Cardiac myosin-binding protein C N-terminal domains differentially affect calcium dissociation from the cardiac thin filament.

Abstract

We have evaluated the effects of myosin-binding protein C (cMyBP-C) N-terminal domains on the kinetics of Ca2+ exchange with cardiac troponin C (cTnC) in reconstituted cardiac thin filaments (CTFs). The CTF is an essential component of the sarcomere, conferring Ca2+ regulation to muscle contraction and relaxation via Ca2+ exchange with cTnC. cMyBP-C is a sarcomeric protein anchored to the thick filament, only in the C-zone, by its C-terminal domains, allowing for its N-terminal domains to interact with the thin and thick filaments. These interactions are further modulated by PKA-mediated phosphorylation of cMyBP-C's M-domain. Stopped-flow combined with fluorescently labeled-cTnC (cTnC-IAANS) was used to measure Ca2+ exchange kinetics with cMyBP-C fragments titrated onto CTFs. The presence of cMyBP-C domains C0 through C2 (C0-C1-M-C2, i.e., C0-C2) slowed Ca2+ dissociation from CTFs in a concentration-dependent manner. Phosphorylation of C0-C2 blunted this effect. Addition of domains C0 through C1 (C0-C1, lacking C2 and M-domain) did not change Ca2+ dissociation. Domains C1 through C2 (C1-M-C2, i.e., C1-C2) also slowed Ca2+ dissociation in a dose-dependent manner, but to a significantly lesser extent than C0-C2. Phosphorylation of C1-C2 blunted this effect; however, this blunting was reduced as compared to phosphorylated C0-C2. Ca2+ association was unchanged upon titration with all cMyBP-C fragments tested. In control experiments, Ca2+ dissociation from cTnC in cTn alone was not affected by cMyBP-C, indicating that cMyBP-C effects on the CTF are likely mediated through actin and/or tropomyosin. These results suggest a new role for cMyBP-C in the regulation of contraction by modulating Ca2+ dissociation from cTnC. We propose that tuning Ca2+ dissociation in the C-zone where cMyBP-C is located, and further fine-tuning by phosphorylation, synchronizes the timing of contraction/relaxation across the span of the sarcomere.