2.1 Experimental design
The dorsiflexors of the left limb of young (12 weeks of age; n=14) and aged (30 months of age; n=14) male Fischer 344 Brown x Norway rats were subjected to repetitive loading exercise. Seven animals from each age group were randomly assigned to a diet supplemented with Vitamin E (DL-alpha tocopheryl acetate; 30,000 mg/kg) and Vitamin C (L-ascorbic acid; 2% by weight) or normal non-supplemented (NS) rat chow containing 126 mg/kg of Vitamin E and 0% Vitamin C. All animals had free access to rat chow and water. The antioxidant or NS diets began seven days before the first exercise session. The non-supplemented animals were a subset of animals described in another study . All experimental procedures carried approval from the Institutional Animal Use and Care Committee from West Virginia University School of Medicine. The animal care standards were followed by adhering to the recommendations for the care of laboratory animals as advocated by the American Association for Accreditation of Laboratory Animal Care (AAALAC) and fully conformed to the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings."
2.2 Muscle function
Maximal isometric muscle force, positive work, and negative work were assessed in the left exercised and right control limbs on a custom-built dynamometer . The dorsiflexor muscle group was activated indirectly through electrical stimulation of the common peroneal nerve via platinum stimulating electrodes (Grass Medical Instruments, Quincy MA, USA). Muscle stimulation for all protocols was via 120 Hz square wave pulses at 200 µs pulse duration, and 4 volts. Dorsiflexor isometric force was measured at an ankle angle of 90 degrees, using a stimulation duration of 300 ms. Positive and negative work was averaged from the first three individual stretch-shortening contractions in each exercise session . The stretch-shortening contraction was performed by activating the dorsiflexor muscles for 300 ms then moving the load cell fixture from 70 degrees to 140 degrees at an angular velocity of 500°/s. The load cell fixture was immediately returned to 70 degrees. Activation was continued for 300 ms after cessation of the movement.
2.3 Unilateral repetitive loading exercise
Repetitive loading consisted of 3 sessions per week for 4.5 weeks, of 80 stretch/shortening (i.e., eccentric / concentric) contraction cycles per exercise session . This method has been previously shown to produce a hypertrophic response in muscles of young adult rats although aging attenuates the hypertrophic response to these loading conditions.
2.4 Muscle preparation
Forty-eight hours after the last exercise session, the tibialis anterior of both loaded and control limbs were removed with the animal under anesthesia (2% isoflurane). The rats were then euthanized, via an overdose of ketamine/xylazine (30%/70%, v/v). The muscles were washed in phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, and a pH of 7.4), blotted dry, and then weighed. A section of the muscle was obtained for the determination of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). The remaining muscle was snap frozen in liquid nitrogen and stored at −80°C.
2.5 RNA Isolation
Sixty micrograms of frozen muscle was homogenized in 1ml of Tri-Reagent (Molecular Research Center, Cincinnati, OH) with a motorized blade homogenizer. The RNA was isolated according to our standard procedures . The RNA was treated with DNAse I using a DNA-free kit (Ambion, Austin, TX) and quantified using a BioRad SmartSpec 3000. The RNA samples were quantified if their 260:280 ratios were 1.7 or greater.
2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Two micrograms of total RNA were reversed transcribed using 1.0µl of random primers, 1.0µl of 10mM dNTP, and 1.0µl of SuperScript II reverse transcriptase (Invitrogen/Life Technologies, Bethesda MD) as previously described . The resulting complimentary DNA (cDNA) was stored at −80°C or used for PCR analyses.
Primers for the genes of interest were designed as follows: CuZnSOD sense-5’-AGGCCGTGTGCGTGCTGA-3’; anti-sense-5’-CCCAATCACACCACAAGCCA-3’; GPx-1 sense-5’-CCTCGTGGCCTGGTGGTCCT-3’; anti-sense-5’- AGGGGTTGCTAGGCTGCTTGGA-3’. The primers for MnSOD and catalase were the same as previously published by our lab . Preliminary experiments were conducted to ensure that the number of PCR cycles were completed in the linear range of amplification for each gene of interest. PCR products were verified by restriction digestion based on predicted PCR sequences. Routine PCR amplification was conducted using PCR buffer, MgCl2, 5mM dNTPs, 100ng/ml of primer pairs, 18S primer pairs, 1.0µl of Taq DNA polymerase, and 1.0 µl of cDNA . Amplification of PCR products were performed over a linear range of application cycles, using a thermocycler using: a denaturing step at 95°C for 45s, an annealing step for 45s, and an extension step at 72°C for 45s. 20µl of each PCR product was separated by electrophoresis on 1.5% agarose gels. The gels were stained with ethidium bromide to visualize the PCR products. The signal from each PCR gene product was expressed as a ratio to the 18S signal from the same PCR product. The PCR signals were recorded via a digital camera (Kodak 290) and the signals were quantified in arbitrary units as optical density x band area, using 1D Kodak image analysis software (Eastman Kodak Company, Rochester, NY).
2.7 Muscle Protein Fractionation
Cytoplasmic and nuclear protein fractions were obtained from 75 mg of frozen tibialis anterior using methods as reported previously by our lab . Muscle samples were homogenized in 500 µl of ice-cold lysis buffer (10 mM NaCl, 1.5 mM MgCl2, 20mM HEPES at pH 7.4, 20% glycerol, 0.1% Triton X-100, and 10µM dithioreitol) with a mechanical homogenizer. A lower concentration of dithioreitol was used than in previous studies, to prevent interference with subsequent enzyme activity assays. Muscle homogenates were centrifuged at 800g for 5-minutes at 4°C. The supernatants were collected and centrifuged three times at 3500g for 5-minutes at 4°C. The resulting supernatant was collected as the nuclei-free cytosolic fraction and divided into two equal portions; the first portion was frozen at −80°C until needed, and a protease inhibitor cocktail containing 104mM 4-[2-aminoethyl]-benzenesulfonylflouride hydrochloride (AEBSF), 0.8mM aprotinin, 2mM leupeptin, 4mM bestatin, 1.5 mM pepstatin A and 1.4 mME-64 (Sigma-Aldrich, St. Louis, Mo, USA) was added to the second portion before it was frozen at −80°C. Protein concentrations for each sample were determined in triplicate via a DC protein concentration assay (Bio-Rad, Hercules, CA). The cytosolic fraction was used in the following assays: H2O2 concentration, catalase activity, GPX activity, CuZnSOD and MnSOD activity, and western immunoblots.
2.8 Western immunoblots
The protein contents of glutathione peroxidase-1 (GPx-1), catalase, copper-zinc superoxide dismutase, (CuZnSOD) and manganese superoxide dismutase (MnSOD) were measured in the cytosolic protein fractions. Thirty µg of protein was loaded into each well of a 4–12% gradient polyacrylamide gel (Novex, Invitrogen) and separated by routine SDS-polyacrylamide gel electrophoresis (PAGE) for 1.5 hours at 20°C and transferred to a nitrocellulose membrane. The membranes were blocked in 5% non-fat milk protein (NFM) for 1-hour at room temperature then incubated in appropriate dilutions of primary antibodies (diluted in 1% NFM in Tris-buffered saline with 0.05% Tween-20 (TBS-T) overnight at 4°C. The membranes were washed in TBS-T followed by incubation in appropriate dilutions of secondary antibodies (diluted in 5% NFM in TBS-T) that were conjugated to horseradish peroxidase. The protein signals were developed using a chemiluminescent substrate (ECL Advanced, Amersham Bioscience) and visualized by exposing the membranes to X-ray films (BioMax MS-1; Eastman Kodak). Digital records were captured by a Kodak 290 camera and protein bands quantified using 1-D analysis software (Eastman Kodak, USA). The bands were quantified as optical density (OD) x band area and expressed in arbitrary units.
2.9 Hydrogen peroxide (H2O2) levels
A commercially available H2O2 kit (#FLOH 100-3; Cell Technology, Mountain View, CA) was used to measure H2O2 in muscle homogenates. The assay was performed with several modifications to the manufacturer’s directions. Briefly, 50µL of controls, samples, or H2O2 dilutions were mixed with 50µL of the reaction cocktail in each well to initiate the reaction. The plate was incubated in the dark for 10 minutes, at 20°C and fluorescence was detected with an excitation at 530nm and measured at 590nm. All analyses were done in duplicate and the samples were normalized to muscle protein concentration in each sample via a DC protein concentration assay (Bio-Rad, Hercules, CA).
2.10 GSH and GSH/GSSG Ratio
A commercially available kit (#21040, Percipio Biosciences, Inc., Burlingame, CA) was used to measure Glutathione (GSH), and oxidized glutathione (GSSG) in muscle homogenates. The GSH/GSSG ratio was determined from these data. The assay was conducted with several modifications to the manufacturer’s directions. Briefly, muscle tissue (~40 mg) was homogenized immediately after dissection in 530 µl cold 5% metaphosphoric acid (MPA) for the GSH sample and for the GSSG sample ~40 mg of muscle tissue was homogenized immediately after dissection in 500 µl cold 5% metaphosphoric acid (MPA) and 30µl of M2VO scavenger. Homogenates were then frozen in liquid nitrogen and stored at −80°C until analyzed.
The assay was conducted as described by the manufacturer. Briefly, cold 5% MPA was added to each sample mixed, and centrifuged at 1000g for 10 minutes. 50µl of sample and the appropriate buffer and 50µl of 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) in Na·PO4 and 50µl of glutathione reductase in Na·PO4 were mixed and incubated at room temperature. 50µl of NADPH was added and the absorbance of each sample was read every 60 sec at 412 nm for three minutes. The protein concentration for each sample was determined via a DC protein concentration assay (BIO RAD). Signals from each sample were normalized to the corresponding protein content of that sample.
2.11 Oxidative DNA damage as measured by 8-hydroxy-2’-deoxyguanosine (8-OHdG)
A commercially available enzyme linked immunoassay (#21026; Percipio Biosciences, Inc., Burlingame, CA) was used to measure oxidized DNA in isolated muscle DNA samples. DNA was extracted from the muscle via DNeasy Tissue Kit (Qiagen, Valencia, CA). DNA was used if it had a minimum 260:280 ratio of 1.8. The assay was performed with several modifications to the manufacturer’s directions. Briefly, 50 µl DNA was incubated with the primary antibody, washed, and then incubated in secondary antibody. The chromogen (3,3',5,5'-tetramethylbenzidine) was added to each well, and incubated at room temperature in the dark for 15 minutes. The reaction was terminated and the samples were read at an absorbance of 450 nm. Samples were normalized to the DNA concentration measured via a plate reader (ND-1000, NanoDrop, Wilmington, DE). All analyses were done in duplicate.
2.12 Lipid peroxidation
A commercially available kit (#21020, Percipio Biosciences, Inc., Burlingame, CA) was used to measure malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) in muscle homogenates. MDA+HAE was used as an indicator of lipid peroxidation in skeletal muscle samples. The assay was conducted with minor modifications from that recommended by the manufacturer. Briefly, ~100 mg of muscle was homogenized in ice-cold PBS, containing 5 µL 0.5 M butylated hydroxytoluene (BHT) in acetonitrile per 1 ml of tissue homogenate. The muscle homogenate was centrifuged at 3000g at 4°C and the supernatant was used for the assay and protein determination. The muscle sample was incubated in the appropriate reagents according to the manufacturer’s instructions, and centrifuged at 15,000g. An absorbance reading of the supernatant was obtained at 586nm. Samples were normalized for differences in the amount of muscle protein in each sample as determined by a DC protein concentration assay (Bio-Rad, Hercules, CA).
2.13 Catalase Activity
A commercially available catalase activity assay kit (#219265, EMD/Calbiochem, San Diego, CA), was used to measure catalase activity in muscle homogenates. The assay was conducted according to the manufacturer’s recommendations. All analyses were completed in duplicate and samples were read at absorbance of 520nm. The data were normalized to muscle protein in each sample via a DC protein concentration assay (Bio-Rad, Hercules, CA).
2.14 Manganese Superoxide Dismutase (MnSOD) and Copper-Zinc Superoxide Dismutase (CuZnSOD)
A commercially available kit (#574601, EMD/Calbiochem, San Diego, CA) was used to measure total and MnSOD activity in muscle homogenates. CuZnSOD was calculated by subtracting the value for MnSOD activity from the total SOD activity. The assay was performed with minor modifications to the manufacturer’s directions and all samples and standards were measured in duplicate. Briefly, the muscle was homogenized in 20mM HEPES buffer, pH 7.2, containing 1mM EGTA, 210mM mannitol, and 70 mM sucrose and centrifuged at 1000g for 10 minutes. The assay was performed in a 96-well plate with each sample being treated with and without 10µL of 12 mM potassium cyanide. Potassium cyanide was used to inhibit CuZnSOD, resulting in the detection of only MnSOD activity. The reagents and samples were protected from white light and incubated at 26°C for 20 minutes with periodic shaking. The absorbance was measured at 450 nm using a 96-well plate reader (Dynex Tech., Chantilly VA., USA).
2.15 Glutathione Peroxidase (GPx)
A commercially available cellular GPx assay (#35319, EMD/Calbiochem, San Diego, CA) was used to measure GPx activity in the cytosolic fractions of the muscle homogenates. The assay was preformed with several modifications to the manufacturer’s directions. Briefly, a portion of each muscle was homogenized in a buffer containing 50mM Tris-HCl, pH 7.5, 5 mM EDTA, 1mM DTT. The homogenate was centrifuged at 10,000g for 15 min at 4°C and the supernatant was used for the assay. All reagents and samples were equilibrated to 25°C and the remaining assay procedures followed manufacturer’s guidelines. The absorbance was measured at 340 nm using a 96-well plate reader (DYNEX Technologies, Chantilly Va., USA). Each sample and control was performed in duplicate.
2.16 Statistical analyses
Statistical analyses were performed using an SPSS 18.0 software package. Statistical significance of the data was calculated by a multiple analyses of variance (MANOVA). When significant F scores were indentified from the MANOVA, subsequent protected one-way analysis of variance followed by Tukey post-hoc tests were used to identify differences between means. Statistical significance was accepted at p≤0.05. Data are reported as mean ± standard error mean (SEM).