Cardiac Myosin Binding-Protein C (cMyBP-C) Phosphorylation affect Cross-Bridge Function

Abstract

To understand the functional significance of phosphorylation that takes place in the M domain of cMyBP-C, chemically skinned papillary muscle fibers of transgenic mice were studied by sinusoidal length alterations and concomitant tension transients. Muscle fibers were maximally activated at pCa 4.55 in the solution that mimic physiological conditions (5 mM MgATP, 8 mM Pi, 200 mM total ionic strength with K-acetate) in myocytes. WT mice possess phosphorylation sites S273, S282, and S302. SAS is a single mutant S282A, and ADA and DAD are triple mutants S273A/S282D/S302A and S273D/S282A/S302D, respectively. D models for phosphorylation (phosphomimetic), and A models for non-phosphorylation (phosphor-ablation). Isometric tension and stiffness of DAD were respectively ∼0.5x of those of WT, but tension and stiffness of t/t (cMyBP-C null), ADA, and SAS were respectively similar to WT. The fast rate constant 2πc of DAD and t/t was ∼0.6x of WT, but that of ADA and SAS was similar to WT. The intermediate rate constant 2πb of DAD and SAS was ∼1.3x of WT, but that of ADA and t/t was similar to WT. These results demonstrate that cMyBP-C M domain phosphorylation affects the cross-bridge kinetics at ATP binding and phosphate release steps, indicating that phosphorylation affects myosin structure and its interaction with actin. However, pCa-tension and pCa-stiffness studies demonstrated that pCa50 (Ca2+ sensitivity) and nH (cooperativity) were respectively not different among mutants and WT groups, indicating that phosphorylation of cMyBP-C has a minimal effect on the regulatory system. The decreased amount of isometric tension only in DAD indicates that phosphorylation of S273 and S302 are most significant and they diminish the force generation capability, presumably owing to the extra electrostatic interaction of the M domain of cMyBP-C with actin thin filament, which may serve as a break.