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

• 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
• 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

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.

项目总结