title: Analysis of superoxide production in single skeletal muscle fibers.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.forename: Ladora V
authors.initials: LV
authors.lastname: Thompson
authors.forename: Marian
authors.initials: M
authors.lastname: Navratil
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Arriaga
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000378:metabolism; D002457:Cell Extracts; D002478:Cells, Cultured; D020374:Chromatography, Micellar Electrokinetic Capillary; D005240:Feasibility Studies; D007202:Indicators and Reagents / Q000737:chemistry; D007834:Lasers; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000737:chemistry; D051381:Rats; D011916:Rats, Inbred F344; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; D013481:Superoxides; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D000968:Antimycin A
references.citation: BMC Physiol. 2005 Feb 14;5(1):4
references.pmid: 15710036
references.citation: Biophys J. 1999 Jan;76(1 Pt 1):469-77
references.pmid: 9876159
references.citation: J Gerontol A Biol Sci Med Sci. 2008 May;63(5):461-6
references.pmid: 18511748
references.citation: Mol Cell Biochem. 2005 Aug;276(1-2):15-20
references.pmid: 16132680
references.citation: Anal Chem. 2005 Jul 1;77(13):4278-81
references.pmid: 15987137
references.citation: Biosens Bioelectron. 2010 Mar 15;25(7):1729-34
references.pmid: 20047824
references.citation: Am J Physiol. 1994 Feb;266(2 Pt 2):R375-80
references.pmid: 8141392
references.citation: Free Radic Biol Med. 2010 Apr 15;48(8):983-1001
references.pmid: 20116425
references.citation: Anal Chem. 1998 Jun 1;70(11):2243-7
references.pmid: 9624898
references.citation: J Chromatogr A. 2008 Mar 14;1184(1-2):144-58
references.pmid: 18054026
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2008;1:191-227
references.pmid: 20636079
references.citation: Am J Physiol Cell Physiol. 2007 Apr;292(4):C1353-60
references.pmid: 17122413
references.citation: Anal Bioanal Chem. 2003 Jan;375(1):28-9
references.pmid: 12520431
references.citation: Nature. 2000 Nov 9;408(6809):239-47
references.pmid: 11089981
references.citation: Genetics. 2009 Sep;183(1):175-84
references.pmid: 19546321
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Mar 25;769(1):97-106
references.pmid: 11936700
references.citation: Anal Chem. 2009 Mar 15;81(6):2410-3
references.pmid: 19219984
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):15038-43
references.pmid: 17015830
references.citation: J Biol Chem. 2004 Nov 19;279(47):49064-73
references.pmid: 15317809
references.citation: Nat Protoc. 2008;3(6):941-7
references.pmid: 18536642
references.citation: Biochem Biophys Res Commun. 2002 Nov 15;298(5):750-4
references.pmid: 12419317
references.citation: Langmuir. 2008 Nov 18;24(22):13218-24
references.pmid: 18950212
references.citation: Free Radic Biol Med. 2009 Apr 1;46(7):905-13
references.pmid: 19168125
references.citation: Nat Protoc. 2008;3(6):965-76
references.pmid: 18536644
references.citation: J Physiol. 2007 May 15;581(Pt 1):309-18
references.pmid: 17331997
references.citation: Anal Chem. 2000 Feb 15;72(4):872-7
references.pmid: 10701276
references.citation: Annu Rev Biophys Biomol Struct. 2000;29:239-63
references.pmid: 10940249
references.citation: Free Radic Biol Med. 2005 Sep 1;39(5):651-7
references.pmid: 16085183
references.citation: Anal Chem. 1997 Jul 1;69(13):2292-8
references.pmid: 9212703
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2008;1:165-90
references.pmid: 20636078
references.citation: Histochemistry. 1984;81(4):379-83
references.pmid: 6239843
references.citation: Nitric Oxide. 2002 Aug;7(1):36-41
references.pmid: 12175818
references.citation: Anal Chem. 2002 Feb 1;74(3):497-503
references.pmid: 11838666
references.citation: In Vitro Cell Dev Biol Anim. 1995 Nov;31(10):773-9
references.pmid: 8564066
references.citation: Talanta. 2005 Jul 15;67(1):212-6
references.pmid: 18970157
references.citation: Am J Physiol Endocrinol Metab. 2007 Feb;292(2):E571-6
references.pmid: 17018773
references.citation: Free Radic Biol Med. 2009 Nov 1;47(9):1267-75
references.pmid: 19748570
references.citation: Anal Chem. 2008 Dec 1;80(23):9141-9
references.pmid: 19551983
references.citation: J Appl Physiol (1985). 2007 Apr;102(4):1664-70
references.pmid: 17082364
references.citation: Trends Biochem Sci. 2000 Oct;25(10):502-8
references.pmid: 11050436
references.citation: Anal Chem. 1998 Dec 1;70(23):4974-84
references.pmid: 21644679
references.citation: Exp Gerontol. 2009 Jan-Feb;44(1-2):106-11
references.pmid: 18657920
references.citation: Annu Rev Pharmacol Toxicol. 2007;47:143-83
references.pmid: 17029566
references.citation: Exp Cell Res. 2008 Jan 1;314(1):164-72
references.pmid: 17964571
references.citation: J Cell Biol. 1986 Jun;102(6):2273-80
references.pmid: 3711146
references.citation: Antioxid Redox Signal. 2008 Aug;10(8):1463-74
references.pmid: 18407749
references.citation: Nat Protoc. 2008;3(1):8-21
references.pmid: 18193017
references.citation: In Vitro Cell Dev Biol Anim. 2002 Feb;38(2):66-72
references.pmid: 11928997
references.citation: Anal Chem. 2010 Feb 15;82(4):1411-9
references.pmid: 20102164