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A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation
Published on: May 9, 2012
Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in
Carlos Henríquez-Olguín1, Francisco Altamirano2, Denisse Valladares3
1Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago 8389100, Chile; Laboratorio Ciencias del Ejercicio, Clínica MEDS, Santiago, Chile.
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 Ca2+ 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 Ca2+ 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: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H2O2 increased NF-κB activity; after administration of 100 and 200 μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 μM and 200 μM, 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 Ca2+ 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.
Insights
Duchenne muscular dystrophy myotubes show impaired NF-κB activation and IL-6 expression due to altered calcium and ROS signaling. This suggests a dysregulation contributing to abnormal muscle response in the disease.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Duchenne muscular dystrophy (DMD) results from dystrophin gene mutations, leading to muscle cell dysfunction and increased reactive oxygen species (ROS).
- NF-κB signaling and Interleukin-6 (IL-6) expression are crucial in cellular responses, including inflammation and muscle function.
Purpose of the Study:
- To investigate alterations in NF-κB activation and IL-6 expression in dystrophic mdx myotubes following membrane depolarization.
- To elucidate the roles of intracellular calcium (Ca2+) and ROS in modulating NF-κB and IL-6 responses in DMD.
Main Methods:
- Utilized myotube cultures from wild-type (wt) and mdx mice.
- Induced membrane depolarization via electrical stimulation.
- Assessed p65 phosphorylation, NF-κB transcriptional activity, and IL-6 expression.
- Measured intracellular Ca2+ transients and ROS production.
- Investigated the effects of NOX2 inhibitors and hydrogen peroxide (H2O2).
- Examined lipopolysaccharide (LPS)-induced responses.
Main Results:
- Electrical stimulation increased NF-κB and IL-6 in wt myotubes but had an opposite effect in mdx myotubes.
- DMD myotubes exhibited reduced Ca2+ transients and elevated ROS production, linked to NOX2 activity.
- ROS levels critically influenced NF-κB activity, with mdx myotubes showing reduced activation in response to H2O2.
- LPS stimulation effectively activated NF-κB and IL-6 in both wt and mdx myotubes, indicating pathway-specific dysregulation.
Conclusions:
- DMD myotubes display impaired NF-κB and IL-6 responses to electrical stimulation, stemming from dysregulated Ca2+ release and excessive ROS production.
- These signaling deficits in dystrophin-deficient muscle cells contribute to abnormal responses to contraction and exercise.
- The findings highlight a critical link between calcium handling, ROS generation, and NF-κB/IL-6 signaling in the pathophysiology of Duchenne muscular dystrophy.
