Editorial: Release of inorganic phosphate from the myosin active site in actomyosin energy transduction

development in cardiac myofibrils. Tests of the results against six single-pathway models of the cross-bridge cycle, suggest either slow Pi binding or slow force reversal. However, the models do not consider strain-dependence of transition rates which might alter the conclusions (cf. (Månsson, 2025) and (link 4)). Nonetheless, the non-linear negative correlation described by Stehle (link 5) adds to the list of phenomena to explain.Li et al. (link 6) used quantum dots (QDs), whose fluorescence is reduced upon increased local temperature, to study critical illness myopathy (CIM; (Friedrich et al., 2018)) in a rat intensive care model. They show that myosin extracted from muscle fibre bundles rich in type I myosin, from CIM animals, exhibited universal increase in heat production consistent with decrease in myosin efficiency. Beyond insights into the CIM pathogenesis, novel methodology is presented for analyzing free energy changes during actomyosin cycling dominated by Pi-release from myosin.Finally, a theoretical paper (link 7) considers the mechanism of action of a small molecule myosin-activating compound, omecamtiv mecarbil, highlighting the central role of Pi-release in the mechanism of myosin-active drugs.Different papers in this topic present intricate experimental and modelling data to support specific models for the coupling between Pi-release and force-generation (Table 1). However, conclusive evidence is lacking. Limitations include different assumptions used in data interpretation and absence of methodological means for direct observation of critical details. As pointed out by Debold et al. (link 2), a key goal would be to visualize the location of Pi on myosin while simultaneously detecting the lever arm position. A recent time-resolved cryo-EM study (Klebl et al., 2025) may be a step towards that goal. Another issue which complicates data interpretation is the difficulty of calibrating all model parameter values against experimental results (links 4, 7) to create coherence between work on isolated proteins and large myosin ensembles interacting with actin filaments, outside or inside an organized sarcomere. Moreover, macro-level experiments, e.g. from single muscle fibres, are affected by biomechanical complexities, e.g. sarcomere non-uniformities (cf. (Stehle, 2017;Rassier and Månsson, 2025)), not considered in modelling. Moreover, from a physiological point of view, accumulation of Pi occurs together with proton accumulation and temperatures above 30 o C (for mammals), with myosin types presenting differing kinetics changes under fatigue (Karatzaferi et al., 2017). This emphasizes the importance of assessing the effects of varied [Pi] while considering modulating and synergistic roles of other factors, particularly protons and temperature but also ADP and myosin light chain phosphorylation (Karatzaferi et al., 2004;Karatzaferi et al., 2008;Debold, 2012).Beyond uncertainties regarding whether Pi-release occurs before or after the power stroke or if there is loose coupling between power stroke and Pi-release, there are other outstanding issues. This includes the possible role of Pi-binding on myosin outside the active site and what happens upon Pi rebinding to myosin. Moreover, the possibility of different mechanisms in different myosin isoforms was brought up (e.g. in links 1, 5) as well as the need to obtain more experimental data under coherent conditions to calibrate model parameter values in a consistent way. Finally, modelling using simple kinetic schemes vs. more complex mechanokinetic models requires further consideration. To conclude, the present research topic gives an insightful overview of current understanding and suggests interesting paths forward. Pi-release is assumed to be necessary for the power-stroke to occur (cf. (Llinas et al., 2015)) a) Single-step Pi-release (e.g. (Smith, 2014)) b) Multi-step Pi-release (e.g. (Moretto et al., 2022)) 2. Powerstroke first Pi-release occurs first after the forcegenerating power-stroke (e.g. (Dantzig et al., 1992)) a) Pi-rebinding e.g. in muscle fatigue leads to reversal of the power-stroke (Dantzig et al., 1992) b) Pi-rebinding leads to detachment into a post-powerstroke Pi-bound state (e.g. (Marang et al., 2025) Each of several power-stroke transitions into different "mechanical states" are governed by strain in the myosin elasticity, independent of whether Pi and ADP or only ADP is at the active site. Pi-release can occur from any mechanical state but varying conformations lead to different kinetics.Two elements that seem to be integral parts of the model (but that may possibly be omitted) are myosin slippage transitions between actin sites and a branched pathway with irreversible detachment (with certain probability) into a prepower-stroke Pi-bound state