The super-relaxed (SRX) state, characterized by low ATPase activity, has been studied heavily in the context of thick filament regulation during normal and pathological physiology. Mohan et al. (2024 https://doi.org/10.1016/j.jbc.2023.105565) demonstrated that the mantATP displacement assay could not be used to estimate the presence of any SRX in tissue purified HMM from cardiac, fast or slow skeletal muscle. Here, we extend these studies to whole myosin, either soluble (in 0.5 M KCl) or in synthetic thin filaments (0.1 M KCl). In all cases the displacement of mantATP by unlabeled ATP results in a single exponential decrease in the mant fluorescence signal. This is compatible with any SRX present being in relatively rapid equilibrium with the disordered relaxed (DRX) state of myosin heads. Our result for HMM has been recently confirmed by Cail et al. 2025 (https://doi.org/10.1016/j.jbc.2025.108412) for human cardiac HMM expressed in mouse C2C12 cell lines. These authors suggest that direct comparison of the mantATP displacements by HMM and S1 could reveal the % SRX present. Assuming that mant release by HMM heads in a DRX conformation ( k DRX ) was identical to that by S1 and turnover by SRX heads is approximately zero. Then k HMM / k S1 = SRX/(SRX + DRX) = α, and the observed rate of mant displacement is α· k DRX . We tested this assumption by comparing mantATP displacements for S1 and myosin as a function of salt concentration (0.02 M–0.5 M), conditions where the any SRX and is expected to diminish as salt concentration increases. We also demonstrate that the mantATP displacement assay is suitable to use with myosin or myofibrils derived from human iPSCs, opening up the potential of mantATP based assays of cardiac patient-derived iPSCs.
Kao et al. (2026) studied this question.