Quantification of hormone-induced atrophy of large myotubes from C2C12and L6 cells: atrophy-inducible and atrophy-resistant C2C12myotubes

Abstract

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C2C12and rat L6 cells were used. C2C12myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5–1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C2C12myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C2C12myotubes. In both cell lines, 3,3′,5-triiodo-l-thyronine (T3) did not induce a significant size reduction. Expression of the major T3receptor (T3Rβ1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C2C12myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C2C12myotubes. Therefore, C2C12myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C2C12cells produce atrophy-inducible and atrophy-resistant myotubes.