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.

Abstract

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. 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. 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 <i>γ</i>C0C7 (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 (<i>r</i>)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 <i>γ</i>C0C7 reduced myofilament Ca²⁺ sensitivity and increased cross-bridge cycling (<i>k</i>_tr) at submaximal [Ca²⁺]. Acute loss of <i>γ</i>C0C7 also induced auto-oscillatory contractions at submaximal [Ca²⁺]. Ligation of <i>r</i>C0C7 (exogenous [recombinant] N'-terminal domains C0 to C7 of cardiac myosin binding protein-C)-sc returned pCa₅₀ and <i>k</i>_tr to control values and abolished oscillations, but phosphorylated (p)-<i>r</i>C0C7-sc did not completely rescue these effects. 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)-<i>r</i>C0C7-sc was less effective at damping contractile oscillations, results suggest that spontaneous oscillatory contractions may contribute to enhanced contractility in response to inotropic stimuli.