Helix Three (H3) of the M-Domain of Cardiac Myosin Binding Protein-C is not Essential for Functional Effects in Permeabilized Rat Trabeculae

Abstract

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Cardiac specific phosphorylation sites in the M-domain regulate binding of cMyBP-C to myosin and actin. Immediately downstream of the phosphorylation sites is a bundle of three α-helixes that are well conserved across all species and isoforms of MyBP-C, however the functional significance of these helices is unknown. Helix 3 (H3) bears high homology to the inhibitory peptide of troponin I, suggesting it as a potential binding site for interactions with actin. To understand the functional significance of H3, we produced a recombinant N-terminal protein containing domains C1, M and C2 (C1C2) with the H3 sequence deleted (H3KO). We then assessed effects of the mutant H3KO protein on contractile forces in permeabilized rat trabeculae and compared them to effects of the wild-type C1C2. Results showed that 5µM C1C2 significantly increased Ca2+-sensitivity of force (ΔpCa50=0.29±0.03), whereas 5µM H3KO had no effect (ΔpCa50=0.02±0.01) and 10µM H3KO only modestly increased Ca2+-sensitivity (ΔpCa50=0.11±0.02,). Similarly, whereas 10µM C1C2 activated force in the absence of Ca2+ (pCa 9.0), H3KO activated force in the absence of Ca2+ only at concentrations >20µM. Together, these results establish that H3 is not absolutely required for the functional effects of the N-terminal domains of cMyBP-C to either increase Ca2+-sensitivity of force or to activate tension in sarcomeres, but that H3 contributes to the overall efficacy of the N-terminus in mediating these effects. Potentially, H3 could dock the N-terminus of cMyBP-C with actin or extend/stabilize cMyBP-C interactions with actin or other regulatory proteins. This work was supported by NIH HL-080367 (SPH), NDSEG and AHA graduate fellowships (KLB), and AHA undergraduate fellowship (JKK).