Structural Dynamics of Cardiac Muscle Contraction

描述（由申请人提供）：本项目的主要目标是准备申请人的职业生涯作为一个独立的研究人员在分子生物物理学的肌肉，特别是对心脏。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，这是申办者实验室的特别优势。 该培训计划的重点是一个研究项目，该项目是赞助商的NIH资助的研究的核心，该项目提出了有关蛋白质结构动力学和磷酸化在骨骼肌，心脏和平滑肌功能中的作用的基本问题。本项目的重点是心肌肌球蛋白结合蛋白-C（cMyBP-C）在收缩调节中的作用。cMyBP-C的蛋白激酶A（PKA）依赖性磷酸化通过减轻cMyBP-C的抑制作用而加速心肌收缩的动力学，但关于这些功能性作用是否是由于对肌球蛋白和/或肌动蛋白的结构性作用存在争议。因此，该项目的重点是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。 有三个具体目标：（1）开发光谱方法，包括特异性结合肌球蛋白或肌动蛋白的自旋标记物的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。该方法基于申办方实验室先前的工作。(2)使用Aim 1的方法定量测定cMyBP-C的PKA依赖性磷酸化对肌球蛋白和肌动蛋白结构动力学的影响。(3)使用时间分辨磷光各向异性（TPA）和荧光共振能量转移（FRET），与肌动蛋白或肌球蛋白在溶液中的探针，以确定纯化的重组cMyBP-C结构的影响，对肌动蛋白和肌球蛋白丝的结构动力学。 有相当多的证据表明，cMyBP-C在调节健康的心脏功能和疾病中的收缩功能缺陷中起主要作用。在了解这些疾病机制之前，有必要解决有关这种蛋白质对肌球蛋白和肌动蛋白的结构影响的基本问题。该项目将建立在申请人过去在肌肉结构和功能调节机制方面的经验基础上，同时向他介绍新的光谱技术。该项目的重点是心肌，其中MyBP-C已被最彻底地研究。在未来，本项目中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。 公共卫生相关性：该项目为申请人定义了心肌基础研究的培训计划，因为他正在走向独立的研究生涯。研究的目的是了解一种特殊的蛋白质，肌球蛋白结合蛋白C，在心脏功能中的作用。由于这种蛋白质的缺陷会导致人类心脏病，因此这项研究可能会对心脏病的治疗方法产生重大影响。

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.