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.

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Monitoring of Hydrogen Peroxide and Other Reactive Oxygen and Nitrogen Species Generated by Skeletal Muscle

Hydrogen Peroxide and Cell Signaling, Part C - Methods in Enzymology ◽

10.1016/b978-0-12-405881-1.00017-3 ◽

2013 ◽

pp. 279-300 ◽

Cited By ~ 1

Author(s):

Malcolm J. Jackson

Keyword(s):

Skeletal Muscle ◽

Hydrogen Peroxide ◽

Reactive Oxygen ◽

Nitrogen Species

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Quantitative Redox Biology of Exercise

International Journal of Sports Medicine ◽

10.1055/a-1157-9043 ◽

2020 ◽

Vol 41 (10) ◽

pp. 633-645

Author(s):

Michalis G. Nikolaidis ◽

Nikos V. Margaritelis ◽

Antonios Matsakas

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

Reactive Oxygen ◽

Research Area ◽

Nitrogen Species ◽

Algebraic Equations ◽

Redox Biology ◽

Nitric Oxide Concentration ◽

Whole Muscle

AbstractBiology is rich in claims that reactive oxygen and nitrogen species are involved in every biological process and disease. However, many quantitative aspects of redox biology remain elusive. The important quantitative parameters you need to address the feasibility of redox reactions in vivo are: rate of formation and consumption of a reactive oxygen and nitrogen species, half-life, diffusibility and membrane permeability. In the first part, we explain the basic chemical kinetics concepts and algebraic equations required to perform “street fighting” quantitative analysis. In the second part, we provide key numbers to help thinking about sizes, concentrations, rates and other important quantities that describe the major oxidants (superoxide, hydrogen peroxide, nitric oxide) and antioxidants (vitamin C, vitamin E, glutathione). In the third part, we present the quantitative effect of exercise on superoxide, hydrogen peroxide and nitric oxide concentration in mitochondria and whole muscle and calculate how much hydrogen peroxide concentration needs to increase to transduce signalling. By taking into consideration the quantitative aspects of redox biology we can: i) refine the broad understanding of this research area, ii) design better future studies and facilitate comparisons among studies, and iii) define more efficiently the “borders” between cellular signaling and stress.

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The production of reactive oxygen and nitrogen species by skeletal muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.01102.2006 ◽

2007 ◽

Vol 102 (4) ◽

pp. 1664-1670 ◽

Cited By ~ 125

Author(s):

Malcolm J. Jackson ◽

Deborah Pye ◽

Jesus Palomero

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Potential Role ◽

Reactive Oxygen ◽

Oxygen Species ◽

Nitrogen Species ◽

Redox Biology ◽

Controlled Systems ◽

Potential Source

Skeletal muscle has been recognized as a potential source for generation of reactive oxygen and nitrogen species for more than 20 years. Initial investigations concentrated on the potential role of mitochondria as a major source for generation of superoxide as a “by-product” of normal oxidative metabolism, but recent studies have identified multiple subcellular sites, where superoxide or nitric oxide are generated in regulated and controlled systems in response to cellular stimuli. Full evaluation of the factors regulating these processes and the functions of the reactive oxygen species generated are important in understanding the redox biology of skeletal muscle.

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GPx-Mimetic Copper Vanadate Nanozyme Mediates the Release of Nitric Oxide from S-Nitrosothiols

Faraday Discussions ◽

10.1039/d1fd00067e ◽

2021 ◽

Author(s):

Sourav Ghosh ◽

Punarbasu Roy ◽

Sanjay Prasad ◽

Govindasamy Mugesh

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

Hydroxyl Radical ◽

Cellular Signaling ◽

Reactive Oxygen ◽

Nitrogen Species ◽

Redox Biology ◽

Copper Vanadate

Although reactive oxygen and nitrogen species (ROS/RNS) such as hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radical (OH.), superoxide (O2-) etc. play crucial roles in redox biology and cellular signaling,...

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Use of Microdialysis to Study Interstitial Nitric Oxide and Other Reactive Oxygen and Nitrogen Species in Skeletal Muscle

Methods in Enzymology - Nitric Oxide, Part E ◽

10.1016/s0076-6879(05)96043-6 ◽

2005 ◽

pp. 514-525 ◽

Cited By ~ 2

Author(s):

Malcolm J. Jackson

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Reactive Oxygen ◽

Nitrogen Species

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Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2009.08.006 ◽

2009 ◽

Vol 1788 (10) ◽

pp. 2142-2149 ◽

Cited By ~ 58

Author(s):

M. Fidorra ◽

A. Garcia ◽

J.H. Ipsen ◽

S. Härtel ◽

L.A. Bagatolli

Keyword(s):

Fluorescence Microscopy ◽

Lipid Domains ◽

Equilibrium Thermodynamic ◽

Giant Unilamellar Vesicles ◽

Unilamellar Vesicles ◽

Microscopy Imaging ◽

Imaging Approach ◽

Quantitative Fluorescence

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Oxidized LDL induces mitochondrially associated reactive oxygen/nitrogen species formation in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00015.2005 ◽

2005 ◽

Vol 289 (2) ◽

pp. H852-H861 ◽

Cited By ~ 86

Author(s):

Jaroslaw W. Zmijewski ◽

Douglas R. Moellering ◽

Claire Le Goffe ◽

Aimee Landar ◽

Anup Ramachandran ◽

...

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

Endothelial Cells ◽

Oxidized Ldl ◽

Critical Role ◽

Significant Proportion ◽

Reactive Oxygen ◽

Oxidized Lipids ◽

Nitrogen Species ◽

Nitric Oxide Synthase Inhibitor

Exposure of cells to complex mixtures of oxidized lipids such as those found in oxidized low-density lipoprotein (oxLDL) induce reactive oxygen and nitrogen species (ROS/RNS) formation. The source of the ROS/RNS within cells is unknown; it is thought they may be involved in redox cell signaling. Although this possibility was initially overlooked, it is becoming clear that mitochondria, which are a source of superoxide and hydrogen peroxide, may play a critical role in the response of cells on exposure to oxidized lipids. In this study, we tested the possibility that mitochondria are a potential source of oxLDL-dependent formation of ROS/RNS in endothelial cells. Using confocal microscopy, we demonstrated that a significant proportion of oxLDL-dependent dichlorodihydrofluorescein (DCF) fluorescence is colocalized to mitochondria. In support of this concept, rho0 endothelial cells showed a substantial decrease in ROS/RNS formation stimulated by oxLDL. In contrast, mostly nonmitochondrial DCF fluorescence was detected in cells exposed to an extracellular source of hydrogen peroxide. The exposure of cells to a nitric oxide synthase inhibitor and urate resulted in a decrease in oxLDL-induced DCF fluorescence that was restored by addition of nitric oxide donors to the medium. Taken together, these results suggest that oxLDL-dependent DCF fluorescence is mitochondrially associated and may be due to the formation of peroxynitrite.

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Signaling of Reactive Oxygen and Nitrogen Species in Diabetes Mellitus

Oxidative Medicine and Cellular Longevity ◽

10.4161/oxim.3.6.14415 ◽

2010 ◽

Vol 3 (6) ◽

pp. 361-373 ◽

Cited By ~ 49

Author(s):

Igor Afanas'ev

Keyword(s):

Diabetes Mellitus ◽

Hydrogen Peroxide ◽

Free Radical ◽

Reactive Oxygen ◽

Diabetes Mellitus Type 1 ◽

Free Radical Scavengers ◽

Adapter Proteins ◽

Nitrogen Species ◽

Radical Scavengers ◽

Enzymatic Cascades

Disorder of physiological signaling functions of reactive oxygen species(ROS) superoxide and hydrogen peroxide and reactive nitrogen species (RNS) nitric oxide and peroxynitrite is an important feature of diabetes mellitus type 1 and type 2. It is now known that hyperglycemic conditions of cells are associated with the enhanced levels of ROS mainly generated by mitochondria and NADPH oxidase. It has been established that ROS stimulate many enzymatic cascades under normal physiological conditions, but hyperglycemia causes ROS overproduction and the deregulation of ROS signaling pathways initiating the development of diabetes mellitus. On the other hand the deregulation of RNS signaling leads basically to a decrease in NO formation with subsequent damaging disorders. In the present work we will consider the pathological changes of ROS and RNS signaling in enzyme/gene regulated processes catalyzed by protein kinases C and B (Akt/B), phosphatidylinositol 3′-kinase (PI3-kinase), extracellular signal-regulated kinase 1/2 (ERK1/2) and some others. Furthermore we will discuss a particularly important role of several ROS-regulated genes and adapter proteins such as the p66shc, FOXO3a and Sirt2. The effects of low and high ROS levels in diabetes will be also considered. Thus the regulation of damaging ROS levels in diabetes by antioxidants and free radical scavengers must be one of promising treatment of this disease, however, because of the inability of traditionalantioxidative vitamin E and C to interact with superoxide and hydrogen peroxide,new free radical scavengers such as flavonoids, quinones and synthetic mimetics of superoxide dismutase (SOD) should be intensively studied.

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