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

Circ Res. 2020 Mar 13;126(6):737-749. doi: 10.1161/CIRCRESAHA.119.315760. Epub 2020 Feb 13.

Abstract

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.

Keywords: SpyCatcher; SpyTag; cardiac myosin binding protein-C; cardiomyocyte; myosin; phosphorylation; sarcomere.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Calcium Signaling*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Gene Editing / methods*
  • Mice
  • Mice, Inbred C57BL
  • Myocardial Contraction*
  • Protein Domains
  • Protein Engineering / methods*
  • Sarcomeres / metabolism*
  • Sarcomeres / physiology

Substances

  • Carrier Proteins
  • myosin-binding protein C
  • Endopeptidases
  • TEV protease