Short-term caloric restriction, resveratrol, or combined treatment regimens initiated in late-life alter mitochondrial protein expression profiles in a fiber-type specific manner in aged animals

Abstract

Aging is associated with a loss in muscle known as sarcopenia that is partially attributed to apoptosis. In aging rodents, caloric restriction (CR) increases health and longevity by improving mitochondrial function and the polyphenol resveratrol (RSV) has been reported to have similar benefits. In the present study, we investigated the potential efficacy of using short-term (6 weeks) CR (20%), RSV (50 mg/kg/day), or combined CR+ RSV (20% CR and 50 mg/kg/day RSV), initiated at late-life (27 months) to protect muscle against sarcopenia by altering mitochondrial function, biogenesis, content, and apoptotic signaling in both glycolytic white and oxidative red gastrocnemius muscle (WG and RG, respectively) of male Fischer 344 × Brown Norway rats. CR but not RSV attenuated the age-associated loss of muscle mass in both mixed gastrocnemius and soleus muscle, while combined treatment (CR + RSV) paradigms showed a protective effect in the soleus and plantaris muscle (P < 0.05). Sirt1 protein content was increased by 2.6-fold (P < 0.05) in WG but not RG muscle with RSV treatment, while CR or CR + RSV had no effect. PGC-1α levels were higher (2-fold) in the WG from CR-treated animals (P < 0.05) when compared to ad-libitum (AL) animals but no differences were observed in the RG with any treatment. Levels of the anti-apoptotic protein Bcl-2 were significantly higher (1.6-fold) in the WG muscle of RSV and CR + RSV groups compared to AL (P < 0.05) but tended to occur coincident with elevations in the pro-apoptotic protein Bax so that the apoptotic susceptibility as indicated by the Bax to Bcl-2 ratio was unchanged. There were no alterations in DNA fragmentation with any treatment in muscle from older animals. Additionally, mitochondrial respiration measured in permeabilized muscle fibers was unchanged in any treatment group and this paralleled the lack of change in cytochrome c oxidase (COX) activity. These data suggest that short-term moderate CR, RSV, or CR + RSV tended to modestly alter key mitochondrial regulatory and apoptotic signaling pathways in glycolytic muscle and this might contribute to the moderate protective effects against aging-induced muscle loss observed in this study.

Keywords: Aging; Apoptosis; Biogenesis; Caloric restriction; Sarcopenia; Sirtuins.

Fig. 1

AMPK activation and Sirtuin 1 (Sirt1) protein content. AMPK activation was measured in white gastrocnemius (WG; A) and red gastrocnemius (RG; C) muscle in 27 month old AL = ad-libitum fed (n = 11), CR = caloric restricted (n = 4), RSV = resveratrol (n = 4), and CR + RSV = caloric restricted + resveratrol (n = 12). Kinase activation status was determined as the ratio of the phosphorylated form (p-AMPKα) of the protein compared to the total protein (AMPKα) content for each sample. Sirt1 protein in WG (B) and RG (D) muscle. Representative protein blots are shown along with the corresponding Ponceau S-stained membrane used to correct for loading. A graphical summary of the data is shown below (n = 4–11). Significance was set at P < 0.05 and all data are represented as mean ± SE. Data are expressed as arbitrary units (AU). *P < 0.05 vs. AL.

Fig. 2

PGC-1α and cytochrome c protein content. PGC-1α protein was measured in white gastrocnemius (WG; A) and red gastrocnemius (RG; C) muscles and cytochrome c protein in WG (B) and RG (D) in 27 month old AL = ad-libitum, CR = caloric restricted, RSV = resveratrol, and CR + RSV = caloric restricted + resveratrol. Representative Western blots and corresponding Ponceau S-stained membranes, along with a graphical summary of the data is shown (n = 4–11). Significance was set at P < 0.05 and all data are represented as mean ± SE. Data are expressed as arbitrary units (AU). *P < 0.05 vs. AL; †P < 0.05 vs. CR group and RSV group.

Fig. 3

Apoptosis Inducing Factor (AIF) and Manganese Superoxide Dismutase (MnSOD) protein content. AIF protein was measured via immunoblotting in white gastrocnemius (WG; A) and red gastrocnemius (RG; C) muscles and MnSOD protein in WG (B) and RG (D). AL = ad libitum, CR = caloric restricted, RSV = resveratrol, and CR + RSV = caloric restricted + resveratrol. Representative blots are shown above with the corresponding Ponceau S-stained membrane below. A summary of repeated experiments is graphically illustrated (n = 4–11). Significance was set at P < 0.05 and all data are represented as mean ± SE. Data are expressed as arbitrary units (AU). *P < 0.05 vs. AL.

Fig. 4

Protein levels of pro-apoptotic Bax and anti-apoptotic Bcl-2. Bax protein was measured via immunoblotting in white gastrocnemius (WG; A) and red gastrocnemius (RG; C) muscles and Bcl-2 protein in WG (B) and RG(D). AL = ad-libitum, CR = caloric restricted, RSV = resveratrol, and CR + RSV = caloric restricted + resveratrol. Representative Western blots, and the corresponding Ponceau S-stained membranes are shown above with a graphical summary of the data illustrated below (n = 4–11). Significance was set at P < 0.05 and all data are represented as mean ± SE. Data are expressed as arbitrary units (AU). *P < 0.05 vs. AL; †P < 0.05 vs. RSV group.

Fig. 5

Cell death in aged gastrocnemius muscle. DNA fragmentation was measured in cytosol from WG (A) and RG (B) using a Cell death ELISA kit. The data are presented as an apoptotic index. All data are represented as mean ± SE (n = 4–8).