Regulation of cardiac contractility by site specific phosphorylation of cardiac myosin binding protein C

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

? DESCRIPTION (provided by applicant): More than 5.1 million Americans suffer from the debilitating effects of congestive heart failure (CHF) which is the common pathway for a range of cardiac diseases including hypertrophic cardiomyopathy (HCM). HCM is the most common inherited cardiovascular disease, affecting 1 in 200 people worldwide. Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric accessory protein that binds to regions of the myosin head to depress force generation. Phosphorylation of cMyBP-C increases cardiac contractility following adrenergic stimulation by attenuating the inhibitory interactions of cMyBP-C with myosin. Failure of this mechanism due to decreased adrenergic signaling in heart failure most likely contributes to reduced contractility. In addition, HCM causing mutations in cMyBP-C may contribute to the development of the disease by altering phosphorylation of the protein. cMyBP-C contains three phosphorylatable serines at the N-terminus that exist in a range of phosphorylation states in the sarcomere. However, the functional effects of these partial phosphorylations are not known. To test the hypothesis that the three phosphoserines regulate contraction kinetics in unique ways, we are generating mutated mouse engineered cardiac tissue expressing systematic combinations of serines, aspartes, or alanines to mimic WT, phosphorylation, and dephosphorylation respectively (Aim 1). We will quantify the relative contributions of the three phosphoserines to accelerate contraction kinetics by recording twitch force and kinetics of contraction and relaxation. Furthermore, to determine the whether the mechanisms of differential regulation are mediated by phosphorylation state dependent binding of cMyBP-C to myosin and actin, the phosphoserines of bacterially or yeast expressed N-terminal fragments of cMyBP-C will be mutated in similar systematic combinations to Aim 1. The effect of these mutations on myosin and actin binding will be characterized with yeast two hybrid screening and surface plasmon resonance (Aim 2). Understanding the binding partners of cMyBP-C after post translational modification will identify potential therapeutic targets for heart failure and HCM.