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

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.