Dexamethasone increases protein degradation in cultured myotubes through a Calcium-calmodulin Kinase II (CaMK-II) dependent mechanism

Abstract

Introduction. Previous reports suggest that glucocorticoids are important mediators of muscle protein breakdown in various muscle-wasting conditions, including sepsis. The mechanisms by which glucocorticoids induce muscle proteolysis are poorly understood. Previous studies provided evidence that glucocorticoids may influence cellular calcium homeostasis, and, in other experiments, calcium increased muscle protein breakdown. We tested the hypothesis that glucocorticoid-induced protein degradation in muscle cells is at least in part mediated by calcium and the calcium-activated enzyme CaMK-II. Methods. Cultured L6 myotubes, a rat skeletal muscle cell line, were treated with 1 μM dexamethasone for 24 h. Protein degradation was assessed by measuring the release of TCA-soluble 3H-tyrosine from proteins that had been pre-labeled with the amino acid. The role of calcium in dexamethasone-induced protein degradation was tested by treating the cells with the intracellular calcium chelator BAPTA-AM (20 μM). The involvement of CaMK-II was tested by using the CaMK-II inhibitor KN-93 (20 μM). Results. Treatment of L6 myotubes with dexamethasone resulted in a 12% increase in protein degradation, confirming previous reports from this laboratory. This effect of dexamethasone was blocked by BAPTA-AM and KN-93. KN-93 also inhibited basal protein degradation, in the absence of dexamethasone ( P < 0.001 by ANOVA). Conclusions. This study provides the first evidence that calcium and the calcium-activated enzyme CaMK-II may be involved in glucocorticoid-induced muscle protein degradation. TABLE—ABSTRACT 15 − DEX + DEX % Change with DEX Control 14.2 ± 0.2 15.9 ± 0.2 + 12.0% ∗ BAPTA-AM 13.6 ± 0.2 13.5 ± 0.1 Not significant KN-93 10.0 ± 0.2 10.4 ± 0.2 Not significant Note. Protein degradation (% 24 h) in myotubes treated with dexamethasone, BAPTA-AM,and/or KN-93. Results are means ± SEM with n = 6 per group. ∗ P < 0.001 versus no dex by ANOVA.