Analysis of Superoxide Production in Single Skeletal Muscle Fibers

Due to their high energetic profile, skeletal muscle fibers are prone to damage by endogenous reactive oxygen species (ROS), thereby causing alterations in muscle function. Unfortunately, the complexity of skeletal muscle makes it difficult to measure and understand ROS production by fibers since ot...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2010-06, Vol.82 (11), p.4570-4576
Main Authors: Xu, Xin, Thompson, LaDora V, Navratil, Marian, Arriaga, Edgar A
Format: Article
Language:English
Subjects:
1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt - metabolism
Algorithms
Analytical chemistry
Animals
Antimycin A - metabolism
Cell Extracts
Cells, Cultured
Chemistry
Chromatographic methods and physical methods associated with chromatography
Chromatography, Micellar Electrokinetic Capillary
Comparative analysis
Crystallography
Diffraction
Exact sciences and technology
Feasibility Studies
Indicators and Reagents - chemistry
Lasers
Male
Muscle Fibers, Skeletal - metabolism
Organophosphorus Compounds - chemistry
Other chromatographic methods
Rats
Rats, Inbred F344
Reproducibility of Results
Spectrometric and optical methods
Spectrometry, Fluorescence
Superoxides - metabolism
X-rays
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Summary:Due to their high energetic profile, skeletal muscle fibers are prone to damage by endogenous reactive oxygen species (ROS), thereby causing alterations in muscle function. Unfortunately, the complexity of skeletal muscle makes it difficult to measure and understand ROS production by fibers since other components (e.g., extracellular collagen and vascular vessels) may also generate ROS. Single cell imaging techniques are promising approaches to monitor ROS production in single muscle fibers, but usually the detection schemes for ROS are not specific. Single cell analysis by capillary electrophoresis (aka chemical cytometry) has the potential to separate and detect specific ROS reporters, but the approach is only suitable for small spherical cells that fit within the capillary lumen. Here, we report a novel method for the analysis of superoxide in single fibers maintained in culture for up to 48 h. Cultured muscle fibers in individual nanoliter-volume wells were treated with triphenylphosphonium hydroethidine (TPP-HE), which forms the superoxide specific reporter hydroxytriphenylphosphonium ethidium (OH-TPP-E+). After lysis of each fiber in their corresponding nanowell, the contents of each well were processed and analyzed by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF) making it possible to detect superoxide found in single fibers. Superoxide basal levels as well as changes due to fiber treatment with the scavenger, tiron, and the inducer, antimycin A, were easily monitored demonstrating the feasibility of the method. Future uses of the method include parallel single-fiber measurements aiming at comparing pharmacological treatments on the same set of fibers and investigating ROS production in response to muscle disease, disuse, exercise, and aging.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac100577q