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 (k_tr) 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 k_tr 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. VISUAL OVERVIEW: An online visual overview is available for this article.