The Effects Of Quercetin On Ros Generation And Myotube Area In C2C12 Muscle Cells

PURPOSE: The relationship between intracellular ROS generation and skeletal muscle proteolysis remains unclear. This research tested the hypotheses that elevated intracellular ROS generation induces measurable loss of myonuclei and myotube area in cultured skeletal muscle and these effects are attenuated with quercetin, a natural antioxidant. METHODS: C2C12 mouse muscle cells were grown to 70% confluence then supplemented with 2% horse serum to initiate differentiation into myotubes. At 4 days post-differentiation, myotubes were treated with 5μm quercetin for 18hrs. At 5 days post-differentiation, myotubes were loaded with 10μM 2',7'-dichlorodihydrofluorescein, an intracellular ROS probe. Baseline fluorescence was measured on a microplate reader, then culture media was supplemented with 200μM H2O2. Fluorescence measurements were taken at 6, 12, and 24 hours post- H2O2 administration to examine ROS generation. Immunocytochemistry (antibody staining of myonuclei (DAPI) and myosin heavy chain (MF-20)) and fluorescence microscopy were performed at baseline, 6, 12, and 24hrs to examine myotube morphology. Metamorph™ imaging software was used to quantify myonuclei and myotube area under each experimental condition. RESULTS: Myotubes treated with 200μM H2O2 displayed higher DCFH fluorescence at 6 (42%), 12 (32%), and 24hrs (26%) relative to untreated controls (p<0.001). Conversely, myotubes treated with 5μM quercetin prior to H2O2 exposure displayed lower DCFH fluorescence than did either myotubes treated with H2O2 alone (p<0.001) or untreated controls (p<0.001). Morphological analysis revealed a 21% loss of myotube area at 24hrs in cells treated with H2O2 alone as compared with untreated controls (p = 0.025). Myotubes pre-treated with quercetin displayed only a 13% loss of area at 24hrs; however, this was not different from myotubes treated with H2O2 alone. The ratio of myonuclei/myotube area (1.25±0.3 SEM) was not different between any of the treatment groups at baseline, 6, 12, and 24hrs, which suggests a proportional decrease in myonuclei and myotube area at 24hrs. CONCLUSIONS: Findings demonstrate that H2O2 stimulates increased ROS generation and leads to a significant loss of myotube area. Pre-treatment with quercetin abrogates ROS generation but does not attenuate loss of myotube area.