Revisiting the SRX paradox for myosin and myofibrils

Q: What is the clinical evidence from this study?

Population: Striated muscle myosin and myofibril regulation (super-relaxed state). Intervention: Mavacamten and 2'-deoxyATP (dATP) vs. No drug / 100% mant-ATP control. Primary outcome: Fast-phase mant-ATP single-turnover rate constant (kobs,fast) in porcine cardiac myofibrils (p=<0.05).

Q: What does this research mean for the field?

Mavacamten (0.5 µM) shifts porcine cardiac myofibril ATP turnover toward the slow phase and decreases the fast-phase rate, while isolated monomeric and synthetic filament myosins exhibit only a single turnover phase without evidence of a distinct super-relaxed state. Novelty: ClaimNovelty.CONTRADICTORY. Consensus alignment: ConsensusAlignment.CHALLENGES_CONSENSUS.

Q: What question did this study set out to answer?

To test whether isolated myosin isoforms or synthetic thick filaments display the biochemically defined super relaxed (SRX) state, and to compare their turnover kinetics with intact cardiac myofibrils.

Kerry Y. Kao; Kalen Z. Robeson; Anthony Asencio; Tristan Wasley; Timothy S. McMillen; Joseph D. Powers; Michael A. Geeves; Michael Regnier

doi:10.64898/2026.10.27.762302

October 29, 2026Open Access

Revisiting the SRX paradox for myosin and myofibrils

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Key result

Mavacamten slows fast-phase ATP turnover by ~31% in cardiac myofibrils.

P<0.05

Why the study?

Previous mant-ATP displacement assays in tissue-isolated HMM found no evidence of the biochemically-defined super relaxed (SRX) state, prompting evaluation in isolated monomeric myosin, synthetic myosin filaments, and myofibrils.

Does structural complexity (monomeric myosin, synthetic thick filaments, or intact myofibrils) and treatment with mavacamten or 2'-deoxyATP alter mant-ATP single-turnover displacement kinetics and the presence of a biochemically-defined super-relaxed (SRX) state in striated and cardiac muscle preparations?

Key Points

To test whether isolated myosin isoforms or synthetic thick filaments display the biochemically defined super relaxed (SRX) state, and to compare their turnover kinetics with intact cardiac myofibrils.
Conducted mant-ATP displacement assays on monomeric myosin from rabbit psoas, bovine masseter, and porcine left ventricle, as well as synthetic thick filaments dialyzed to 0.1 M KCl.
Assayed single-turnover kinetics under perturbation with the inhibitor Mavacamten (0 to 10 µM) or the activator mant-2'-deoxyATP (mant-dATP) across varying concentrations.
Measured mant-ATP single-turnover kinetics in native isolated porcine left ventricular cardiac myofibrils in the presence and absence of Mavacamten (0.5 µM) or mant-dATP.
Monomeric myosin and synthetic thick filaments displayed single-exponential turnover kinetics with kobs ranging from 0.0051 to 0.0120 s-1 for monomers and 0.0159 to 0.0449 s-1 for filaments, showing no evidence of a distinct slow SRX phase.
Mavacamten slowed mant-ATP turnover in porcine cardiac myosin by roughly 10-fold in monomers (Ki = 0.48 µM) and 15-fold in synthetic filaments (Ki = 0.37 µM), while mant-dATP accelerated turnover in a biphasic manner without inducing a two-exponential decay.
Cardiac myofibrils exhibited two distinct turnover phases with kobs,fast = 0.0382 ± 0.0043 s-1 and kobs,slow = 0.0043 ± 0.0010 s-1 at an amplitude ratio of 76:24, shifting to 56:44 with 0.5 µM Mavacamten, whereas mant-dATP abolished the slow phase.

Structured PICO

Does structural complexity (monomeric myosin, synthetic thick filaments, or intact myofibrils) and treatment with mavacamten or 2'-deoxyATP alter mant-ATP single-turnover displacement kinetics and the presence of a biochemically-defined super-relaxed (SRX) state in striated and cardiac muscle preparations?

P

Population

In vitro biochemical study using isolated full-length myosin, synthetic thick filaments, and myofibrils from porcine cardiac left ventricular, rabbit psoas, and bovine masseter tissues.

I

Intervention

Mavacamten (0 to 10 µM for isolated myosin/synthetic thick filaments; 0.5 µM or 5 µM for myofibrils) or 2'-deoxyATP (mant-dATP, replacing 0% to 100% of mant-ATP) in stopped-flow kinetic assays at 20 °C

C

Comparator

Control (0 µM mavacamten / no drug, or 100% mant-ATP without dATP) in stopped-flow assay buffer

O

Outcome

Single-turnover mant-ATP displacement kinetics (observed rate constants kobs, fast and slow phase amplitudes, and single- vs double-exponential decay fits) measured via FRET from protein tryptophan to mant-ATP over 300 to 2500 secondssurrogate

Main Result

Absolute Event Rate: 0.0263% vs 0.0382%

p-value: p=<0.05

This biochemical study demonstrates that the slow ATP turnover phase (classically defining the myosin super-relaxed state) is absent in isolated monomeric myosin and synthetic thick filaments, requiring the intact sarcomeric architecture of myofibrils to manifest as distinct, non-interconverting myosin populations.

Limitations

In vitro biochemical assay using animal tissue preparations rather than human clinical subjects
Myofibril preparations are intrinsically more variable than purified proteins with a much smaller total fluorescence signal amplitude (~4-5% vs 20-25%)
The slow phase in myofibrils represents only 1% of the total signal, making detailed titrations of Mavacamten or dATP on slow-phase parameters futile
Bovine masseter monomeric myosin exhibited prolonged slow baseline drift due to light scattering from slow aggregation
Myofibrils are intrinsically more variable than purified proteins and the total fluorescence amplitude observed is much smaller (~4-5% compared to 20-25% for isolated myosin), making detailed analysis more problematic.
Detailed titrations of either mavacamten or dATP with myofibrils proved futile because the slow phase amplitude is very small (~1% of the total signal).
Monomeric bovine masseter muscle myosin exhibited prolonged slow drift of the baseline lasting beyond 1000 s, assumed to be due to a light scattering artifact caused by slow aggregation.

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Implication

Mavacamten modulates porcine myofibril kinetics; leaves open SRX relevance to human disease and precludes practice change.

Abstract

We recently reported that we could find no evidence of the biochemically-defined super relaxed (SRX) state in tissue-isolated HMM, as defined in the original mant-ATP displacement assay. This is consistent with theoretical predictions that any SRX state would have to be in thermal equilibrium with the disordered-relaxed (DRX) form of myosin heads. We repeat the assay here for isolated myosin, from different sources, in its monomeric form or in synthetic myosin filaments. No slow component of the displacement assay was observed, and we again conclude that there is no evidence for the SRX state for isolated myosins. This does not mean that there are no myosins in the ATPase-inhibiting Interacting Heads Motif (IHM) in our assays, just that any IHM is in rapid equilibrium with DRX heads. Mant-ATP displacement assays in the presence of the inhibitor Mavacamten or the activator 2'-deoxyATP (dATP) do provide evidence of an interaction between the two heads of myosin. In contrast, assays with myofibrils do provide evidence of two components in the mant-ATP displacement assay. We propose that these two components result from distinct populations of myosin within the sarcomere thick filament with different fractions of myosin in the IHM structural state.

Discussion

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Cite This Study

Kao et al. (2026) studied Striated muscle myosin and myofibril regulation (super-relaxed state). Mavacamten and 2'-deoxyATP (dATP) vs. No drug / 100% mant-ATP control was evaluated on Fast-phase mant-ATP single-turnover rate constant (kobs,fast) in porcine cardiac myofibrils (p=<0.05).

synapsesocial.com/papers/6ae585344c46c197bbab385d https://doi.org/10.64898/2026.10.27.762302

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Revisiting the SRX paradox for myosin and myofibrils

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Revisiting the SRX paradox for myosin and myofibrils

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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