶合同。对于心肌，主要同工型为β-myHC。在β-MyHC和其他肌动蛋白中已经确定了几十年的突变，并且人们对研究其功能以及它们如何引起诸如家族性肥厚性心肌病或家族性心肌病等疾病的兴趣很大。最近，在β-MyHC中发现了新的突变，并在早期患者中表现出普遍存在。这些患者的临床预后非常差，唯一的选择是心脏移植，因此需要减少侵入性干预措施。了解这些突变如何导致疾病对于发展治疗至关重要，但是过去对这种疾病的研究缺乏人类情境，因为它们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肌球蛋白，使净化人β-myHC具有同质性，并被Leinwand Lab开拓以克服这些障碍。在此提案中，我假设：（1）HCM导致突变始终导致肌球蛋白活性增强，而DCM则导致突变导致活性降低； （2）OmeCamtiv Mecarbil，一种小分子肌球蛋白激活剂，目前正在临床试验中用于治疗 心力衰竭将通过提高ATP - 溶解速率，ADP释放速率和肌动蛋白亲和力来对β-MyHC产生更强大的动力学作用； （3）引起HCM的突变增加了占空比，因此进行更长的跨桥动力学和更长的放松时间，而引起DCM的突变将显示相反的效果。我将通过表达已知疾病的人肌球蛋白来完成这些项目，从而导致突变并进行常规动力学测定，例如ATPase和停止流动动力学，以分析如何影响不同的速率。我还将在孤立的心肌细胞中进行收缩力研究，该研究表达此处提出的突变，并将突变的作用与生化率相关。这些目的应该使对心肌病的全面了解引起突变及其功能和收缩特征。