The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner.

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0-C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1-C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0-C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0-C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0-C3 in proportion to the total number phosphomimetic replacements. Under load, C0-C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0-C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0-C3 on F:V was well fitted by a model that assumed C0-C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.

Roles for cardiac MyBP-C in maintaining myofilament lattice rigidity and prolonging myosin cross-bridge lifetime.

We investigated the influence of cardiac myosin binding protein-C (cMyBP-C) and its constitutively unphosphorylated status on the radial and longitudinal stiffnesses of the myofilament lattice in chemically skinned myocardial strips of the following mouse models: nontransgenic (NTG), effective null for cMyBP-C (t/t), wild-type cMyBP-C expressed into t/t (WT(t/t)), and constitutively unphosphorylated cMyBP-C (AllP-(t/t)). We found that the absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) resulted in a compressible cardiac myofilament lattice induced by rigor not observed in the NTG and WT(t/t). These results suggest that the presence and phosphorylation of the N-terminus of cMyBP-C provides structural support and radial rigidity to the myofilament lattice. Examination of myofilament longitudinal stiffness under rigor conditions demonstrated a significant reduction in cross-bridge-dependent stiffness in the t/t compared with NTG controls, but not in the AllP-(t/t) compared with WT(t/t) controls. The absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) both resulted in a shorter myosin cross-bridge lifetime when myosin isoform was controlled. These data collectively suggest that cMyBP-C provides radial rigidity to the myofilament lattice through the N-terminus, and that disruption of the phosphorylation of cMyBP-C is sufficient to abolish this structural role of the N-terminus and shorten cross-bridge lifetime. Although the presence of cMyBP-C also provides longitudinal rigidity, phosphorylation of the N-terminus is not necessary to maintain longitudinal rigidity of the lattice, in contrast to radial rigidity.