Physiological and functional characterization for high-throughput optogenetic skeletal muscle exercise assays

Abstract

Exercise has long been considered an essential part of human health and longevity. Recent physiological studies have expanded muscles role beyond simply acting as an actuator, revealing muscles exercise-mediated paracrine and endocrine relationships with other organ systems. In vitro engineered skeletal muscle models can address physiological questions about exercise adaptation with the precision of cell biology. Optogenetic tools have enabled a noninvasive approach to stimulating muscle contraction that avoids the potential off-target effects of electrical stimulation techniques. In this article we propose high-throughput culture and optical exercise protocols to generate statistically robust cellular exercise response datasets. We characterize optical rheobase for 2D muscle tissue morphology, finding that optical intensities as low as 5 W mm-2 can trigger contraction. We then analyze bulk RNA sequencing data collected from high throughput, acute exercise protocols and find a rich display of transcriptional behavior that is consistent with experimental observations. The spontaneous contractility of our tissue constructs introduced oxygen diffusion challenges when maintained in a 24 well plate, and our analysis shows divergent myogenic and pathological transcriptional consequences of hypoxia. We believe our techniques provide a practical foundation for conducting future high-precision in vitro exercise studies of skeletal muscle. Translational impactHigh-fidelity, engineered skeletal muscle has potential to elucidate exercise-mediated response pathways at the cell and tissue level. We leverage optogenetic techniques to develop a high-throughput assay that optically stimulates 2D muscle monolayers, avoiding potential cell damage from electrical stimulation. Our culture and exercise protocol generates statistically robust RNA sequencing datasets which reveal myogenic and pathological responses to exercise and in vitro culture conditions, informing practical next steps to cultivate stronger, more physiologically relevant muscle models.