Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

So Jin Park-Holohan; Elisabetta Brunello; Thomas Kampourakis; Martin Rees; Malcolm Irving; Luca Fusi

doi:10.1073/pnas.2023706118

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

So Jin Park-Holohan, Elisabetta Brunello, Thomas Kampourakis, Martin Rees, Malcolm Irving, Luca Fusi

King's British Heart Foundation Centre, King's College London

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Abstract

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

Original language	English
Article number	e2023706118
Journal	Proceedings of the National Academy of Science (USA)
Volume	118
Issue number	16
Early online date	13 Apr 2021
DOIs	https://doi.org/10.1073/pnas.2023706118
Publication status	Published - 20 Apr 2021

Keywords

heart muscle
muscle regulation
myosin motor
myosin-binding protein C

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10.1073/pnas.2023706118Licence: CC BY

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title = "Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing",

abstract = "Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.",

keywords = "heart muscle, muscle regulation, myosin motor, myosin-binding protein C",

author = "Park-Holohan, {So Jin} and Elisabetta Brunello and Thomas Kampourakis and Martin Rees and Malcolm Irving and Luca Fusi",

note = "Funding Information: We are grateful to Marty Rajaratnam (King's College London) for mechanical engineering support, Yin-Biao Sun (King's College London) for providing the wild-type cardiac troponin C, and Birgit Brandmeier (King's College London) for providing DNA vectors for smMLCK and calmodulin. This work and the investigators were supported by the Wellcome Trust/Royal Society and by the British Heart Foundation (BHF). S.J.P.-H. was funded by the BHF (Grant PG/16/19/32072 awarded to Malcolm Irving); E.B. was funded by a BHF Intermediate Basic Science Research Fellowship (Award FS/17/3/32604); T.K. was funded by a BHF Intermediate Basic Science Research Fellowship (Award FS/16/3/31887); M.R. was funded by the BHF (Grant RG/15/8/31480 awarded to Mathias Gautel); and L.F. was funded by a Sir Henry Dale Fellowship awarded by the Wellcome Trust and the Royal Society (Award 210464/Z/18/Z). Publisher Copyright: {\textcopyright} This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).",

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AU - Park-Holohan, So Jin

AU - Brunello, Elisabetta

AU - Kampourakis, Thomas

AU - Rees, Martin

AU - Irving, Malcolm

AU - Fusi, Luca

N1 - Funding Information: We are grateful to Marty Rajaratnam (King's College London) for mechanical engineering support, Yin-Biao Sun (King's College London) for providing the wild-type cardiac troponin C, and Birgit Brandmeier (King's College London) for providing DNA vectors for smMLCK and calmodulin. This work and the investigators were supported by the Wellcome Trust/Royal Society and by the British Heart Foundation (BHF). S.J.P.-H. was funded by the BHF (Grant PG/16/19/32072 awarded to Malcolm Irving); E.B. was funded by a BHF Intermediate Basic Science Research Fellowship (Award FS/17/3/32604); T.K. was funded by a BHF Intermediate Basic Science Research Fellowship (Award FS/16/3/31887); M.R. was funded by the BHF (Grant RG/15/8/31480 awarded to Mathias Gautel); and L.F. was funded by a Sir Henry Dale Fellowship awarded by the Wellcome Trust and the Royal Society (Award 210464/Z/18/Z). Publisher Copyright: © This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

PY - 2021/4/20

Y1 - 2021/4/20

N2 - Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

AB - Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

KW - heart muscle

KW - muscle regulation

KW - myosin motor

KW - myosin-binding protein C

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JO - Proceedings of the National Academy of Science (USA)

JF - Proceedings of the National Academy of Science (USA)

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ER -

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

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