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Traditional work loop studies, that use sinusoidal length trajectories with constant frequencies, lack the complexities of in vivo muscle mechanics observed in modern studies. This study refines methodology of the “avatar” method (a modified work loop) to infer in vivo muscle mechanics using ex vivo experiments with mouse extensor digitorum longus (EDL) muscles. The “avatar” method involves using EDL muscles to replicate in vivo time varying force, as demonstrated by previous studies focusing on guinea fowl lateral gastrocnemius (LG). The present study extends this method by using in vivo length trajectories and electromyographic (EMG) activity from rat medial gastrocnemius (MG) during various gaits on a treadmill. Methodological enhancements from previous work, including adjusted stimulation protocols and systematic variation of starting length, improved predictions of in vivo time varying force production (R2 0.80 – 0.96). The study confirms there are significant influence of length, stimulation, and their interactions on work loop variables (peak force, length at peak force, highest and average shortening velocity, and maximum and minimum active velocity), highlighting the importance of these interactions when muscles produce in vivo forces. We also investigated the limitations of traditional work loops in capturing muscle dynamics in legged locomotion (R2 0.01 – 0.71). While in vivo length trajectories enhanced force prediction, accurately predicting work per cycle remained challenging. Overall, the study emphasizes the utility of the avatar method in elucidating dynamic muscle mechanics and highlights areas for further investigation to refine its application in understanding in vivo muscle function. 

ABSTRACT The work loop technique has provided key insights into in vivo muscle work and power during steady locomotion. However, for many animals and muscles, ex vivo experiments are not feasible. In addition, purely sinusoidal strain trajectories lack variations in strain rate that result from variable loading during locomotion. Therefore, it is useful to develop an ‘avatar’ approach in which in vivo strain and activation patterns from one muscle are replicated in ex vivo experiments on a readily available muscle from an established animal model. In the present study, we used mouse extensor digitorum longus (EDL) muscles in ex vivo experiments to investigate in vivo mechanics of the guinea fowl lateral gastrocnemius (LG) muscle during unsteady running on a treadmill with obstacle perturbations. In vivo strain trajectories from strides down from obstacle to treadmill, up from treadmill to obstacle, strides with no obstacle and sinusoidal strain trajectories at the same amplitude and frequency were used as inputs in work loop experiments. As expected, EDL forces produced with in vivo strain trajectories were more similar to in vivo LG forces (R2=0.58–0.94) than were forces produced with the sinusoidal trajectory (average R2=0.045). Given the same stimulation, in vivo strain trajectories produced work loops that showed a shift in function from more positive work during strides up from treadmill to obstacle to less positive work in strides down from obstacle to treadmill. Stimulation, strain trajectory and their interaction had significant effects on all work loop variables, with the interaction having the largest effect on peak force and work per cycle. These results support the theory that muscle is an active material whose viscoelastic properties are tuned by activation, and which produces forces in response to deformations of length associated with time-varying loads.