Role of Cardiac Myosin Binding Protein-C in the Regulation of Myocardial Contraction

TITLE: Role of Cardiac Myosin Binding Protein-C in the Regulation of Myocardial Contraction ABSTRACT: The overall goal of this project is to understand how cardiac myosin binding protein-C (cMyBP-C) regulates heart muscle contraction and relaxation. cMyBP-C is a critical regulator of cardiac function, with dysfunction of cMyBP-C commonly occurring in heart failure and mutations in cMyBP-C being the most common genetic cause of HCM. However, the regulatory effects of cMyBP-C are complex and most likely involve dynamic interactions with both thick (myosin containing) and thin (actin containing) filaments, making it challenging to sort out what are likely to be reciprocal or interrelated effects of cMyBP-C on each filament. To overcome these and other challenges, we recently developed a novel method that allows us to rapidly remove and replace (“cut and paste”) cMyBP-C at its endogenous position in sarcomeres, thus affording us the opportunity to quickly test cMyBP-C effects on both filament systems. In Aim 1 we will test the functional significance of the “middle domains” of cMyBP-C which were recently implicated as regulators of thick filament relaxation by stabilizing the “interacting heads motif” conformation of myosin on the thick filament as well as novel regulators of cMyBP-C function including Ca2+-Calmodulin (CaM) that we show in exciting new preliminary data may regulate cMyBP-C binding to the thin filament. Because HCM mutations and posttranslational modifications (PTMs) also occur in the middle domains and have been correlated with diastolic dysfunction we will also test effects of selected mutations and PTMs in these domains. Experiments will include measurements of steady state force in permeabilized cardiomyocytes as well as rates of activation and relaxation in myofibrils and the activation state of thick filaments determined by myosin DRX/SRX ratios. In Aim 2 we will use the cut and paste approach combined with X-ray diffraction to determine structural effects of cMyBP-C on the activation/relaxation states of thick and thin filaments in sarcomeres. Specifically, we will test the hypothesis that C-links between thick and thin filaments modulate the on/off states of thick and thin filaments and contribute to thick filament activation in response to passive stretch or active load. Results from these studies will provide new insights into how cMyBP- C regulates heart function during health and disease.