Project Summary Cardiomyopathies are the most common genetic cardiovascular disease worldwide. The presence of cardiac troponin variants accounts for at least 15% of all familial cardiomyopathy cases. Cardiac muscle contraction is regulated by free intracellular Ca2+ concentration via two thin filament regulatory proteins – tropomyosin and troponin complex. Ca2+ binding to troponin displaces tropomyosin from myosin-binding sites and allows formation of myosin cross-bridges, which on their own contribute to thin filament activation. Troponin complex is composed of Ca2+ sensing troponin C, actin-binding troponin I, and Tm-bound troponin T. For decades, the helical approach to electron microscopy reconstruction of the thin filament eliminated information on the structure of the Tn complex. Hence, the complex interactions among components of the thin filament remained unknown. We developed cryo-EM non-helical algorithm to the reconstruction of native cardiac thin filaments to reveal the structure of the whole troponin complex at physiological Ca2+ levels. We show that the thin filament is comprised of an array of Ca2+-free and Ca2+-bound non-equivalent troponin complexes with short-range cooperativity between adjacent units. Troponin variants associated with inherited cardiomyopathies affect thin filament Ca2+- dependent activation. We hypothesize that: (1) dilated (DCM) or hypertrophic (HCM) cardiomyopathy variants in troponin affect thin filament regulation by: (a) altering the equilibrium between Ca2+-free and Ca2+-bound troponin complexes via conformational changes in Ca2+-sensing troponin C unit; and/or (b) altering the distribution of Ca2+-free and Ca2+-bound troponin complexes by changing communication between the adjacent troponins along and across the thin filament. To test our hypothesis we chose 4 strategically located mutations. In Aim 1 we will utilize pathogenic variants in troponin C located in distal parts of Ca2+-sensing troponin N-lobe to evaluate how they affect the equilibrium between Ca2+-free and Ca2+-bound troponin complexes, and if they affect communication between troponin units that may curb activating effect of rigor myosin-S1. In Aim 2 we will focus on highly penetrant pathogenic variants in troponin T located in N-terminus of troponin T, which stabilizes the interaction between tropomyosin cables belonging to adjacent troponin units. We will evaluate how these mutations affect the communication between neighboring troponins and activation by myosin-S1. To reveal how distal parts of the troponin complex (Ca2+ sensing troponin C and N-terminus of troponin T) communicate, we will use a Ca2+ sensitizer, which binds to troponin C to revert effects of a troponin T variant. Our multidisciplinary, multi-PI approach with collective expertise in structural, functional and computational methods will reveal how the complex interactions between components of the thin filament make heartbeats possible. Successful execution of the aims ...