Myosin-binding protein C N-terminal domains slow Ca2+ dissociation from cardiac thin filaments.

The cardiac thin filament (cTF) regulates contraction through Ca2+ binding to, and release from, cardiac troponin C (cTnC), yet how sarcomeric accessory proteins influence these kinetics remains incompletely understood. Cardiac myosin-binding protein C (cMyBP-C) N-terminal domains interact with the thin filament and are regulated by protein kinase A (PKA)-mediated phosphorylation. To determine whether cMyBP-C alters thin filament Ca2+ exchange kinetics, we performed stopped-flow measurements using IAANS-labeled cTnC in reconstituted cTFs. The N-terminal fragments C0-C2 and C1-C2 slowed Ca2+ dissociation in a concentration-dependent manner, whereas C0-C1 produced only a modest effect. PKA-mediated phosphorylation markedly attenuated the slowing by C0-C2 and C1-C2. Thus, N-terminal domain composition and phosphorylation state strongly influence cMyBP-C-mediated regulation of thin filament Ca2+ dissociation. In contrast, the tested cMyBP-C fragments did not alter Ca2+ association kinetics or Ca2+ dissociation from isolated troponin. Cosedimentation measurements further showed that differences in kinetic effects were not explained solely by differences in thin filament occupancy. Together, these results demonstrate that cMyBP-C N-terminal fragments selectively modulate Ca2+ dissociation from cTnC in myosin-free reconstituted cardiac thin filaments. The absence of an effect on isolated troponin is consistent with regulation involving additional thin filament components, including actin and/or tropomyosin, although tropomyosin-dependent and -independent mechanisms remain possible. This work identifies a previously unrecognized effect of cMyBP-C N-terminal domains on cTnC Ca2+ dissociation in the cardiac thin filament.