Calcium influx inhibition by steroids and analogs in C2C12 skeletal muscle cells

Abstract

1 Glucocorticoids, namely a-methylprednisolone (PDN) and de¯azacort, are the only drugs reported to have a bene®cial e ect on the degenerative course of Duchenne muscular dystrophy (DMD). Increased cytosolic calcium concentrations ([Ca 2+ ] c ) have been implicated as one of the pathological events responsible for the degeneration of dystrophic 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 [Ca 2+ ] c and improved myogenesis. Here we have investigated the mechanism underlying the e ects of glucocorticoids on cellular Ca 2+ in¯ux into C2C12 skeletal muscle cells. 2 Long-term incubation of C2C12 myocytes with PDN was necessary to observe a reduction of 45 Ca 2+ in¯ux. PDN was most e ective in inhibiting 45 Ca 2+ uptake when added for 4 days (at the time of fusion of myoblasts into myotubes) and to a lesser extent, when added after fusion. It was ine ective when added to C2C12 cells at the myoblast stage. Short PDN incubation times, at the time of fusion were insu cient to elicit a response. 3 Several steroids were tested for their ability to inhibit 45 Ca 2+ in¯ux in C2C12 myocytes. All four glucocorticoids examined were able to reduce Ca 2+ in¯ux, dexamethasone being the most potent (IC 50 3.14+0.34610 78 M). Mineralocorticoids (aldosterone and 11-deoxycorticosterone) were also able to reduce Ca 2+ in¯ux. 4 The vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived lazaroid U-74389G also elicited a decrease in Ca 2+ in¯ux, but higher concentrations were necessary. Because both glucocorticoids and lazaroids display antioxidant properties, but U-83836E is devoid of glucocorticoid activity, the reduction in Ca 2+ in¯ux was suspected to be triggered via an antioxidant mechanism. 5 To test this hypothesis, we assessed 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 45 Ca 2+ in¯ux. None of these agents had an e ect on 45 Ca 2+ in¯ux. In addition, several oxidants were tested (either acutely or chronically) for their ability to elicit 45 Ca 2+ in¯ux in C2C12 myocytes and were found to be inactive. 6 The involvement of the glucocorticoid receptor on the modulation of Ca 2+ in¯ux was investigated. The glucocorticoid receptor antagonist mifepristone (code name RU38486, 10 76 M) caused a shift of two orders of magnitude of the PDN response. However, neither actinomycin D nor cycloheximide a ected the response to PDN. 7 Results with the phospholipase A 2 inhibitor, manoalide, suggest that glucocorticoid-induced protein synthesis (e.g. enhanced stimulation of lipocortin) does not play a role in the reduction of calcium in¯ux. 8 Our results suggest that steroids elicit a decrease in calcium in¯ux in C2C12 skeletal muscle cells. This decrease is not due to an antioxidant mechanism or to a mechanism which requires gene expression. Since mineralocorticoids and U...

Small Leucine Zipper Protein (sLZIP) Negatively Regulates Skeletal Muscle Differentiation via Interaction with α-Actinin-4

“…There are four unique types of ACTN genes (ACTN1, 2, 3, and 4) that encode for highly homologous proteins. ACTN2 and 3 are enriched in muscle cells, whereas ACTN1 and 4 are widely expressed in other cells (5,6). In nonmuscle cells, ACTN1 and 4, which belong to the cytoskeletal isoforms, are found in actin filament bundles and adherent junctions and are involved in cell shape and motility (5,6).…”

“…ACTN2 and 3 are enriched in muscle cells, whereas ACTN1 and 4 are widely expressed in other cells (5,6). In nonmuscle cells, ACTN1 and 4, which belong to the cytoskeletal isoforms, are found in actin filament bundles and adherent junctions and are involved in cell shape and motility (5,6). ACTN2 and 3, which belong to skeletal, cardiac, and smooth muscle isoforms, are localized in the Z-disc and help in binding to actin filaments (7,8).…”

Mechanisms for Aldosterone and Spironolactone-Induced Positive Inotropic Actions in the Rat Heart

“…9 -12 These actions were classified as nongenomic because of their speed, lack of inhibition by spironolactone, and independence from new protein synthesis. Based on these characteristics, nongenomic effects of aldosterone have been reported from a number of laboratories in renal epithelial cells, 13 vascular smooth muscle cells, 14,15 skeletal muscle cells, 16 and colonic. The molecular basis of these nongenomic effects of aldosterone actions have been ascribed to changes in intracellular pH (pHi), [17][18][19] 20 -25 Although it has been observed that aldosterone does have rapid effects on protein kinase C activity in cultured cardiac cells, 26 and that aldosterone has rapid effects on cardiac sodiumhydrogen exchange, 27 the molecular mechanism of the rapid inotropic effect of aldosterone is still unclear.…”

The Acute Effect of the Antioxidant Drug U-74389G on Red Blood Cell Distribution Width Levels During Hypoxia Reoxygenation Injury in Rats

“…Schmid-Elsaesser R et al 4 showed signifi cantly less neurologic defi cits postoperatively and signifi cantly reduced cortical infarct volumes by the neuroprotective microvascularly acting 21-aminosteroid U-74389G. Passaquin AC et al 5 elicited a benefi cial effect of glucocorticoids in Duchenne muscular dystrophy, attributing it to a reduction of the pathological increase in Ca + + infl ux via an effect on the sarcolemma of C2C12 skeletal muscle cells. Van Table 1.…”