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

Abstract

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 microM H(2)O(2) 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.

FIG. 1. Viability of single mature skeletal muscle fibers in culture

Light microscopic images of isolated fibers after 24 h in culture: (A) Bright field image, 32× original magnification. (B) Epifluorescence image of fibers stained with DAPI, 20× original magnification. (C) Bright-field image showing a viable fiber that excluded Trypan blue and parts of two damaged fibers. 32× original magnification. Scale bar = 10 μm. (D) (i–viii) Bright-field images of the same fiber acquired each day over 7 days. Main image: 32× original magnification. Scale bar = 10 μm. Inset image: 20× original magnification.

FIG. 2. Confocal images of a single mature skeletal muscle fiber

Selected area of a single mature skeletal muscle fiber loaded with the fluorophores CM-DCFH (17.5 μM) and Mitotracker red (20 nM). (A) Bright-field image. (B) and (C) Confocal images of the same optical section (thick = 2.61 μm) showing: CM-DCF fluorescence (B) and Mitotracker red fluorescence (C). 63× original magnification. Scale bar 10 = μm.

FIG. 3. Optimization of measurements of CM-DCF fluorescence in single mature skeletal muscle fibers over a time course

Data from seven individual quiescent fibers are presented. (A) Individual CM-DCF fluorescence values obtained from each fiber over 45 min. (B) Mean CM-DCF fluorescence values from the fibers at different time points (data are presented as mean ± SEM). (C) Rate of change in CM-DCF fluorescence each 15 min over the 45 min experiment (data are presented as mean ± SEM). (D) Fluorescence values relative to the initial fluorescence obtained for each fiber were calculated and these relative fluorescence values are presented for each fiber over the 45 min experimental period. (E) Mean relative fluorescence values from the group of fibers at different time points (data presented as mean ± SEM). (C) Rate of change in relative fluorescence each 15 min over the 45 min experiment (data presented as mean ± SEM).

FIG. 4. Assessment of CM-DCFH loading and comparison of methods to correct for differences in loading of CM-DCFH into single mature skeletal muscle fibers

(A) Change in CM-DCF fluorescence in two fibers during continuous exposure to UV over 15 min. Fiber 1 fluorescence (), fiber 2 fluorescence (–▲–), background fluorescence (). Inset images show the morphology and fluorescence emission of the fibers prior to photo-oxidation, at the point when maximum photo-oxidation was observed and at the end of the UV exposure. (B) Comparison of two approaches to normalize values of fluorescence obtained from fibers. (B-i) Mean changes in fluorescence where data were normalized to the initial value from that fiber (◆), or data are expressed as a proportion of the total CM-DCFH within the fiber (◇). (B-ii) Rate of change in fluorescence where data were normalized to the initial value from that fiber (□), or data are expressed as a proportion of the total CM-DCFH within the fiber (). Values are expressed as mean ± S.E.M. for five fibers.

FIG. 5. CM-DCF fluorescence from single mature skeletal muscle fibers following exposure to H₂O₂ with and without glutathione loading

(A). The relative fluorescence from control fibers that were not exposed to H₂O₂ () compared with fibers exposed to 1 μM H₂O₂ at the 15 min time point (■). (B). Rate of change of relative DCF fluorescence in control fibers (□), fibers exposed to 1 μM H₂O₂ for 30 min starting at the 15 min time point (■) and fibers pre -treated with 5 mM glutathione ethyl ester (GSHEE) for 2 h prior to CM-DCFH DA loading and exposure to 1 μM H₂O₂ for 30 min starting at the 15 min time point (). *Statistically significant compared with control fibers during the same time period; #statistically significant compared with fibers exposed to H₂O₂ during the same time period. Data were analyzed using a one-way ANOVA (p < 0.05), post hoc LSD (p < 0.05) and are shown as mean ± S.E.M, n 5–7 fibers in each group.

FIG. 6. CM-DCF fluorescence from single mature skeletal muscle fibers subjected to a period of electrically stimulated contractile activity with and without prior glutathione loading

(A). The relative fluorescence from control fibers () compared with fibers that underwent contractile activity induced by electrical stimulation over the 15–30 min period (■). (B). Rate of change of relative DCF fluorescence in control fibers (□), fibers subjected to contractile activity induced by electrical stimulation over the 15–30 min period (■), and fibers pre -treated with 5 mM glutathione ethyl ester (GSHEE) for 2 h prior to CM-DCFH DA loading and subjected to contractile activity induced by electrical stimulation over the 15–30 min period (). *Statistically significant compared with control fibers during the same time period; #statistically significant compared with fibers subjected to contractile activity induced by electrical stimulation over 15–30 min period. Data were analyzed using a one-way ANOVA (p < 0.05), post hoc LSD (p < 0.05) and are shown as mean ± S.E.M, n 7–13 fibers in each group.