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

Muscle's function within an organism depends on the feedback between molecular to meter-scale processes. While the motions of muscle's 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 muscle's adaptability and may facilitate its robust function in unpredictable conditions.