Crossbridge Arrangement in Cardiac Thick Filaments Isolated from cMyBP-C Phosphomimetic Mice

Abstract

Although cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, the mechanism by which changes in phosphorylation of cMyBP-C affect crossbridge kinetics remains unknown. cMyBP-C mediated changes in thick filament structure, binding of cMyBP-C to the thin filament, or both remain viable alternatives. In this study, we isolated thick filaments from the hearts of phosphomimetic mice in which the three phosphorylatable serine residues (Ser273, Ser282 and Ser302) in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid to mimic the fully non-phosphorylated (CT-SA mice) or fully phosphorylated (CT-SD mice) state of cMyBP-C, respectively. Thus, the state of phosphorylation of cMyBP-C of every filament was known in this transgenic system. We found that the thick filaments from the CT-SA mouse hearts corresponding to the non-phosphorylated state of cMyBP-C had highly ordered crossbridge arrays, while the filaments from the CT-SD mouse hearts showed a strong tendency towards disorder of the myosin array. These results are consistent with our previous studies of filaments from cMyBP-C knockout mouse hearts. The results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed quasi-helical ordering of the myosin heads on the backbone consistent with slower crossbridge kinetics; while filaments with phosphorylated cMyBP-C show a greater tendency towards disorder of the heads consistent with increased flexibility of the myosin heads, extension of the heads from the backbone, and increased potential interaction with the actin thin filament. Work supported by NIH grants SC1 HL096017 (RWK), AR034711 (RC), R37 HL82900 and P01 HL094291 (RLM). RC also received funding through PPG grant P01 HL059408 (David Warshaw).