CALCIUM INFLUX INHIBITION BY STEROIDS AND ANALOGS IN C2C12 SKELETAL-MUSCLE CELLS

1 Glucocorticoids, namely alpha-methylprednisolone (PDN) and deflazaco rt, are the only drugs reported to have a beneficial effect on the deg enerative course of Duchenne muscular dystrophy (DMD). Increased cytos olic calcium concentrations ([Ca2+](c)) have been implicated as one of the pathological events responsible for the degeneration of dystrophi c skeletal muscles. In previous studies, we have demonstrated that PDN treatment of both normal and dystrophic murine skeletal muscle cells was able to normalize elevated [Ca2+](c) and improved myogenesis. Here we have investigated the mechanism underlying the effects of glucocor ticoids on cellular Ca2+ influx into C2C12 skeletal muscle cells. 2 Lo ng-term incubation of C2C12 myocytes with PDN was necessary to observe a reduction of Ca-45(2+) influx. PDN was most effective in inhibiting Ca-45(2+) uptake when added for 4 days (at the time of fusion of myob lasts into myotubes) and to a lesser extent, when added after fusion. It was ineffective when added to C2C12 cells at the myoblast stage. Sh ort PDN incubation times, at the time of fusion were insufficient to e licit a response. 3 Several steroids were tested for their ability to inhibit Ca-45(2+) influx in C2C12 myocytes. All four glucocorticoids e xamined were able to reduce Ca2+ influx, dexamethasone being the most potent (IC50 3.14+/-0.34x10(-8) M). Mineralocorticoids (aldosterone an d 11-deoxycorticosterone) were also able to reduce Ca2+ influx. 4 The vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived laz aroid U-74389G also elicited a decrease in Ca2+ influx, but higher con centrations were necessary. Because both glucocorticoids and lazaroids display antioxidant properties, but U-83836E is devoid of glucocortic oid activity, the reduction in Ca2+ influx was suspected to be trigger ed via an antioxidant mechanism. 5 To test this hypothesis, we assesse d the action of several antioxidants, such as vitamin E, vitamin C, 2- tert.-butyl-4-methoxyphenol (BHA), 2,6-di-tert.-butyl-4-methyl-phenol (BHT) and nordihydroguaiaretic acid (NDGA), on Ca-45(2+) influx. None of these agents had an effect on Ca-45(2+) influx. In addition, severa l oxidants were tested (either acutely or chronically) for their abili ty to elicit Ca-45(2+) influx in C2C12 myocytes and were found to be i nactive. 6 The involvement of the glucocorticoid receptor on the modul ation of Ca2+ influx was investigated. The glucocorticoid receptor ant agonist mifepristone (code name RU38486, 10(-6) M) caused a shift of t wo orders of magnitude of the PDN response. However, neither actinomyc in D nor cycloheximide affected the response to PDN. 7 Results with th e phospholipase A(2) inhibitor, manoalide, suggest that glucocorticoid -induced protein synthesis (e.g. enhanced stimulation of lipocortin) d oes not play a role in the reduction of calcium influx. 8 Our results suggest that steroids elicit a decrease in calcium influx in C2C12 ske letal muscle cells. This decrease is not due to an antioxidant mechani sm or to a mechanism which requires gene expression. Since mineralocor ticoids and U-83836E also had similar effects, the mechanism could bel ong to the nongenomic effects of corticoids (e.g. membrane stabilizati on). The beneficial effect of glucocorticoids in DMD could be attribut ed to a reduction of the pathological increase in Ca2+ influx via an e ffect on the sarcolemma.