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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Biosensing Techniques/*methods ; Glutathione/*metabolism ; Muscle, Skeletal/*metabolism ; Nitrogen/*metabolism ; Oxygen/*metabolism ; Reactive Nitrogen Species/*metabolism ; Reactive Oxygen Species/*metabolism; Animals ; Female ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; Hydrogen Peroxide/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Mitochondria/metabolism ; Muscle, Skeletal/cytology ; Oxidation-Reduction

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 CO 2 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.

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SAF2017-85762-R Ministry of Economy, Industry and Competitiveness; 18KB7D and 18KC7D University of Salamanca (Spain)

Keywords: C2C12 myoblast/myotube; RONS; biosensors; glutathione redox potential; hydrogen peroxide; nitric oxide; quantitative fluorescence microscopy; redox signaling; single skeletal muscle fiber; skeletal muscle

0 (Reactive Nitrogen Species)
0 (Reactive Oxygen Species)
147336-22-9 (Green Fluorescent Proteins)
BBX060AN9V (Hydrogen Peroxide)
GAN16C9B8O (Glutathione)
N762921K75 (Nitrogen)
S88TT14065 (Oxygen)

Date Created: 20211013 Date Completed: 20211101 Latest Revision: 20211101

20231215

PMC8509583

10.3390/ijms221910876

34639217