RT Journal Article SR Electronic T1 Generating fast-twitch myotubes in vitro using an optogenetic-based, quantitative contractility assay JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.10.04.510824 DO 10.1101/2022.10.04.510824 A1 Katharina Hennig A1 David Hardman A1 David Barata A1 Inês Martins A1 Miguel O. Bernabeu A1 Edgar R. Gomes A1 William Roman YR 2022 UL http://biorxiv.org/content/early/2022/10/05/2022.10.04.510824.abstract AB The composition of fiber types within skeletal muscle impacts the tissue’s physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and upregulation of neuron receptor genes. We observed an upregulation in the expression of distinct myosin heavy chain isoforms (namely, neonatal-Myh8 and fast-Myh), which induced a shift towards a fast fiber phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.Competing Interest StatementThe authors have declared no competing interest.