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

被引:10
作者
Fernandez-Puente, Escarlata [1 ,2 ,3 ]
Palomero, Jesus [1 ,2 ,3 ]
机构
[1] Univ Salamanca, Fac Med, Dept Physiol & Pharmacol, Campus Miguel de Unamuno,Av Alfonso X Sabio, Salamanca 37007, Spain
[2] Inst Neurosci Castilla & Leon INCyL, Salamanca 37007, Spain
[3] Inst Biomed Res Salamanca IBSAL, Salamanca 37007, Spain
关键词
biosensors; quantitative fluorescence microscopy; redox signaling; RONS; hydrogen peroxide; nitric oxide; glutathione redox potential; skeletal muscle; single skeletal muscle fiber; C2C12; myoblast/myotube; OXIDATIVE STRESS; NITRIC-OXIDE; FIBERS; H2O2; EXERCISE; MITOCHONDRIAL; BIOCHEMISTRY;
D O I
10.3390/ijms221910876
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页数:20
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