Heat Stress Prevents Dexamethasone‐induced Skeletal Muscle Atrophy as Demonstrated by Its Regulation of Anabolic and Catabolic Signaling Pathways in C2C12 Myotubes

Skeletal muscle weakness is one side effect of synthetic glucocorticoids and endogenous glucocorticoids levels are increased in diseases involving muscle atrophy. Consistent with causing muscle atrophy, glucocorticoids increase protein degradation by catabolic pathways and inhibit protein synthesis by anabolic pathways. Heat stress is beneficial for muscle atrophy and attenuates glucocorticoid‐induced muscle effects but its mechanisms are unclear. We examined the molecular effects of heat stress against glucocorticoid‐induced muscle atrophy, focusing on both anabolic and catabolic signaling pathways. We induced C2C12 myoblasts to differentiate and treated the established myotubes with the glucocorticoid dexamethasone as an in vitro model system for skeletal muscle atrophy. Prior to dexamethasone treatment, some myotube cultures were subjected to a heat stress protocol, 60 min at 41 °C, while controls were maintained at 37 °C. Myotubes were analyzed morphologically and mRNA and protein levels of key factors in anabolic and catabolic processes were assessed with real‐time RT‐PCR and western blotting, respectively. Statistical tests employed were Mann–Whitney U test to compare two conditions, and Kruskal–Wallis test followed by Steel's test for comparison with controls and by Steel–Dwass test for multiple comparisons. Dexamethasone caused a dose‐dependent decrease in myotube diameter and protein content. It also influenced expression of regulatory factors for both catabolic and anabolic pathways consistent with promoting muscle atrophy. Heat treatment of myotubes, inducing heat stress, 6 h before dexamethasone prevented the morphological, and many of the biochemical, effects of the glucocorticoid. For example, heat stress attenuated increases in “regulated in development and DNA damage responses 1” (REDD1) and “Kruppel‐like factor 15” (KLF15), two genes directly targeted by glucocorticoids that decrease protein synthesis by inhibiting mTORC1. Heat stress also normalized dexamethasone‐induced increases in key factors, including FoxO1/FoxO3a and KLF15, that promote protein degradation through the ubiquitin‐proteasome system. Overall, we describe mechanisms by which heat preconditioning may prevent glucocorticoid‐induced muscle atrophy. These mechanisms include modulating signaling pathways and expression of several genes targeted by glucocorticoids. Our findings have potential clinical impact for many patients because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. Support or Funding Information This work was supported, in part, by Japan Society for the Promotion of Science KAKENHI Grant Numbers 26870691 (WT), 26350639 (MI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and in part by a grant from the Public Advertisement Research Project of Nihon Fukushi University.