This is a resubmission of a project entitled "Structural Dynamics of Cardiac Myosin Binding Protein-C." The primary goal of this project is to prepare me for a career as an independent investigator in molecular biophysics of muscle, with particular emphasis on the heart. My strong predoctoral background in heart and skeletal muscle physiology, using methods of muscle fiber mechanics and X-ray diffraction, has been augmented by training in spectroscopic probe techniques, which are particular strengths of the mentor's laboratory. The mentoring program now focuses on a research project that synthesizes my previous experiences and asks fundamental questions about the role of protein structural dynamics and phosphorylation in the function of muscle. Myosin binding protein-C (CPro) plays a major role in the modulation of cardiac function and in deficits of contractile function in hypertrophic cardiomyopathy (HCM) and heart failure. My Project also focuses on understanding and alleviating HCM disease mechanisms, as it is necessary to resolve fundamental questions about the structure and dynamics of this protein's complexes with myosin and actin as well as the contractile dysfunction that ensues in the CPro-associated HCM disease onset and progression. There are three specific aims of this Resubmission: (1) Use electron paramagnetic resonance (EPR) of spin labels specifically bound to myosin or actin, to measure accurately the effects of genetic ablation of CPro on structural dynamics in skinned cardiac muscle fibers, from several mouse models of human HCM. (2) Use the approach of Aim 1 to determine quantitatively the effects of PKA-dependent phosphorylation of CPro on myosin and actin structural dynamics. Assess baseline and phosphorylated contractile performance in exchanged fibers and intact hearts. (3) Use dipolar electron-electron resonance (DEER) and extend this method to FRET with probes on CPro in solution with myosin or actin, to determine quantitatively the effects of binding, phosphorylation, and HCM mutation on structural dynamics of specific regions of CPro. I will use these mechanistic details to provide the bases for a high-throughput drug assay for small molecule effectors of CPro as potential therapy for HCM and heart failure. My recent PNAS publication revealed exciting new information about the effects of CPro and its phosphorylation on the structural dynamics of labeled actin filaments, and these results will inform all three aims. Feasibility has been established for all three aims, and the proposal below describes the work needed to achieve my goals under continued supervised research experience -- to publish several high-impact peer- reviewed papers within two years, augment my expertise in these areas of research, and prepare to start my own independent lab.
This Project aims to determine the molecular requirements for healthy muscle function, in order to understand and treat the most common genetic cardiac disease, with a clear vision articulated in this Resubmitted research career development plan directed towards translational medicine. The short-term significance of my work will be to definitively answer crucial questions about the function of heart muscle protein in normal and diseased hearts, using specially engineered reporter probes attached to these proteins to visualize how these proteins move in these physiological states. In the long term, this research should greatly improve the human health prospects for understanding genetic heart disease onset, progression, and treatment, including drug screens for novel disease therapy.