Structural, Biochemical, and Mechanical Effects of Myosin Cardiomyopathy Mutations

肌球蛋白心肌病突变的结构、生化和机械效应

基本信息

批准号：
9170417
负责人：
Eva Forgacs
金额：
$ 53.98万
依托单位：
RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9170417
关键词：
ATP Hydrolysis ATP phosphohydrolase Actins Actomyosin Affect Beryllium Binding Biochemical Biological Assay Biological Models Biomechanics Cardiac Cardiac Myosins Cardiomyopathies Cell Line Characteristics Cleaved cell Clinical Management Clinical Treatment Clinical Trials Communication Complement Complex Coupling Data Defect Dilated Cardiomyopathy Disease Dissociation Drug Binding Site Elements Equilibrium Functional disorder Genes Goals Heart Heart Diseases Heterogeneity Human Hydrolysis Hypertrophic Cardiomyopathy In Vitro Infection Inherited Intervention Kinetics Knowledge Lead Link Measurement Measures Mechanics Methods Missense Mutation Modeling Molecular Motion Motor Motor Activity Movement Muscle Cells Mutate Mutation Mutation Analysis Myocardium Myopathy Myosin ATPase Nucleotides Patients Pharmaceutical Preparations Power stroke Production Property Proteins Publishing Recovery Rotation Specimen Striated Muscles Structure Study models Symptoms System Systolic heart failure Techniques Testing Therapeutic Tissues Ventricular adenoviral-mediated arm base beta pleated sheet biophysical properties cell motility clinical phenotype design driving force human disease insight multidisciplinary mutant nucleotide analog optical traps prevent research study single molecule small molecule stroke recovery sudden cardiac death targeted treatment therapeutic development

项目摘要

PROJECT SUMMARY Mutations in the human β-cardiac myosin gene (MYH7) are responsible for a large number of inherited Hypertrophic (HCM) and Dilated Cardiomyopathies (DCM). The objective of the proposal is the biochemical and biophysical characterization of the effects of human cardiac myosin mutations as an essential first step in identifying the changes in the structure and mechanism that culminate in cardiomyopathies. A major hurdle in tackling this problem with human cardiac myosin has been the instability and heterogeneity of the protein obtained from patient tissues, and the lack of an adequate expression system to produce high quality human β-cardiac myosin. We developed a mammalian expression system based on adenoviral infection of a muscle cell line that is now widely accepted as the model for these studies. This approach produces the quantities of the human β-cardiac myosin required for detailed kinetic and structural studies. The crystal structures of the human β-cardiac myosin motor domain reveal a cluster of HCM and DCM mutations in a region linking structural elements that are critical for mechanochemical coupling. We have called this the coupling region. Furthermore, we have shown that the cardiac myosin activator, omecamtiv mecarbil, binds in a narrow cleft in the center of the coupling region and acts by influencing the mechanochemical coupling mechanism. We propose to study eight HCM/DCM mutations with severe clinical phenotypes residing within the coupling region to quantify the effects on structure and mechanism of the mutations by: (1) steady-state and transient kinetic assays to quantify subtle changes in the catalytic mechanism; (2) motility assays to evaluate ensemble motor dynamics, and single molecule force measurements to measure unitary mechanical characteristics; and (3) crystallographic structure analysis and modeling to complement the biomechanical measurements and guide interpretation. The effect of omecamtiv mecarbil on the coupling mechanism of mutated cardiac myosin will provide insights into the potential for clinical management of the disease. Understanding the molecular basis of mechanical changes resulting from cardiac myosin mutations will aid in the development of therapeutic approaches to mitigate the dysfunction leading to cardiomyopathies.

项目概要人类 β-心肌肌球蛋白基因 (MYH7) 的突变导致大量遗传性疾病肥厚型 (HCM) 和扩张型心肌病 (DCM) 该提案的目标是生化。人类心肌肌球蛋白突变影响的生物物理表征是重要的第一步确定导致心肌病的结构和机制的变化是治疗心肌病的一个主要障碍。人类心肌肌球蛋白解决了这个问题，即蛋白质的不稳定性和异质性从患者组织中获得，并且缺乏足够的表达系统来产生高质量的人类我们开发了一种基于肌肉腺病毒感染的哺乳动物表达系统。这种方法产生的细胞系现已被广泛接受为这些研究的模型。详细动力学和结构研究所需的人β-心肌肌球蛋白。人β-心肌肌球蛋白运动结构域揭示了连接区域中的一系列 HCM 和 DCM 突变对于机械化学耦合至关重要的结构元素我们称之为耦合区域。此外，我们还发现心肌肌球蛋白激活剂 omecamtiv mecarbil 结合在狭窄的裂隙中。耦合区域的中心并通过影响力化学耦合机制来发挥作用。提议研究耦合区域内具有严重临床表型的八种 HCM/DCM 突变通过以下方式量化突变对结构和机制的影响：（1）稳态和瞬态动力学 (2) 运动测定来评估整体运动动力学和单分子力测量，以测量单一机械特性；以及 (3) 晶体结构分析和建模以补充生物力学测量和指导 omecamtiv mecarbil 对突变心肌肌球蛋白偶联机制的影响。深入了解该疾病的临床管理潜力。心肌肌球蛋白突变引起的机械变化将有助于治疗药物的开发减轻导致心肌病的功能障碍的方法。