In Situ Detection and Measurement of Intracellular Reactive Oxygen Species in Single Isolated Mature Skeletal Muscle Fibers by Real Time Fluorescence Microscopy

Abstract

[EN] Reactive oxygen species (ROS) produced by skeletal muscle stimulate adaptive responses to activity and me-diate some degenerative processes. ROS activity is usually studied by measuring indirect end-points of theirreactions with various biomolecules. In order to develop a method to measure the intracellular ROS genera-tion in real-time in mature skeletal muscle fibers, these were isolated from the flexor digitorum brevis(FDB)muscle of mice and cultured on collagen-coated plates. Fibers were loaded with 5- (and 6-) chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate (CM-DCFH DA) and measurements of 5- (and 6-) chloromethyl-2 ,7 -dichlorofluorescin (CM-DCF) fluorescence from individual fibers obtained by microscopy over 45 min. Thesensitivity of this approach was demonstrated by addition of 1 MH2O2to the extracellular medium. Con-tractions of isolated fibers induced by field electrical stimulation caused a significant increase in CM-DCFfluorescence that was abolished by pre-treatment of fibers with glutathione ethyl ester. Thus, CM-DCF fluo-rescence microscopy can detect physiologically relevant changes in intracellular ROS activity in single iso-lated mature skeletal muscle fibers in real-time, and contractions generated a net increase that was abolishedwhen the intracellular glutathione content was enhanced. This technique has advantages over previous ap-proaches because of the maturity of the fibers and the analysis of single cells, which prevent contributionsfrom nonmuscle cells.