PROJECT SUMMARY In deadly and common familial hypertrophic and dilated cardiomyopathies, structural variation at the single protein level leads to adverse ventricular remodeling, systolic dysfunction, and diastolic dysfunction. Muscle contraction is driven by interactions among motor proteins, structural filaments, and regulatory proteins within sarcomeres. Structural perturbations to the contractile machinery disrupt the kinetics of these interactions and give rise to systems-level dysfunction in cardiac tissue. This project will investigate the mechanisms of cardiac contractile dysfunction across multiple biologically relevant spatial and temporal scales using a combined computational and experimental platform. The proposed work focuses specifically on structural perturbations that impact the essential interaction between actin and myosin. The overarching hypothesis of this work is that structural perturbations along a structural communication pathway within the upper and lower 50 kDa domains of myosin modulate the electrostatic potential and surface area of myosin’s actin binding surface and modulate the association of myosin heads onto thin filaments. Recent stopped flow kinetics and x-ray diffraction-based measurements have shown that cardiomyopathy mutations and the small molecule 2’-deoxy-ATP modulate actomyosin affinity. I have used computational simulations to show that these mutations and small molecules alter the structure and dynamics of the upper 50 kDa domain of myosin. However, a general description of the ‘rules’ by which mutations and/or small molecules modulate actomyosin interaction requires further study. These computational predictions also require rigorous testing using in vitro methods. The goals of this work are to establish a mechanistic framework that explains how structural perturbations to myosin affect its interaction with actin and to modify actin-myosin interactions with small molecules designed to modulate myosin structure. These goals will be accomplished by simulating the impact of mutations on myosin structure, myosin recruitment, and actomyosin interaction (Aim 1), testing computational predictions in single molecules and contractile organelles from stem cell-derived cardiomyocytes (Aim 2), and developing small molecules designed to modulate actin-myosin interactions by targeting structural communication pathways in myosin (Aim 3). The project will utilize machine-learning infused computational workflows and state-of-the-art stem cell technologies to accelerate translational cardiomyopathy research with a combined computational/experimental platform. The proposed training program will provide me with new skills in stem cell biology, protein biochemistry, and muscle mechanics that increase the scope of my research. I will be mentored by a diverse team led by Dr. Michael Regnier, an accomplished researcher in muscle biology who has significant experience in leading collaborative, multiscale, and interdisciplinary...