Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament

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Abstract

Key points

Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.
Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.
Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.
Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.
Thick filament regulation is a promising target for novel therapeutics in heart disease.

Abstract

Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Key points

Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.
Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.
Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.
Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.
Thick filament regulation is a promising target for novel therapeutics in heart disease.

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Most cited references 57

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Regulation of contraction in striated muscle.

A M Gordon, E. Homsher, M Regnier (2000)

Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

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Mechanism of blebbistatin inhibition of myosin II.

Mihály Kovács, Judit Tóth, Csaba Hetényi … (2004)

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.

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Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Sheri Hartman, John Finer, David Morgans … (2011)

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

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Author and article information

Contributors

Malcolm Irving:

ORCID: http://orcid.org/0000-0001-6419-3583

malcolm.irving@kcl.ac.uk

Journal

Journal ID (nlm-ta): J Physiol

Journal ID (iso-abbrev): J. Physiol. (Lond.)

Journal ID (doi): 10.1111/(ISSN)1469-7793

Journal ID (publisher-id): TJP

Journal ID (hwp): jphysiol

Title: The Journal of Physiology

Publisher: John Wiley and Sons Inc. (Hoboken )

ISSN (Print): 0022-3751

ISSN (Electronic): 1469-7793

Publication date (Electronic): 21 November 2017

Publication date (Print): 01 January 2018

Publication date PMC-release: 21 November 2017

Volume: 596

Issue: 1 ( doiID: 10.1113/tjp.2018.596.issue-1 )

Pages: 31-46

Affiliations

[ ¹ ] Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence King's College London London SE1 1UL UK

Author notes

[*] [* ] Corresponding author M. Irving: The Randall Centre for Cell and Molecular Biophysics and BHF Centre of Research Excellence, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK. Email: malcolm.irving@ 123456kcl.ac.uk

Author information

Malcolm Irving http://orcid.org/0000-0001-6419-3583

Article

Publisher ID: TJP12679

DOI: 10.1113/JP275050

PMC ID: 5746517

PubMed ID: 29052230

SO-VID: 4032c75d-12c5-4215-957e-5d146df73062

License:

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

History

Date received : 02 August 2017

Date accepted : 06 October 2017

Page count

Figures: 7, Tables: 1, Pages: 16, Words: 10325

Funding

Funded by: British Heart Foundation

Award ID: FS/09/001/26329

Award ID: FS/16/3/31887

Award ID: PG/12/52/29713

Custom metadata

source-schema-version-number 2.0

component-id tjp12679

cover-date 1 January 2018

details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_NLMPMC version:5.2.8 mode:remove_FC converted:28.12.2017

ScienceOpen disciplines: Human biology

Keywords: cardiac myosin,muscle contraction,cardiac muscle regulation,fluorescence polarization,omecamtiv mecarbil

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ScienceOpen disciplines: Human biology

Keywords: cardiac myosin, muscle contraction, cardiac muscle regulation, fluorescence polarization, omecamtiv mecarbil

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament

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Regulatory T cell (Treg) therapy

Most cited references 57

Regulation of contraction in striated muscle.

Mechanism of blebbistatin inhibition of myosin II.

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

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