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Comparative Study
. 2013 Aug 15;305(4):R351-8.
doi: 10.1152/ajpregu.00530.2012. Epub 2013 May 22.

Aging increases the oxidation of dichlorohydrofluorescein in single isolated skeletal muscle fibers at rest, but not during contractions

Jesus Palomero  1 , Aphrodite Vasilaki, Deborah Pye, Anne McArdle, Malcolm J Jackson
Affiliations
Comparative Study

Aging increases the oxidation of dichlorohydrofluorescein in single isolated skeletal muscle fibers at rest, but not during contractions

Jesus Palomero et al. Am J Physiol Regul Integr Comp Physiol. .

Abstract

An increase in the activity of reactive oxygen species (ROS) has been implicated in the mechanisms of loss of skeletal muscle that occurs during aging, but few studies have attempted to directly assess activities in intact muscle fibers. The current project used the nonspecific fluorescent probe for ROS and reactive nitrogen species, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCFH), in single, isolated, mature skeletal muscle fibers from adult and old mice in addition to biochemical measurements of key regulatory proteins for ROS in muscles of these animals. Data confirmed the changes in key regulatory processes for ROS (increased glutathione peroxidase 1 and catalase activities and reduced total glutathione content) previously reported in muscle from old mice and showed increased CM-DCFH oxidation in muscle fibers from old mice at rest and indicate that these changes are likely due to an increase in generation of oxidants rather than a lack of scavenging capacity. The increased CM-DCFH oxidation persisted even when cellular defenses against oxidants were increased by loading fibers from young and old mice with glutathione. During contractile activity, and in contrast to the increase observed in fibers from young mice, there was no further increase in CM-DCFH oxidation in muscle fibers from old mice. These data also suggest that the defect in short-term adaptations to contractions that occurs in old mice may be related to a diminished, or absent, increase in the muscle generation of ROS and/or reactive nitrogen species that normally accompanies contractile activity in young mice.

Keywords: muscle; reactive oxygen; single fiber.

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Figures

Fig. 1.
Fig. 1.
A: mass of the tibialis anterior muscle in young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from young animals, n = 50–59. B: example of transverse sections from muscles of young (i) and old (ii) mice stained with hematoxylin and eosin. C: cross-sectional area of single fibers obtained from the flexor digitorum brevis (FDB) muscle of young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from young animals, n = 100 fibers.
Fig. 2.
Fig. 2.
A: total glutathione (GSH) content of gastrocnemius muscles from young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from young animals, n = 31–32. B: oxidized glutathione (GSSG) content of gastrocnemius muscles from young and old mice. Data are presented as means ± SE, n = 31–32. C: ratio of total/oxidized glutathione in gastrocnemius muscles from young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from young animals, n = 31–32. D: glutathione peroxidase 1 (GPx1) activities of gastrocnemius muscles from young and old mice. Data are presented as means± SE. *P < 0.05 compared with values from young animals, n = 8. E: catalase (CAT) activities of gastrocnemius muscles from young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from young animals, n = 8. F: total superoxide dismutase (SOD) activities of gastrocnemius muscles from young and old mice. Data are presented as means ± SE, n = 8.
Fig. 3.
Fig. 3.
A: rate of change in 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (CM-DCF) fluorescence in single quiescent FDB fibers from young and old mice. Data are presented as means ± SE. *P < 0.05 compared with values from fibers of young animals over the same time period, n = 15–16. B: rate of change in CM-DCF fluorescence in single FDB fibers from young mice that were either at rest throughout the experiment or subjected to a 15-min period of electrically stimulated contractions during the 15- to 30-min time period. Data are presented as means ± SE. *P < 0.05 compared with values from nonstimulated fibers over the same time period, n = 15–6. C: rate of change in CM-DCF fluorescence in single FDB fibers from old mice that were either at rest throughout the experiment or subjected to a 15-min period of electrically stimulated contractions during the 15- to 30-min time period. Data are presented as means ± SE. *P < 0.05 compared with values from nonstimulated fibers over the same time period, n = 16–13.
Fig. 4.
Fig. 4.
A: rate of change in CM-DCF fluorescence in single FDB fibers from young mice that were exposed to 5 μM hydrogen peroxide (H2O2) during the 15- to 45-min time period. Data are presented as means ± SE. *P < 0.05 compared with values from fibers that were not exposed to H2O2 over the same time period, n = 5–6. B: rate of change in CM-DCF fluorescence in single FDB fibers from old mice that were exposed to 5 μM H2O2 during the 15- to 45-min time period. Data are presented as means± SE. *P < 0.05 compared with values from fibers that were not exposed to H2O2 over the same time period, n = 4. C: rate of change in CM-DCF fluorescence in single FDB fibers from young and old mice that were pretreated with 1 mM glutathione ethyl ester (GSHEE) for 18 h prior to loading with CM-DCFH. Data are presented as means ± SE. *P < 0.05 compared with values from fibers from young mice at the same time interval, n = 3–4.
Fig. 5.
Fig. 5.
Fluorescence intensities from fibers loaded with 5-chloromethylfluorescein diacetate (CMFDA) as an indicator of intracellular GSH content. Before CMFDA loading, fibers were maintained in culture conditions for 1) 2 h (n = 7); 2) 24 h (n = 5); 3) 24 h and pretreated with GSHEE (5 mM for 4 h) (n = 8); and 4) 24 h and pretreated with GSHEE (5 mM for 6 h) (n = 11). Data are presented as means ± SE. *P < 0.05 compared with values from untreated fibers maintained in culture for 24 h.
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