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.
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.