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Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene.

ABSTRACT: Chronic low-frequency contraction of skeletal muscle, either induced by a slow motor nerve or through direct electrical stimulation, generally induces expression of proteins associated with the slow phenotype, while repressing the corresponding fast isoforms. Contractions thereby counteract the primarily transcriptional effect of thyroid hormone (T3) which results in the selective induction and stimulation of expression of fast isoforms. We studied the regulation of expression of the fast-type sarcoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic component of the fast phenotype. Previous work suggested that reduction of SERCA1 expression by contractile activity might result from interference with the T3-dependent transcriptional stimulation of the SERCA1 gene. The present study was set up to test this unexpected mode of action of contractile activity. We show that electrical stimulation of C2C12 mouse myotubes, which results in synchronous contractions at the imposed frequency, reduces basal but virtually abolishes T3-dependent SERCA1 expression. T3-dependent expression of a reporter gene driven by the SERCA1 promoter was similarly affected by electrical stimulation. This is the first demonstration that the counteracting effects on muscle gene expression of electrically induced contractions and T3 may interact at the transcriptional level.

SUBMITTER: Thelen MH

PROVIDER: S-EPMC1218143 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Project description:The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblasts with caffeine or high [K+]o. The role of [Ca2+]i in the regulation of Ca(2+)-ATPase levels was investigated by varying [Ca2+]i through exposure of cells to different concentrations of extracellular Ca2+ (0.2-1.8 mM) and ionomycin (0.1-0.25 microM). At subnormal [Ca2+]i (55 nM) the T3-induced net synthesis of Ca(2+)-ATPase was virtually abolished, and at supranormal [Ca2+]i (195 nM) it was greatly depressed. Intermediate stimulation of net Ca(2+)-ATPase synthesis was found at [Ca2+]i of 95 and 165 nM, with an optimum at approx. 125 nM. Similar but less pronounced effects were found for the basal Ca(2+)-ATPase levels. In contracting primary rat myotubes, Ca(2+)-ATPase levels were significantly lower than in tetrodotoxin-arrested myotubes. The same results were obtained in the presence of T3. Since the mean [Ca2+]i in contracting cells is higher than in resting cells, these data agree with those obtained in the L6 cells with ionomycin. A major conclusion of this study is the existence of a [Ca2+]i optimum, near resting levels, for the expression of the fast-type Ca(2+)-ATPase in the L6 muscle cell line.

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Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene.

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