Cardiac Myosin Binding Protein-C Slows Cross-Bridge Kinetics And Increases Mgatp Sensitivity

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that augments filament stiffness and modulates cardiac contractility through its phosphorylation. We investigated the effect of cMyBP-C on myosin cross-bridge kinetics in demembranated papillary muscle strips from non-transgenic (NTG) and transgenic homozygous mice lacking cMyBP-C (t/t). Both groups expressed ∼100% β-myosin heavy chain isoform due to hypothyroidism. Sinusoidal length perturbation analysis at maximal Ca2+-activation (pCa 4.8 at 17° C, 0 mM Pi) showed that MgATP-dependent myosin detachment rate (g) increased hyperbolically with MgATP (0.025 to 5 mM), with saturating values of 2.5±0.2 and 3.9±0.2 s−1 (P<0.01) for NTG and t/t, respectively. Kinetic analysis of g vs. [MgATP] resulted in a lower [MgATP]50 for NTG than t/t (187±45 vs. 361±58 μM, P<0.05). As myosin attachment time (ton) is inversely related to g, ton correspondingly decreased as [MgATP] increased, with saturating values of 71.3±5.0 and 44.2±2.3 ms (P<0.01) for NTG vs. t/t, respectively. At saturating MgATP, ton decreased as [Pi] increased in both groups (0-12 mM), although ton remained ∼25% longer at each [Pi] for NTG vs. t/t. Protein kinase-A (PKA) incubation reduced ton ∼20% at each [Pi] for NTG, but did not affect ton for t/t. The longer ton for NTG at saturating MgATP suggests a longer lived MgADP myosin cross-bridge state when cMyBP-C is present. The lower [MgATP]50 for NTG further suggests that cMyBP-C elevates the nucleotide binding pocket affinity for MgATP in addition to MgADP. cMyBP-C phosphorylation by PKA may diminish this affinity resulting in a shorter ton. These results support a functional role for cMyBP-C slowing myosin kinetics, possibly through altered strain-dependent myosin kinetics due to cMyBP-C supporting a stiffness of the thick filament or myofilament lattice that is diminished with PKA phorphorylation of cMyBP-C.