Effects of phosphomimetic mutations in the M-domain of cardiac myosin binding protein-c.

Abstract

The long-term goal of the proposed research is to characterize the function of phosphorylation sites in the M-domain of Cardiac Myosin Binding Protein-C in damping spontaneous oscillatory contractions (SPOC) in skinned cardiomyocytes. The M-domain is considered the primary regulatory domain of cMyBP-C and aids in binding of cMyBP-C to both myosin and actin. Phosphorylation of the M-domain reduces affinity for both. Reduced phosphorylation in the M-domain is common in many cases of hypertrophic cardiomyopathy (HCM), and cMyBP-C mutations that reduce cMyBP-C expression are present in approximately 50% of cases of HCM. Recently, we developed a “cut-and-paste” method that allows us to remove the endogenous domains C0-C7 followed bycovalent replacement with a desired recombinant protein containing SpyCatcher. Using this method, we discovered that removal of the C0-C7 domains induced SPOC in skinned cardiomyocytes and addition of recombinant full-length protein (rC0C7Sc) damped SPOC. Here, we tested whether phosphorylation of specific sites in the M-domain (S273, S282, and S302) reduce cMyBP-C's ability to damp SPOC. Specifically, the functional effects of three recombinant proteins were tested in detergent permeabilized cardiomyocytes. These proteins contained a phosphomimetic S->D mutation at all three phosphorylation sites (DDD) and at S282 and S302 individually. Results showed that DDD, S282D and S302D damped SPOC and returned both Ca2+ sensitivity of tension and ktr back to values of endogenous cMyBP-C. Future studies will investigate recombinant proteins containing a combination of phospho-null mutations treated with PKA, in order to control which specific sites in the M-domain become phosphorylated. We seek to determine whether individual or a combination of phosphorylation in the M-domain impose limits to cMyBP-C's ability to damp SPOC. This work supported by NIH HL080367 and HL140925 and Predoctoral Fellowship from the American Heart Association 827268.