In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy

Reactive oxygen species (ROS) produced by skeletal muscle stimulate adaptive responses to activity and mediate some degenerative processes. ROS activity is usually studied by measuring indirect end-points of their reactions with various biomolecules. In order to develop a method to measure the intracellular ROS generation 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. The sensitivity of this approach was demonstrated by addition of 1 mu M H2O2 to the extracellular medium. Contractions of isolated fibers induced by field electrical stimulation caused a significant increase in CM-DCF fluorescence that was abolished by pre-treatment of fibers with glutathione ethyl ester. Thus, CM-DCF fluorescence microscopy can detect physiologically relevant changes in intracellular ROS activity in single isolated mature skeletal muscle fibers in real-time, and contractions generated a net increase that was abolished when the intracellular glutathione content was enhanced. This technique has advantages over previous approaches because of the maturity of the fibers and the analysis of single cells, which prevent contributions from nonmuscle cells.