Structural Dynamics of Cardiac Muscle Contraction

心肌收缩的结构动力学

• 批准号: 8211618
• 负责人: Brett A Colson
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-06 至 2013-01-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8211618
• 关键词: Ablation; Actins; Actomyosin; Affect; Anisotropy; Baculoviruses; Basic Science; Binding; Binding Sites; Biological Models; Biophysics; Bundling; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Cells; Cyclic AMP-Dependent Protein Kinases; Defect; Disease; Doctor of Philosophy; Electron Spin Resonance Spectroscopy; F-Actin; Fiber; Filament; Fluorescence Resonance Energy Transfer; Frequencies; Funding; Future; Genetic; Goals; Health; Heart; Heart Diseases; Human; Insecta; Kinetics; Knock-out; Knockout Mice; Label; Laboratories; Length; Measures; Mechanics; Mediating; Methods; Microfilaments; Molecular; Molecular Biology; Muscle; Muscle Contraction; Muscle Fibers; Muscle function; Myocardium; Myopathy; Myosin ATPase; Myosin Regulatory Light Chains; Optics; Phosphorylation; Physiology; Play; Protein Isoforms; Proteins; Recombinant Proteins; Recombinants; Regulation; Research; Research Personnel; Research Project Grants; Resolution; Role; Skeletal Muscle; Skin; Smooth Muscle; Solutions; Spectrum Analysis; Spin Labels; Structural Protein; Structure; Techniques; Therapeutic; Time; Training; Training Programs; Troponin I; United States National Institutes of Health; Work; base; career; experience; flexibility; heart function; muscular structure; myosin-binding protein C; phosphorescence; public health relevance; research study; therapeutic development; time use

DESCRIPTION (provided by applicant): The primary goal of this project is to prepare the applicant for a career as an independent investigator in molecular biophysics of muscle, with particular emphasis on the heart. His strong background in heart and skeletal muscle physiology, using methods of muscle fiber mechanics, x-ray diffraction, and molecular biology, will be augmented by training in spectroscopic probe techniques, which are particular strengths of the sponsor's laboratory. The training program focuses on a research project that is central to the sponsor's NIH-funded research, which asks fundamental questions about the role of protein structural dynamics and phosphorylation in the function of skeletal, cardiac, and smooth muscle. This project focuses on the role of cardiac myosin binding protein-C (cMyBP-C) in modulation of contraction. Protein kinase A (PKA)-dependent phosphorylation of cMyBP-C accelerates the kinetics of cardiac muscle contraction by relieving inhibitory effects of cMyBP-C, but there is controversy concerning whether these functional effects are due to structural effects on myosin and/or actin. Therefore, this project focuses on spectroscopic analysis of both myosin and actin, as affected by cMyBP-C and its phosphorylation. There are three specific aims: (1) Develop spectroscopic methods, involving EPR of spin labels specifically bound to myosin or actin, to measure accurately the effects of genetic ablation of cMyBP-C on structural dynamics in skinned cardiac muscle fibers. This approach is based on previous work in the sponsor's laboratory. (2) Use the approach of Aim 1 to determine quantitatively the effects of PKA-dependent phosphorylation of cMyBP-C on myosin and actin structural dynamics. (3) Use time-resolved phosphorescence anisotropy (TPA) and fluorescence resonance energy transfer (FRET), with probes on actin or myosin in solution, to determine the effects of purified recombinant cMyBP-C constructs, on the structural dynamics of actin and myosin filaments. There is considerable evidence that cMyBP-C plays a major role in the modulation of cardiac function in health and in deficits of contractile function in disease. Before these disease mechanisms can be understood, it is necessary to resolve fundamental questions about the structural effects of this protein on myosin and actin. This project will build on the applicant's past experience in regulatory mechanisms of muscle structure and function, while introducing him to new spectroscopic techniques. This project focuses on cardiac muscle, where MyBP-C has been most thoroughly studied. In the future, the approaches developed in this project will be applicable to the study of this protein in skeletal muscle, and to the understanding of muscle disease and the development of therapeutic approaches. PUBLIC HEALTH RELEVANCE: This project defines a training program for the applicant in basic research on cardiac muscle, as he progresses toward an independent research career. The research goal is to understand the role of a specific protein, myosin-binding protein C, in the function of the heart. Since defects in this protein cause human heart disease, this research is likely to have substantial impact on therapeutic approaches to heart disease.

描述（由申请人提供）：该项目的主要目标是为申请人成为肌肉分子生物物理学的独立研究者做好准备，特别强调心脏。他在心脏和骨骼肌生理学方面的强大背景，使用肌纤维力学，x射线衍射和分子生物学的方法，将通过光谱探针技术的培训来增强，这是赞助商实验室的特别优势。培训计划的重点是一个研究项目，该项目是美国国立卫生研究院资助的研究的核心，该研究提出了关于蛋白质结构动力学和磷酸化在骨骼、心脏和平滑肌功能中的作用的基本问题。本项目主要研究心肌肌球蛋白结合蛋白c （cMyBP-C）在收缩调节中的作用。蛋白激酶A （PKA）依赖的cMyBP-C磷酸化通过减轻cMyBP-C的抑制作用来加速心肌收缩动力学，但关于这些功能作用是否由于对肌球蛋白和/或肌动蛋白的结构作用而存在争议。因此，本项目重点研究受cMyBP-C及其磷酸化影响的肌球蛋白和肌动蛋白的光谱分析。有三个具体目标：(1)发展光谱方法，包括与肌凝蛋白或肌动蛋白特异性结合的自旋标签的EPR，以准确测量cMyBP-C基因消融对皮肤心肌纤维结构动力学的影响。这种方法是基于先前在赞助商实验室的工作。(2)利用Aim 1的方法定量确定pka依赖的cMyBP-C磷酸化对肌球蛋白和肌动蛋白结构动力学的影响。(3)利用时间分辨磷光各向异性（TPA）和荧光共振能量转移（FRET），用探针在溶液中的肌动蛋白或肌球蛋白上，测定纯化的重组cMyBP-C构建物对肌动蛋白和肌球蛋白细丝结构动力学的影响。有相当多的证据表明，cMyBP-C在健康时的心功能调节和疾病时的收缩功能缺陷中起主要作用。在了解这些疾病机制之前，有必要解决有关该蛋白对肌凝蛋白和肌动蛋白的结构作用的基本问题。本项目将以申请人过去在肌肉结构和功能调控机制方面的经验为基础，同时向申请人介绍新的光谱技术。这个项目的重点是心肌，MyBP-C已经被研究得最彻底。在未来，本项目开发的方法将适用于骨骼肌中这种蛋白质的研究，以及对肌肉疾病的理解和治疗方法的开发。