Characterization of Reactive Oxygen Species Signaling changes in a cell culture model of skeletal muscle ageing, and its application to screening pharmacokinetically-relevant exposures of dietary polyphenols for bioactivity.

Age-related frailty is a significant health and social care burden, however treatment options are limited. There is currently a lack of suitable cell culture model for screening large numbers of test compounds to identifying those which can potentially promote healthy skeletal muscle function. This paper describes the characterization of reactive oxygen and nitrogen species (RONS) signalling changes in young and aged myoblasts and myotubes using the C2C12 cell line, and the application of aged myoblast and myotube cultures to assess the effect of dietary polyphenols on RONS signalling. Aged myoblasts and myotubes were observed to have significantly increased reactive oxygen species levels (p<0.01 and p<0.001 respectively), increases in nitric oxide levels (p<0.05 for myoblasts and myotubes), and lipid peroxidation markers (p<0.05 for myoblasts and myotubes). A panel of nine polyphenols were assessed in aged myoblasts and myotubes using concentrations and incubation times consistent with known pharmacokinetic parameters for these compounds. Of these, although several polyphenols were seen to reduce single markers of RONS signalling, only kaempferol and resveratrol consistently reduced multiple markers of RONS signalling with statistical significance in both cell models. Modulation of cellular enzymatic antioxidant activities (superoxide and catalase) was assessed as a possible mechanism of action for these polyphenols, and although both superoxide and catalase activities were significantly reduced in aged (versus young) myotubes (p<0.01 and p<0.05 respectively), no effect of polyphenol treatment on these enzyme activities were observed. Overall, this research has shown the utility of the C2C12 model, as both myoblasts and myotubes, as a suitable cell model for screening compounds for modulating RONS signalling in aged muscle, and that resveratrol and kaempferol (using pharmacokinetically-informed exposures) can modulate RONS signalling in skeletal muscle cells after an acute exposure.