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

Abstract

[EN] Hydrogen peroxide (H2O2) is generated in cells and plays an important role as a signalling molecule. It has been reported that H2O2 is involved in physiological and pathological processes in skeletal muscle. However, H2O2 detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H2O2 specifcally in real time using fuorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in diferent models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fbres isolated from C57BL/6J mice, and to measure intracellular H2O2 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 fuctuations in intracellular H2O2 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 H2O2 in skeletal muscle fbres. In conclusion, the expression of the HyPer biosensor in the diferent experimental models combined with fuorescence microscopy techniques is a powerful methodology to monitor and register intracellular H2O2 specifcally in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H2O2 in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specifc biosensors for diferent reactive oxygen and nitrogen species or metabolites involved in cellular functions.