PROJECT SUMMARYHypertrophic cardiomyopathy (HCM) is a relatively common disease affecting more than 1 in 500 individualsand the leading cause of sudden death in young individuals and athletes. HCM is an unmet medical need withno FDA-approved treatments. ~40% of all HCM cases are associated with mutations in the gene encodingcardiac myosin-binding protein C (MyBP-C). MyBP-C is a thick filament-associated protein that is critical fornormal myocardial performance; it is centrally positioned in the sarcomere to regulate interactions betweenmyosin cross-bridges and actin thin filaments that are responsible for force development. We have previouslydemonstrated that increased phosphorylation of MyBP-C enhances actin-myosin interactions leading toaccelerated contraction kinetics in myocardium whereas reduced phosphorylation led to reduced actin-myosinproximity and decelerated contraction. However it is not understood how MyBP-C phosphorylation alters thestructural dynamics of its interactions with actin and/or myosin to modulate force development in normalmyocardium or how mutations alter functions that ultimately contribute to HCM pathogenesis. We havedeveloped innovative biophysical tools that for the first time enable evaluation of: (1) the structural dynamics ofMyBP-C (2) how it interacts with actin and/or myosin in muscle and (3) how these interactions are affected byphosphorylation and known pathologic mutations. We will test the central hypothesis that phosphorylation andHCM mutations of N-terminal MyBP-C alter functionally significant structural properties of MyBP-C andinteractions with actin and myosin. Aim 1 will evaluate the effects of phosphorylation HCM mutations andbinding to actin or myosin on MyBP-C structural dynamics. Spectroscopic approaches will be employed to detectconformational changes (structure) within MyBP-C due to phosphorylation HCM mutation and actin/myosinbinding (function). Molecular dynamics (MD) simulations will be applied as a complementary approach. Aim 2will determine how MyBP-C phosphorylation and HCM mutants affect proximities and dynamics of keymyocardial proteins. We will utilize site-directed probe technologies in skinned (demembranated) cardiac fibersto determine how phosphorylation/mutants affect protein structure/interactions in situ to regulate contractility.The proposed studies capture structural dynamics in real time and resolve interactions in real myocardial spaceusing novel high-resolution approaches. These aims are a stepwise progression developing a new paradigm forstudying normal and mutant MyBP-C during the contractile cycle. This paradigm involves monitoring distancesbetween points on proteins and the order (or disorder) of those distances under physiological conditions ininteracting proteins and functioning myocardium. Not all HCM mutants impact the same functions of MyBP-C.Time-resolved fluorescence data components thin/thick filament dynamics mechanics and simulations will beused to separate mutants into identifiable bins setting the stage for identifying mechanistic-based therapies tospecifically treat different classes of mutations.