C2C12 transcriptome changes in response to hydrogen peroxide

In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis. Keywords: Gene Expression, C2C12 Myotubes, Oxidative Stress, Adaptation Cell Culture: C2C12 myoblasts (ATCC, Rockville, MD) were cultured in DMEM supplemented with 10% fetal calf serum at 37°C in 5% CO2. Myoblast differentiation was initiated by switching to differentiation medium (DMEM supplemented with 2% heat-inactivated horse serum) and allowed to continue for 96 hrs. Differentiated myotubes were treated for 2 hours with H2O2 (Sigma-Aldrich, St. Louis, MO) 0, 1, 10, 100, or 1000 μM in DMEM supplemented with 0.5% heat-inactivated horse serum. cDNA microarrays: Total RNA was obtained from treated C2C12 myotubes with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s recommended protocol.Oligonucleotide microarray studies with Affymetrix Mouse Genome 430A gene chips (n=20 chips) were conducted as described earlier. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Treatment comparisons were crossed so that each H2O2 sample was compared to each control. The software uses the one-sided Wilcoxon’s signed rank test to estimate “increase/no change/ decrease” difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.75 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.