Genetically encoded biosensors to monitor intracellular reactive oxygen and nitrogen species and glutathione redox potential in skeletal muscle cells

Abstract

Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded bio-sensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochon-drial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for reg-istration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluc-tuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.