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Regulation of Skeletal Muscle Reactive Oxygen Species During Exercise
Published in Peter M. Tiidus, Rebecca E. K. MacPherson, Paul J. LeBlanc, Andrea R. Josse, The Routledge Handbook on Biochemistry of Exercise, 2020
Catherine A. Bellissimo, Christopher G.R. Perry
Limited evidence of increased mitochondrial ROS during contraction: Some of the key studies that argued against mitochondria being a source of ROS used a redox sensitive mito-roGFP fluorescent protein to determine the effects of single-fibre contraction on mitochondrial redox potential (108). Changes in fluorescence of this protein reflect a change in either mitochondrial ROS generation or redox buffering activity. Following 15 minutes of contractions of mouse muscle fibres in situ, no changes in fluorescence were observed, suggesting contraction did not alter the fluorescence of this reporter protein (98). This same method was used following 20 minutes of moderate-intensity treadmill exercise, whereby no increase in mito-roGFP fluorescence was observed in mouse skeletal muscle (57). Similar conclusions were made by inhibiting superoxide-release channels on mitochondria while monitoring fluorescence of a superoxide-sensitive fluorophore loaded in the cytoplasm of mouse single muscle fibres (130). These inhibitors had no effect on cytoplasmic superoxide responses to contraction, indicating that mitochondrial superoxide release was not affected by contraction. However, mitochondrial superoxide can also be dismutated to membrane-permeable H2O2 that diffuses into the cytoplasm and is thought to do so at a very high rate independent of the exporting superoxide itself. As such, mitochondrial H2O2 emission is not monitored with this approach, although it is possible that a lack of change in superoxide export means it is also unlikely that there was an increase in superoxide sufficient to accelerate H2O2 emission. Overall, the results are consistent with the lack of change in mitochondrial redox potential (mito-roGFP fluorescence) after single-fibre contraction and suggests that mitochondrial ROS is not elevated after exercise.
High-throughput screening in multicellular spheroids for target discovery in the tumor microenvironment
Published in Expert Opinion on Drug Discovery, 2020
Blaise Calpe, Werner J. Kovacs
Genetically encoded fluorescent redox sensors have been developed for H2O2 and to monitor the redox state of GSSG/2GSH, NADH/NAD+, NADPH/NADP+, and TRXSS/TRXSH2 [84–86]. These redox indicators include redox-sensing fluorescent proteins such as Redoxfluor, Hyper, Peredox, and redox-sensitive yellow fluorescent protein (rxYFP) and green fluorescent proteins (roGFPs) [87]. RoGFP is more commonly used than rxYFP for two reasons: 1) unlike rxYFP, roGFP is ratiometric and thus provides a more quantitative measurement; 2) roGFP variants are pH resistant, whereas rxYFP is pH sensitive in the physiological pH range. Importantly, redox potentials differ in subcellular compartments and these reporters can be genetically targeted to specific subcellular compartments. RoGFPs are not directly oxidized by ROS, but equilibrate with the glutathione redox couple (GSH/GSSG) through the action of endogenous glutaredoxins (GRXs) [88]. For example, the redox sensor Grx1-roGFP, a fusion protein consisting of roGFP and human glutaredoxin 1, allows for ratiometric measurements and qualifies it as reporter for imaging of compound-mediated effects in real-time [89]. Importantly, this sensor was used in MTS, providing a proof of concept for redox-based imaging in 3D culture [90].