Age-dependent increase in oxidative stress in gastrocnemius muscle with unloading

Abstract

Oxidative stress increases during unloading in muscle from young adult rats. The present study examined the markers of oxidative stress and antioxidant enzyme gene and protein expressions in medial gastrocnemius muscles of aged and young adult (30 and 6 mo of age) Fischer 344 x Brown Norway rats after 14 days of hindlimb suspension. Medial gastrocnemius muscle weight was decreased by approximately 30% in young adult and aged rats following suspension. When muscle weight was normalized to animal body weight, it was reduced by 12% and 22% in young adult and aged rats, respectively, after suspension. Comparisons between young adult and aged control animals demonstrated a 25% and 51% decline in muscle mass when expressed as absolute muscle weight and muscle weight normalized to the animal body weight, respectively. H(2)O(2) content was elevated by 43% while Mn superoxide dismutase (MnSOD) protein content was reduced by 28% in suspended muscles compared with control muscles exclusively in the aged animals. Suspended muscles had greater content of malondialdehyde (MDA)/4-hydroxyalkenals (4-HAE) (29% and 58% increase in young adult and aged rats, respectively), nitrotyrosine (76% and 65% increase in young adult and aged rats, respectively), and catalase activity (69% and 43% increase in young adult and aged rats, respectively) relative to control muscles. Changes in oxidative stress markers MDA/4-HAE, H(2)O(2), and MnSOD protein contents in response to hindlimb unloading occurred in an age-dependent manner. These findings are consistent with the hypotheses that oxidative stress has a role in mediating disuse-induced and sarcopenia-associated muscle losses. Our data suggest that aging may predispose skeletal muscle to increased levels of oxidative stress both at rest and during unloading.

Figures

Fig. 1.

Muscle mass change during hindlimb suspension. A: muscle weight. The extent of muscle loss following hindlimb suspension was estimated by monitoring the muscle mass loss between the suspended and the control medial gastrocnemius muscle wet weight. The data are presented as means ± SE. The main effects of age and suspension were analyzed by a 2 × 2 ANOVA. B: muscle weight after normalized to animal body weight. The extent of muscle loss following hindlimb suspension was further examined by examining the decrease in medial gastrocnemius muscle wet weight after normalized to the animal's body weight between the suspended and control groups. Data are presented as means ± SE. The main effects of age and suspension were analyzed by a 2 × 2 ANOVA.

Fig. 2.

Malondialdehyde (MDA)/4-hydroxyalkenal (4-HAE) content. The level of lipid peroxidation was estimated by measuring the content of MDA/4-HAE. The measured μM MDA/4-HAE is normalized to the total mg protein content of the sample used in the assay. Normalized data are presented as means ± SE. The main effects of suspension and interaction (age × suspension) were analyzed by a 2 × 2 ANOVA.

Fig. 3.

H2O2 content. The H2O2 content was determined by a fluorometric assay. The fluorescence unit is normalized to the total mg protein content of the sample used in the assay. Normalized data are presented as means ± SE. The main effects of age and interaction (age × suspension) were analyzed by a 2 × 2 ANOVA.

Fig. 4.

Nitrotyrosine content. The content of nitrotyrosine was measured in cytosolic fraction by an immuno-dot blot. The data are expressed as optical density (OD) × resulting dot area and expressed in arbitrary units. Insets: representative blots for nitrotyrosine in control and suspended muscles isolated from young adult and aged animals. Data are presented as means ± SE. The main effects of age and suspension were analyzed by a 2 × 2 ANOVA.

Fig. 5.

mRNA content of antioxidant enzymes. The mRNA contents of Mn superoxide dismutase (MnSOD), Cu-Zn superoxide dismutase (CuZnSOD), and catalase were determined by RT-PCR with 18S rRNA as an internal control. A: data are expressed as a ratio of mRNA expression of interested gene/18S in arbitrary units. B: representative results for the MnSOD, CuZnSOD, and catalase mRNA. Data are presented as means ± SE. The main effect of age was analyzed by a 2 × 2 ANOVA.

Fig. 6.

Protein content of antioxidant enzymes. The protein contents of MnSOD and CuZnSOD were assessed in the total cytosolic protein fraction by immunoblot analysis. A: data are expressed as OD × resulting band area, and expressed in arbitrary units. B: representative blots for MnSOD and CuZnSOD in control and suspended muscles isolated from young adult and aged animals. Data are presented as means ± SE. The main effects of age and interaction (age × suspension) were analyzed by a 2 × 2 ANOVA.

Fig. 7.

Enzymatic activity of antioxidant enzymes. The MnSOD, catalase, and glutathione peroxidase (GPx) enzymatic activities were estimated by spectrophotometric assays. Data are expressed as units/mg protein and presented as means ± SE. The main effects of suspension and age were analyzed by a 2 × 2 ANOVA.

Fig. 8.

The relationship between medial gastrocnemius muscle mass and MDA/4-HAE, H2O2 and nitrotyrosine. Negative significant correlation coefficients (r) were established between the relative muscle mass of medial gastrocnemius and the contents of MDA/4-HAE (A), H2O2 (B), and nitrotyrosine (C) in young adult and aged control and suspended animals (n = 38).

Fig. 9.

The relationship between medial gastrocnemius muscle mass and MnSOD mRNA and protein contents. Positive significant correlation coefficients (r) were established between the relative muscle mass of medial gastrocnemius and the contents of MnSOD mRNA (A) and protein (B) in young adult and aged control and suspended animals (n = 38).

Fig. 10.

The relationship between medial gastrocnemius muscle mass and catalase mRNA and activity. A positive correlation coefficient (r) was established between the relative muscle mass of medial gastrocnemius and the catalase mRNA (A), whereas a negative significant r was established between the relative muscle mass and the catalase activity (B) in young adult and aged control and suspended animals (n = 38).