Phosphorylation of Cardiac Myosin Binding Protein-C Acts as On/Off Switch to Modulate cMyBP-C's Inhibition of Actomyosin Power Generation in the Load-Clamp Laser Trap Assay

Abstract

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains- C0-C10, binds myosin LMM via its C-terminus, while its N-terminus binds both myosin S2 and actin. These N-terminal interactions can be attenuated by phosphorylation of serines within the C1-C2 linker. In vivo, cMyBP-C exists in a range of phosphorylation states, which may alter its regulation of actomyosin. Therefore, N-terminal fragments (C0-C3) of cMyBP-C were bacterially expressed with one or more phosphorylatable serines (S273, S282, S302) mutated to either alanines to mimic dephosphorylation or aspartic acids as phosphomimetics. Using C0-C3 fragments that were effectively mono-, di- or tri-phosphorylated, we characterized how the extent of cMyBP-C phosphorylation modulated the the force:velocity relationship generated by a small ensemble (∼8 motors) of skeletal muscle myosin in the load-clamped laser trap assay. Mono-phosphorylated C0-C3 inhibited unloaded velocity by 70% as did unphosphorylated C0-C3, while di- and tri-phosphorylated constructs were indistinguishable and less inhibitory (30%). Maximal force was unaffected by the extent of phosphorylation. Thus, the un- and mono-phosphorylated C0-C3 constructs depressed the force-velocity relationship, which was well fitted by a modified Hill equation containing an assumed viscous load due to C0-C3 binding to actin (Weith et al., 2011). In addition, both un- and mono- phosphorylated C0-C3 reduced peak power by ∼40%, while neither di- nor tri-phosphorylated C0-C3 had any effect on peak power. cMyBP-C phosphorylation appears to modulate cMyBP-C's inhibition of actomyosin power generation in an “on/off” manner.