PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) is a disease affecting more than 1 in 500 individuals and is an unmet medical need with limited FDA-approved treatments. ~40% of HCM cases are associated with mutations in the gene encoding cardiac myosin-binding protein C (MyBP-C). MyBP-C is a thick filament-associated protein that is critical for normal myocardial performance and centrally positioned in the sarcomere to regulate interactions between myosin and actin responsible for force development. We previously demonstrated that increased phosphorylation of MyBP-C, enhances actin-myosin interactions to accelerate contraction kinetics in myocardium, whereas the decreased MyBP-C phosphorylation, reduces actin-myosin proximity and decelerate contraction. However, it is remains unknown how MyBP-C functions under varied states, including myofilament activation, phosphorylation, and HCM mutations. The structural dynamics of MyBP-C and its interactions with actin and/or myosin to modulate force development in myocardium are key to understanding this mechanism of action. We have developed innovative biophysical tools that, for the first time, enable determination of these mechanisms by evaluation of: (1) MyBP-C structural dynamics, (2) how it interacts with actin and myosin in relaxed and activated muscle, (3) how these interactions are affected by phosphorylation and (4) known pathologic mutations. We will test the central hypothesis that MyBP-C function is determined by dynamic structural changes of its domains that determine interactions of MyBP-C with thin (actin) and thick (myosin) filaments and that the equilibrium of these interactions is affected by phosphorylation and HCM mutations. The proposed aims further develop our innovative biophysical tools to measure structural dynamics underlying MyBP- C regulation of contraction in normal and diseased states. These tools include site-directed fluorescence spectroscopy, computational simulations, thin and thick filament function, and mechanical measurements. We will examine how the activation state of the myocardium (Aim 1), phosphorylation of MyBP-C (Aim 2), and HCM mutations (Aim 3) affect MyBP-C’s structural dynamics and interactions to modulate cardiac contractility. The proposed studies resolve interactions in real myocardial space and capture structural dynamics in real time using high-resolution approaches during the contractile cycle. This involves monitoring distances between points on proteins and the order (or disorder) of those distances under physiological conditions, in interacting proteins and functioning myocardium. Fluorescence lifetime data components, thin and thick filament activation, mechanics, and simulations will be used to define models of MyBP-C regulation. The proposed aims offer unprecedented mechanistic resolution of MyBP-C for its functions in health and HCM disease. This mechanistic understanding is critical to lay the foundation for determining the qualitative and qu...