Signal Transduction
Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes*

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Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]rest was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]rest. Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca2+]rest was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca2+]rest using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca2+]rest, is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

Calcium Intracellular Release
Dystrophin
Muscular Dystrophy
NF-κB Transcription Factor
Nitric-oxide Synthase
Inositol trisphosphate Receptors
Resting Calcium
Ryanodine Receptors

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*

This work was supported, in whole or in part, by National Institutes of Health Grants AR43140 and AR052354 (to P. D. A. and J. R. L.). This work was also supported by Grants Fondo Nacional de Investigación Cientifica y Tecnológica 1110467, Fondo de Financiamiento de Centros de Excelencia en Investigación 15010006, and Asotiation Francaise Contre les Myopathies 14562 (to E. J.), Grant AT-24100066 from Comisión Nacional de Investigación Cientifica y Tecnológica, Vicerrectoría de Asuntos Académicos, Universidad de Chile, and Programa de Mejoramiento de lo Calidad y Equidad de la Educación travel support UCH 0714, Universidad de Chile (to F. A.).

This article contains supplemental Figs. S1–S3.