Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres

Hydrogen peroxide (H(2)O(2)) is generated in cells and plays an important role as a signalling molecule. It has been reported that H(2)O(2) is involved in physiological and pathological processes in skeletal muscle. However, H(2)O(2) detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H(2)O(2) specifically in real time using fluorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in different models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fibres isolated from C57BL/6J mice, and to measure intracellular H(2)O(2) in real time in these cells. The results show that the expression of the HyPer biosensor in skeletal muscle cells is possible. In addition, we demonstrate that HyPer is functional and that this biosensor detects changes and fluctuations in intracellular H(2)O(2) in a reversible manner. The HyPer2 biosensor, which is a more advanced version of HyPer, presents improved properties in terms of sensitivity in detecting lower concentrations of H(2)O(2) in skeletal muscle fibres. In conclusion, the expression of the HyPer biosensor in the different experimental models combined with fluorescence microscopy techniques is a powerful methodology to monitor and register intracellular H(2)O(2) specifically in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H(2)O(2) in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specific biosensors for different reactive oxygen and nitrogen species or metabolites involved in cellular functions.