A Novel "Cut and Paste" Method for In Situ Replacement of cMyBP-C Reveals a New Role for cMyBP-C in the Regulation of Contractile Oscillations.

RATIONALE: cMyBP-C (cardiac myosin-binding protein-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere. OBJECTIVE: The goal of this study was to validate a novel hybrid genetic/protein engineering approach for rapid manipulation of cMyBP-C in sarcomeres in situ. METHODS AND RESULTS: We designed a novel cut and paste approach for removal and replacement of cMyBP-C N'-terminal domains (C0-C7) in detergent-permeabilized cardiomyocytes from gene-edited Spy-C mice. Spy-C mice express a TEVp (tobacco etch virus protease) cleavage site and a SpyTag (st) between cMyBP-C domains C7 and C8. A cut is achieved using TEVp which cleaves cMyBP-C to create a soluble N'-terminal γC0C7 (endogenous [genetically encoded] N'-terminal domains C0 to C7 of cardiac myosin binding protein-C) fragment and an insoluble C'-terminal SpyTag-C8-C10 fragment that remains associated with thick filaments. Paste of new recombinant (r)C0C7 domains is achieved by a covalent bond formed between SpyCatcher (-sc; encoded at the C'-termini of recombinant proteins) and SpyTag. Results show that loss of γC0C7 reduced myofilament Ca2+ sensitivity and increased cross-bridge cycling (ktr) at submaximal [Ca2+]. Acute loss of γC0C7 also induced auto-oscillatory contractions at submaximal [Ca2+]. Ligation of rC0C7 (exogenous [recombinant] N'-terminal domains C0 to C7 of cardiac myosin binding protein-C)-sc returned pCa50 and ktr to control values and abolished oscillations, but phosphorylated (p)-rC0C7-sc did not completely rescue these effects. CONCLUSIONS: We describe a robust new approach for acute removal and replacement of cMyBP-C in situ. The method revealed a novel role for cMyBP-C N'-terminal domains to damp sarcomere-driven contractile waves (so-called spontaneous oscillatory contractions). Because phosphorylated (p)-rC0C7-sc was less effective at damping contractile oscillations, results suggest that spontaneous oscillatory contractions may contribute to enhanced contractility in response to inotropic stimuli.

Point mutations in the tri-helix bundle of the M-domain of cardiac myosin binding protein-C influence systolic duration and delay cardiac relaxation.

Cardiac myosin binding protein-C (cMyBP-C) is an essential regulatory protein required for proper systolic contraction and diastolic relaxation. We previously showed that N'-terminal domains of cMyBP-C stimulate contraction by binding to actin and activating the thin filament in vitro. In principle, thin filament activating effects of cMyBP-C could influence contraction and relaxation rates, or augment force amplitude in vivo. cMyBP-C binding to actin could also contribute to an internal load that slows muscle shortening velocity as previously hypothesized. However, the functional significance of cMyBP-C binding to actin has not yet been established in vivo. We previously identified an actin binding site in the regulatory M-domain of cMyBP-C and described two missense mutations that either increased (L348P) or decreased (E330K) binding affinity of recombinant cMyBP-C N'-terminal domains for actin in vitro. Here we created transgenic mice with either the L348P or E330K mutations to determine the functional significance of cMyBP-C binding to actin in vivo. Results showed that enhanced binding of cMyBP-C to actin in L348P-Tg mice prolonged the time to end-systole and slowed relaxation rates. Reduced interactions between cMyBP-C and actin in E330K-Tg mice had the opposite effect and significantly shortened the duration of ejection. Neither mouse model displayed overt systolic dysfunction, but L348P-Tg mice showed diastolic dysfunction presumably resulting from delayed relaxation. We conclude that cMyBP-C binding to actin contributes to sustained thin filament activation at the end of systole and during isovolumetric relaxation. These results provide the first functional evidence that cMyBP-C interactions with actin influence cardiac function in vivo.