Mediation of endogenous antioxidant enzymes and apoptotic signaling by resveratrol following muscle disuse in the gastrocnemius muscles of young and old rats

Abstract

Hindlimb suspension (HLS) elicits muscle atrophy, oxidative stress, and apoptosis in skeletal muscle. Increases in oxidative stress can have detrimental effects on muscle mass and function, and it can potentially lead to myonuclear apoptosis. Resveratrol is a naturally occurring polyphenol possessing both antioxidant and antiaging properties. To analyze the capacity of resveratrol to attenuate oxidative stress, apoptosis and muscle force loss were measured following 14 days of HLS. Young (6 mo) and old (34 mo) rats were administered either 12.5 mg·kg(-1)·day(-1) of trans-resveratrol, or 0.1% carboxymethylcellulose for 21 days, including 14 days of HLS. HLS induced a significant decrease in plantarflexor isometric force, but resveratrol blunted this loss in old animals. Resveratrol increased gastrocnemius catalase activity, MnSOD activity, and MnSOD protein content following HLS. Resveratrol reduced hydrogen peroxide and lipid peroxidation levels in muscles from old animals after HLS. Caspase 9 abundance was reduced and Bcl-2 was increased, but other apoptotic markers were not affected by resveratrol in the gastrocnemius muscle after HLS. The data indicate that resveratrol has a protective effect against oxidative stress and muscle force loss in old HLS animals; however, resveratrol was unable to attenuate apoptosis following HLS. These results suggest that resveratrol has the potential to be an effective therapeutic agent to treat muscle functional decrements via improving the redox status associated with disuse.

Fig. 1.

Experimental protocol. Resveratrol was administered via oral gavage at a dose of 12.5 mg·kg⁻¹·day⁻¹ for 21 days. One milliliter of 0.01% carboxymethylcellulose was used as a vehicle control. Following 7 days of resveratrol administration, animals were randomly assigned to 14 days of hindlimb suspension (HLS) or continued ambulation. Following the experimental protocol, the gastrocnemius muscles were dissected and assessed for muscle wet weight, oxidative stress, and apoptotic indices. MWW, muscle wet weight.

Fig. 2.

Enzymatic activities and protein contents of CuZnSOD, MnSOD, and catalase in response to aging and HLS. A: CuZnSOD activity was assessed using a spectrophotometric assay and is expressed as units of enzymatic activity (U)·ml⁻¹·mg protein⁻¹. B: CuZnSOD protein content was determined via immunoblotting; a representative blot is shown. C: MnSOD activity was assessed using a spectrophotometric assay and is expressed as units of enzymatic activity (U)·ml⁻¹·mg protein⁻¹. D: MnSOD protein content was determined via immunoblotting; a representative blot is shown. E: catalase activity was assessed using a spectrophotometric assay and is expressed as milliunits of enzymatic activity (mU)/mg protein. F: catalase protein content was determined via immunoblotting; a representative blot is shown. YC, young control; YS, young suspended; OC, old control; OS, old suspended; YVC, young vehicle control, YVCS, young vehicle control suspended, YRC, young resveratrol control; YRS, young resveratrol suspended; OVC, old vehicle control; OVCS, old vehicle control suspended; ORC, old resveratrol control; ORS, old resveratrol suspended. *P ≤ 0.05, vehicle control vs. age-matched and treatment-matched group (Effect of resveratrol).**P ≤ 0.05, nonsuspended treatment vs. matched control (suspension effect). #P ≤ 0.05, young treatment matched (aging effect).

Fig. 3.

Resveratrol attenuated increases in hydrogen peroxide (H₂O₂) concentration and lipid peroxidation associated with HLS in old animals. A: H₂O₂ concentrations were determined fluorometrically in gastrocnemius muscle homogenate. Data are expressed as μmol·H₂O₂⁻¹·μg protein⁻¹. Significance was set at P ≤ 0.05, and all data are expressed as means ± SE. B: MDA and HAE levels were evaluated as a combined marker of lipid peroxidation and expressed in μM[MDA/HAE]/mg protein. See Fig. 2 for definitions of abbreviations. **P ≤ 0.05, nonsuspended control vs. suspended animals (Suspension effect). #P ≤ 0.05, old vs. young treatment matched (aging effect). *P ≤ 0.05, vehicle control vs. age-matched and treatment matched groups (Effect of resveratrol).

Fig. 4.

The effects of resveratrol administration and HLS on apoptotic signaling in young and old animals. A: Bax protein content was determined via immunoblotting; a representative blot is shown. B: Bcl-2 protein content was determined via immunoblotting; a representative blot is shown. C: Caspase 9 activity was assessed using a fluorometric assay. D: caspase 3 activity was assessed using a fluorometric assay. E: cell death ELISA. See Fig. 2 for definitions of abbreviations. *P ≤ 0.05, vehicle control vs. age-matched and treatment-matched groups (effect of resveratrol).**P ≤ 0.05, nonsuspended control vs. suspended animals (suspension effect). #P ≤ 0.05, old vs. young treatment matched (aging effect).

Fig. 5.

Gastrocnemius muscle wet weight and plantar flexor maximal isometric force. The gastrocnemius muscles were dissected, immediately blotted, and weighed as a gross estimation of muscle size and represented in grams (g; A), or as ratio to the animal's body weight (mg/g; B). Pre and post-HLS force measurements were assessed. Three isometric contractions were recorded per animal for both pre and post-HLS assessments. The average of the three contractions at each time point was used to determine the decrease in isometric force output following HLS. Significance was set at (P ≤ 0.05), and all data are expressed as means ± SE. *P ≤ 0.05, vehicle control vs. age-matched and treatment-matched groups (effect of resveratrol).**P ≤ 0.05, nonsuspended control vs. suspended animals (suspension effect). #P ≤ 0.05, old vs. young treatment-matched (aging effect).