Asencio, A. M. | bioRxiv Science

bioRxiv2026

Revisiting the SRX paradox for myosin and myofibrils

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.

biochemistry↗

bioRxiv2020

In vivo x-ray diffraction and simultaneous EMG reveal the time course of myofilament lattice dilation and filament stretch

Muscles function within an organism depends on the feedback between molecular to meter-scale processes. While the motions of muscles contractile machinery are well described in isolated preparations, only a handful of experiments have documented the kinematics of the lattice occurring when multi-scale interactions are fully intact. We used time-resolved x-ray diffraction to record the kinematics of the myofilament lattice within a normal operating context: the tethered flight of Manduca sexta. Since the primary flight muscles of Manduca sexta are synchronous, we used these results to reveal the timing of in vivo cross-bridge recruitment, which occurred 24 (s.d. 26) ms following activation. In addition, the thick filaments stretched an average of 0.75 (s.d. 0.32)% and thin filaments stretched 1.11 (s.d. 0.65)%. In contrast to other in vivo preparations, lattice spacing changed an average of 2.72 (s.d. 1.47)%. Lattice dilation of this magnitude significantly impacts shortening velocity and force generation, and filament stretching tunes force generation. While kinematics were consistent within individual trials, there was extensive variation between trials. Using a mechanism-free machine learning model we searched for patterns within and across trials. While lattice kinematics were predictable within trials, the model could not create predictions across trials. This indicates that the variability we see across trials may be explained by latent variables occurring in this naturally functioning system. The diverse kinematic combinations we documented mirror muscles adaptability and may facilitate its robust function in unpredictable conditions.

biophysics↗