TY - JOUR
T1 - In situ FRET-based localization of the N-terminus of myosin binding protein-C in heart muscle cells
AU - Chandler, Jessica
AU - Treacy, Conor
AU - Ameer-Beg, Simon
AU - Ehler, Elisabeth
AU - Irving, Malcolm
AU - Kampourakis, Thomas
N1 - Funding Information:
ACKNOWLEDGMENTS. We would like to thank Mathias Gautel and Mark Holt for help and advice. This study was supported by grants from the British Heart Foundation. J.C. was supported by a PhD studentship from the British Heart Foundation Centre of Research Excellence at King’s College London. T.K. was supported by a British Heart Foundation Intermediate Basic Science Research Fellowship (FS/16/3/31887). Portions of this work were developed from the doctoral dissertation of Jessica Chandler.
Publisher Copyright:
Copyright © 2023 the Author(s).
PY - 2023/3/21
Y1 - 2023/3/21
N2 - Cardiac myosin binding protein-C (cMyBP-C) is a thick filament–associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer–fluorescence lifetime imaging (FRET–FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with β-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.
AB - Cardiac myosin binding protein-C (cMyBP-C) is a thick filament–associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer–fluorescence lifetime imaging (FRET–FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with β-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.
UR - http://www.scopus.com/inward/record.url?scp=85150112868&partnerID=8YFLogxK
U2 - 10.1073/pnas.2222005120
DO - 10.1073/pnas.2222005120
M3 - Article
SN - 0027-8424
VL - 120
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 12
M1 - e2222005120
ER -