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.
Highlights
IN OLDER PEOPLE, DECLINING muscle mass and function lead to instability, increased risk for falls, and residential care [60]
The hematoxylin and eosin–stained cross-sections shown in Fig. 1B show an apparent increase in the interstitial space between fibers in muscle from old mice, but no other marked changes in fiber structure were apparent
The main findings of this study were that skeletal muscle fibers from muscles of mice that showed age-related changes in muscle mass demonstrated increased oxidation of the nonspecific probe for reactive oxygen and nitrogen species, CM-DCFH
Summary
Mice were killed by an overdose of anesthetic (ketamine hydrochloride and medatomidine hydrochloride) by intraperitoneal injection Both tibilais anterior and gastrocnemius muscles were removed, weighed, and stored at Ϫ80°C for further analysis, and the flexor digitorum brevis (FDB) muscles were removed for isolation of intact single fibers. After CM-DCFH-DA loading and 30 min of incubation, fibers were washed twice with D-PBS and Eagle’s minimum essential medium without phenol red (to avoid interference with fluorescence imaging) was added to fibers to maintain these cells during the fluorescence microscopy imaging. For CMFDA loading, fibers were incubated with CMFDA 5 M in D-PBS for 30 min, washed twice with D-PBS and maintained in MEM without phenol red for fluorescence microscopy.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: American Journal of Physiology-Regulatory, Integrative and Comparative Physiology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
