Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells

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 biosensors 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 mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration 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 fluctuations. 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.     

Dynamic Changes in Reactive Oxygen Species in the Shoot Apex Contribute to Stem Cell Death in Arabidopsis thaliana

In monocarpic plants, stem cells are fated to die. However, the potential mechanism of stem cell death has remained elusive. Here, we reveal that the levels of two forms of reactive oxygen species (ROS), superoxide anion free radical (O2·−) and hydrogen peroxide (H₂O₂), show dynamic changes in the shoot apex during the plant life cycle of Arabidopsis thaliana. We found that the level of O2·− decreased and disappeared at four weeks after bolting (WAB), while H₂O₂ appeared at 3 WAB and showed a burst at 5 WAB. The timing of dynamic changes in O2·− and H₂O₂ was delayed for approximately three weeks in clv3-2, which has a longer lifespan. Moreover, exogenous application of H₂O₂ inhibited the expression of the stem cell determinant WUSCHEL (WUS) and promoted the expression of the developmentally programmed cell death (dPCD) marker gene ORESARA 1 (ORE1). These results indicate that H₂O₂ triggers an important signal inducing dPCD in stem cells. Given that O2·− plays roles in maintaining WUS expression and stem cell activity, we speculate that the dynamic shift from O2·− to H₂O₂ in the shoot apex results in stem cell death. Our findings provide novel insights for understanding ROS-mediated regulation during plant stem cell death.