Development of an in vitro model to study muscle maladaptation to overtraining
Authors: Sirago Giuseppe , Lanfranchi Clément , Carrard Justin , Gremeaux Vincent , Zanou Nadège , Place Nicolas 
Published: January 2025

Introduction Physical training stimulates skeletal muscle mitochondrial biogenesis and protein synthesis, inducing beneficial adaptations and promoting performance. However, in some cases when the training load is higher than required, the outcome may lead to non-functional overreaching – a state in which performance may be transiently reduced. This condition is relatively common especially among endurance athletes, and if protracted would result in the development of the overtraining syndrome (OTS), whose impact on skeletal muscle metabolism and function is still unknown. Muscle soreness, increased inflammation and reduced muscle mass are often reported. It has been reported that regeneration of skeletal muscle fibres is impaired, probably through a change in energy metabolism (Grobler et al., 2004). For instance, altered mitochondrial structure and function have been suggested (Grobler et al., 2004). The aim of the present project is to determine the molecular mechanisms underlying skeletal muscle maladaptation’s in response to simulated exercise in vitro.
Methods Using a model developed in our laboratory (Zanou et al., 2021), we electrically stimulated C2C12 myotubes (a mouse skeletal muscle cell line) to simulate endurance exercise (Zanou et al., 2021). For simulated training (s-T) the stimulation was applied once daily, for three consecutive days. For simulated overtraining (s-OT), the stimulation was applied three times per day for three consecutive days. Mitochondrial biogenesis biomarkers have been measured, alongside proteomics analysis and mitochondrial respiration.
Results In the in vitro s-T model, we observed the expected beneficial adaptations to training, which include myotube hypertrophy, increased fast and slow-twitch myosin heavy chain protein content, and increased mitochondrial respiration. Our proteomics data highlighted many positive adaptations, including upregulation of the muscle contractile and mitochondrial proteins. Immunofluorescence staining of F-actin and of the mitochondrial outer membrane protein Tom20, clearly showed robust F-actin structure and regular mitochondrial distribution in response to s-T. Interestingly, s-OT model presented myotube atrophy, and a decrease in fast and slow-twitch myosin heavy chain protein content. Mitochondrial respiration was increased in s-T and showed a clear trend versus reduction in s-OT. Our proteomics data revealed many downregulated proteins pathways in s-OT including muscle contractile and mitochondrial pathways. Immunofluorescence staining also revealed disrupted F-actin structure and aberrant mitochondrial aggregation in response to s-OT.
Conclusions s-T recapitulates training positive adaptations, whereas s-OT recapitulates many of the muscle maladaptation observed in overtraining. Our next studies in human OTS muscle will validate key findings from our in vitro s-OT model and guide our mechanistic investigations into the molecular mechanisms of OTS.
References
Grobler, L. A., Collins, M., Lambert, M. I., Sinclair-Smith, C., Derman, W., St Clair Gibson, A., & Noakes, T. D. (2004). Skeletal muscle pathology in endurance athletes with acquired training intolerance. British Journal of Sports Medicine, 38(6), 697–703. https://doi.org/10.1136/bjsm.2003.006502
Zanou, N., Dridi, H., Reiken, S., Imamura de Lima, T., Donnelly, C., De Marchi, U., Ferrini, M., Vidal, J., Sittenfeld, L., Feige, J. N., Garcia-Roves, P. M., Lopez-Mejia, I. C., Marks, A. R., Auwerx, J., Kayser, B., & Place, N. (2021). Acute RyR1 Ca²⁺ leak enhances NADH-linked mitochondrial respiratory capacity. Nature Communications, 12(1), 7219. https://doi.org/10.1038/s41467-021-27422-1