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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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| Published in: | Analytical chemistry (Washington) 2010-06, Vol.82 (11), p.4570-4576 |
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| creator | Xu, Xin Thompson, LaDora V Navratil, Marian Arriaga, Edgar A |
| description | 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. |
| doi_str_mv | 10.1021/ac100577q |
| format | article |
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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. 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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. 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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.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>20446672</pmid><doi>10.1021/ac100577q</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Analytical chemistry (Washington), 2010-06, Vol.82 (11), p.4570-4576 |
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| source | American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list) |
| 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 |
| title | Analysis of Superoxide Production in Single Skeletal Muscle Fibers |
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