Structural Dynamics of Cardiac Muscle Contraction

心肌收缩的结构动力学

• 批准号: 8060162
• 负责人: Brett A Colson
• 金额: $ 4.84万
• 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/8060162
• 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; 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做好准备作为肌肉分子生物物理学的独立研究者的职业，尤其强调心脏。他使用肌肉纤维力学，X射线衍射和分子生物学方法的心脏和骨骼肌生理的强烈背景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殊优势。 该培训计划的重点是赞助商NIH资助的研究至关重要的研究项目，该项目询问了有关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。该项目着重于心脏肌球蛋白结合蛋白-C（CMYBP-C）在收缩调节中的作用。 CMYBP-C的蛋白激酶A（PKA）依赖性磷酸化通过缓解CMYBP-C的抑制作用来加速心脏肌肉收缩的动力学，但是关于这些功能是否是由于对肌蛋白和/或肌动蛋白的结构效应引起的争议。因此，该项目侧重于对肌球蛋白和肌动蛋白的光谱分析，受CMYBP-C及其磷酸化的影响。 有三个特定的目的：（1）开发光谱方法，涉及特异性与肌球蛋白或肌动蛋白的自旋标签的EPR，以准确测量CMYBP-C遗传消融对皮肤心肌纤维中结构动力学的影响。这种方法基于赞助商实验室的先前工作。 （2）使用目标1的方法来定量确定CMYBP-C对肌球蛋白和肌动蛋白结构动力学的磷酸化的影响。 （3）使用时间分辨的磷光各向异性（TPA）和荧光共振能量转移（FRET），并在溶液中对肌动蛋白或肌球蛋白上的探针来确定纯化的重组CMYBP-C构建体对肌动蛋白和肌球蛋白丝质丝的结构动力学的影响。 有大量证据表明，CMYBP-C在心脏功能的调节中在健康和疾病中收缩功能缺陷中起着重要作用。在理解这些疾病机制之前，有必要解决有关该蛋白对肌球蛋白和肌动蛋白的结构影响的基本问题。该项目将基于申请人过去在肌肉结构和功能的调节机制方面的经验，同时将他引入新的光谱技术。该项目的重点是心肌，其中MYBP-C进行了最彻底的研究。将来，该项目中开发的方法将适用于骨骼肌中该蛋白质的研究，以及对肌肉疾病和治疗方法的发展的理解。 公共卫生相关性：该项目定义了申请人在心脏肌肉基础研究中的培训计划，因为他迈向了独立的研究职业。研究目标是了解特定蛋白质，肌球蛋白结合蛋白C在心脏功能中的作用。由于该蛋白质的缺陷会导致人心脏病，因此这项研究可能会对心脏病的治疗方法产生重大影响。