Evidence for a role of actin-binding in the regulatory effects of cardiac myosin binding protein c

Cardiac myosin binding protein C (cMyBP-C) is a 140 kD protein that regulates interactions between actin and myosin in sarcomeres of cardiac muscle. Mutations in the gene encoding cMyBP-C (MYBPC3) are a leading cause of hypertrophic cardiomyopathy (HCM), which affects 1:500 people worldwide and is the leading cause of sudden cardiac death in young adults. Its main roles, to slow crossbridge cycling and limit muscle shortening, are regulated by the phosphorylation of a cluster of 3-4 serine residues within the regulatory M-domain. However, the precise mechanism(s) and specific residues underlying how cMyBP-C influences cardiac muscle contraction remain unclear. cMyBP-C has been proposed to act as an internal load on the sarcomere by binding to the myosin backbone at its C-terminus and binding to either myosin S2 and/or actin at its N-terminus, but to which and when is unknown. The overall goal of this thesis was to begin developing a mechanism by which cMyBP-C regulates muscle contraction through binding to actin. We have previously shown that the N-terminus alone, specifically the M-domain, can increase calcium sensitivity of force and rate of tension redevelopment in skinned rat myocardium. Here we present an in-depth analysis of the M-domain to determine what regions and specific residues are important for conferring its functional effects and whether or not they can bind actin specifically. In in vitro studies, we found that residues near phosphorylation sites were important to function, but that residues downstream, within a structural region defined by three alpha-helices, were important for both functional effects and binding to actin. One particular mutation, L348P, which is a known human hypertrophic cardiomyopathy variant, showed a gain in function and increased actin binding. We hypothesized that in vivo this mutation would also bind actin with a higher affinity causing an increase in the internal load on the sarcomere. Using a transgenic mouse model that expressed L348P cMyBP-C, we showed slowed shortening velocity and increased stiffness, consistent with an increased load. We believe that by increasing actin binding in vivo we were able to enhance the already well-studied roles of cMyBP-C, suggesting that actin-binding is both physiologically relevant and involved in the mechanism by which cMyBP-C regulates internal load during normal cardiac contraction.