Sarcomere integrated biosensor detects myofilament-activating ligands in real time during twitch contractions in live cardiac muscle

Anthony D Vetter, Ashley A. Martin, Brian R. Thompson, David D. Thomas, Joseph M. Metzger

Research output: Contribution to journalArticlepeer-review

Abstract

The sarcomere is the functional unit of cardiac muscle, essential for normal heart function. To date, it has not been possible to study, in real time, thin filament-based activation dynamics in live cardiac muscle. We report here results from a cardiac troponin C (TnC) FRET-based biosensor integrated into the cardiac sarcomere via stoichiometric replacement of endogenous TnC. The TnC biosensor provides, for the first time, evidence of multiple thin filament activating ligands, including troponin I interfacing with TnC and cycling myosin, during a cardiac twitch. Results show that the TnC FRET biosensor transient significantly precedes that of peak twitch force. Using small molecules and genetic modifiers known to alter sarcomere activation, independently of the intracellular Ca2+ transient, the data show that the TnC biosensor detects significant effects of the troponin I switch domain as a sarcomere-activating ligand. Interestingly, the TnC biosensor also detected the effects of load-dependent altered myosin cycling, as shown by a significant delay in TnC biosensor transient inactivation during the isometric twitch. In addition, the TnC biosensor detected the effects of myosin as an activating ligand during the twitch by using a small molecule that directly alters cross-bridge cycling, independently of the intracellular Ca2+ transient. Collectively, these results aid in illuminating the basis of cardiac muscle contractile activation with implications for gene, protein, and small molecule-based strategies designed to target the sarcomere in regulating beat-to-beat heart performance in health and disease.

Original languageEnglish (US)
Pages (from-to)49-61
Number of pages13
JournalJournal of Molecular and Cellular Cardiology
Volume147
DOIs
StatePublished - Oct 2020

Bibliographical note

Funding Information:
We thank Drs. J.M. Muretta and M.A. Sanders for helpful discussions and K.W. Prins and H. Cohen for assistance. We thank A. M?ln?si-Csizmadia for the gift of para-nitroblebbistatin. This work was supported by grants from NIH (HL132874; HL122323), AHA (16PRE30480002) and UMN LHI and IBP.

Funding Information:
We thank Drs. J.M. Muretta and M.A. Sanders for helpful discussions and K.W. Prins and H. Cohen for assistance. We thank A. Málnási-Csizmadia for the gift of para-nitroblebbistatin. This work was supported by grants from NIH ( HL132874 ; HL122323 ), AHA ( 16PRE30480002 ) and UMN LHI and IBP.

Publisher Copyright:
© 2020 Elsevier Ltd

Keywords

  • Myofilament
  • Myosin, contraction, calcium
  • Troponin

PubMed: MeSH publication types

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

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