Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells

Carlos Henríquez-Olguín, Francisco Altamirano, Denisse Valladares, José R. López, Paul D. Allen, Enrique Jaimovich

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca<sup>2+</sup> increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca<sup>2+</sup> transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47<sup>phox</sup> and gp91<sup>phox</sup> were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50μM H<inf>2</inf>O<inf>2</inf> increased NF-κB activity; after administration of 100 and 200μM H<inf>2</inf>O<inf>2</inf> there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H<inf>2</inf>O<inf>2</inf>, with a significant effect of 100μM and 200μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca<sup>2+</sup> release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

Original languageEnglish (US)
Pages (from-to)1410-1419
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1852
Issue number7
DOIs
StatePublished - Jul 1 2015

Keywords

  • Calcium
  • Duchenne muscular dystrophy
  • Interleukin-6
  • Membrane depolarization
  • NF-κB
  • Reactive oxygen species

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine

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