Ishemia-Reperfusion enhances GAPDH nitration in aging skeletal muscle

The effect of I/R on GAPDH pool levels in plantaris/FHL from young and aged mice

Glycolytic muscle mass decreases following I/R injury, particularly in muscle of old rats, suggesting a decrease in protein content [2]. Furthermore, the decrease in force production after I/R injury is greater for old muscle suggesting that decreased force production is related to decreased expression of glycolytic and contractile proteins [2]. Based on these observations we determined the protein pool levels of GAPDH for young and old mouse glycolytic skeletal muscle subjected to I/R injury (Figures 1, 2). Prior to normalization of the Western blot data, the comparison of GAPDH protein levels between young control and ischemic muscle showed no statistically significant difference after 2 hours ischemia with no reperfusion (Figure 1A, 1B Group I), after 1 day reperfusion (Group II), or after 7 days reperfusion (Figure 1A, 1B Group V). At the 3 and 5 day time points (Groups II and IV) the GAPDH protein levels were significantly decreased in the I/R tissue, with an average decrease of 89.4% (p = .0004) at 3 days and 71.4% (p = 0.007) at 5 days (Figure 1B).

Figure 1. GAPDH pool levels in plantaris/FHL lysate from young mice. Protein (30 μg) from total muscle cell lysates of plantaris/FHL from young mice (6-7 months) subjected to two hours of ischemia were separated by SDS-PAGE, transferred to PVDF membrane, and blotted with anti-GAPDH antibody. (A) Group I: No reperfusion, showed no significant difference. Group II: One day reperfusion shows that GAPDH levels decreased, but not significantly. Groups III and IV: Three and five days reperfusion, respectively, showed a significant reduction in GAPDH immunoreactive pool levels (p = 0.0004 and p = 0.007, respectively). Group V: Seven days reperfusion showed no significant difference between control and I/R muscle samples. (B) A bar graph showing the statistical differences in GAPDH pool levels in response to 2 hours of ischemia and 1-7 days of reperfusion.

Similar analyses of the GAPDH protein levels in old muscle showed a significant decrease after reperfusion at days 1 (Group I; 61.9%, p = 0.0005), 3 (Group II; 83.1%, p = 0.014), 5 (Group III; 83.3%, p = 0.007), and 7 (Group IV; 73.8%, p = 0.013) of reperfusion (Figure 2A, 2B). The no reperfusion time point was not available for the old animals.

Figure 2. GAPDH pool levels in plantaris/FHL lysate from old mice. Protein (30 μg) from total muscle cell lysates of plantaris/FHL from old mice (24-27 months) subjected to two hours of ischemia were separated by SDS-PAGE, transferred to PVDF membrane, and blotted with anti-GAPDH antibody. (A) Groups I, one day; II, 3 days, III, 5 days and IV, 7 days of reperfusion all showed statistically significant decreases in GAPDH immunoreactive pool levels. (B) A bar graph showing the statistical differences in GAPDH pool levels in response to 2 hours of ischemia and 1-7 days of reperfusion.

Although statistically significant differences in GAPDH pool level were seen at several time points in both age groups, a confounding variable in the data was the question of protein degradation in the ischemic muscle as related to the I/R injury. An examination of the post-transfer gels after Coomassie staining revealed an increase in low molecular weight bands (< 16 kD) in the ischemic samples after reperfusion. Further, comparison of the total protein staining of the one-dimensional gel electrophoresis revealed that the average decrease of GAPDH over all days of reperfusion in the young was 16.2% (p = 0.003 compared to young control).

In the old muscle, there was a 16.6% decrease compared to old controls (p < 0.0001) as indicated by staining the I/R samples, after equalizing protein input prior to loading of the gels. Significantly, the samples from the young animals which had received 2 hours of ischemia but no reperfusion showed no statistically significant differences in protein loading between control and ischemic muscle. In order to correct for differences in the amount of protein actually present in the gel, a normalization factor based on the Coomassie staining of the post-transfer gels was used (as described in Methods).

After normalizing for background levels of protein degradation, the pool level of GAPDH in glycolytic skeletal muscle from young mice was still significantly decreased at 3 and 5 days of reperfusion (p = 0.001 and 0.005 respectively). There were no significant alterations at 2 hours of ischemia without reperfusion at day 1 or at day 7 of reperfusion. After normalization for protein degradation the GAPDH protein pool levels in skeletal muscle from the old mice were still significantly decreased at 1, 3, 5, and 7 days reperfusion (p = 0.001, 0.015, 0.011, and 0.007, respectively).

Total RNA from control and ischemic plantaris following 5 days of reperfusion

We examined the possibility that altered GAPDH transcription may play a role in the decrease in GAPDH pool levels. Total RNA from plantaris muscle was isolated from young and old mice subjected to 2 hours of ischemia and 5 days of reperfusion. An equal percentage of total RNA from each sample was analyzed using a chip-based gel electrophoresis assay to determine the quality of isolated RNA. Surprisingly, the yield of total RNA from the ischemic samples was substantially higher compared to the contralateral control, regardless of age (Figure 3A and B). The data indicate that the bulk of the increase in total RNA was from rRNA as measured by18s and 28s ribosomal subunits (Figure 3A and B).

Figure 3. Analysis of total RNA from control and ischemic plantaris muscle following 5 days of reper-fusion. An equal percentage of total RNA from (A) six separate samples of young (6-7 months) and (B) 3 separate samples of old (24-27 months) plantaris muscle was analyzed by capillary gel electrophoresis. The initial peak is a marker (M) included for calibration. Control and I/R samples are the contralateral and ischemic muscle from the same animal. Samples are paired from left to right, beginning with control. C = control; T = I/R; small ribosomal subunit (18s) and large ribosomal subunit (28s).

PCR analysis of GAPDH transcript levels from control and I/R plantaris following 5 days of reperfusion

To determine whether mRNA levels are affected, GAPDH-specific primers were used to perform PCR on an equal percent of the total cDNA (Figure 4). Significant differences were detected in the number of cycles to threshold (Ct) between young ischemic and young controls, with an average difference of 6.2 cycles (p = 0.0009).

Figure 4. PCR analysis of GAPDH transcript levels from control and I/R plantaris muscle following 5 days of reperfusion. PCR with GAPDH specific probes was performed using an equal percentage of total cDNA (reverse transcribed from mRNA using igo(d)T primers) isolated from each sample of (A) young (6-7 months) and (B) old (24-27 months) plantaris muscle. Control = black; I/R = gray; T = Threshold (arrow shows the threshold).

The difference between the old controls and old ischemics was also significant (p = 0.049), with an average difference of 5.4 cycles (Figure 4B). These data clearly show that there is a decrease in the GAPDH transcript levels in the I/R muscles. The difference in GAPDH transcript levels in response to I/R between the young and the old was not statistically significant. However, the data (threshold levels) suggest that the average level of GAPDH mRNA in the young muscle is approximately twice the level in the old (Figure 4).

Detection of nitrotyrosine modified proteins

Previous studies have shown functional impairment of glycolytic skeletal muscle following I/R injury [2]. Oxidative modification of proteins critical for muscle function is a possible mechanism explaining the decline in muscle force production. As levels of reactants necessary to form nitrotyrosine are known to increase during the reperfusion stage of I/R injury, we tested the hypothesis that protein tyrosine nitration would increase following reperfusion. Relative levels of protein tyrosine nitration were assayed in skeletal muscle extract from young (Figure 5) and old (Figure 6) mice following I/R by one-dimensional gel electrophoresis and Western blotting with antibody specific for nitrotyrosine. The corresponding contralateral muscles were used as a control for the basal level of nitration (Figures 5, 6). Following normalization for back-ground protein degradation, there were no statistically significant differences in total nitrotyrosine for the young, although nitration was increased at every time point following reperfusion (Figure 5; range 22% to 67% increase).

Figure 5. Western blot analysis of total tyrosine nitration of plantaris/FHL lysate from young mice following I/R. (A) Tyrosine nitration of whole cell lysate of glycolytic skeletal muscle from young (6-7 months) mice with 2 hours ischemia and variable lengths of reperfusion was examined by Western blot. C = contralateral control; Is = I/R; Group I = no reperfusion; Group II = 1 day reperfusion; Group III = 3 days reperfusion; Group IV = 5 days reperfusion, and Group V = 7 days reperfusion. (B) Densitometric analysis shows no statistically significant differences in levels of overall nitration.

Figure 6. Western blot analysis of total tyrosine nitration of plantaris/FHL lysate from old mice following I/R. (A) Tyrosine nitration of whole cell lysate of glycolytic skeletal muscle from old mice (24-27 months) with 2 hours ischemia and variable lengths of reperfusion was examined by Western blot. C = contralateral control; Is = I/R; Group I = 1 day reperfusion; Group II = 3 days reperfusion; Group III = 5 days reperfusion, and Group IV = 7 days reperfusion. (B) Densitometric analysis shows statistically significant differences were observed at 1, 3, and 5 days of reperfusion.

In contrast to the young, an analysis of total tyrosine nitration in whole cell lysates of old skeletal muscle following I/R revealed statistically significant increases at the 1, 3, and 5 day time points (Figure 6; p = 0.008, 0.048 and 0.03, respectively). Total nitration was increased at all time points, but the increase was not significant at the 7 day time point (Figure 6).

While evaluating overall levels of an oxidative modification is useful as an indication of oxidative stress in a tissue, knowing which specific proteins are targeted by posttranslational modifications can further provide an understanding of the consequences of the modifications. Therefore, experiments were undertaken to identify those proteins which were targets of tyrosine nitration in I/R injury. Two-dimensional gel electrophoresis was used to resolve lysate proteins from young (6 months) and old (24-27 months) muscles previously assayed by one dimensional gel electrophoresis and western blot (Figure 7). The five day reperfusion time points were selected for analysis. Anti-nitrotyrosine western blotting was used to select spots from a second gel of the same sample (focused concurrently with the gel used for Western blotting) for identification via MALDI-TOF mass spectrometry. A list of identified proteins is included in Table 1. Protein scores greater than 53 were considered to be significant. Of the proteins identified by mass spectrometry in the old muscle following I/R, approximately 30% were also identified in the young following I/R. It was noted that several enzymes in the glycolytic pathway were identified as nitrated, based on Western blot selection for MALDI-TOF. Three of these enzymes, GAPDH, triose phosphate isomerase, and fructose bis-phosphate aldolase A, were identified from both young and old. Since glycolysis is necessary to maintain adequate ATP levels during I/R injury, any effect on the activities of these enzymes might have a significant effect on ATP levels and consequently on energy availability.

Figure 7. Nitrotyrosine western blot analysis of 2D gel electrophoresis of young and old plantaris/FHL lysate following 5 days of reperfusion. Plantaris/FHL lysates (180 μg) from (A) young (6-7 months) or (B) old (24-27 months) I/R tissues were separated by 2D gel electrophoresis. Nitrotyrosine immunoblotting was used to select nitrotyrosine modified proteins from a duplicate gel for mass spectrometry and identification.

Identification of nitrated GAPDH in young and old muscle

After identifying the nitrated proteins from I/R samples, we compared the 37 kD band corresponding to the molecular weight of GAPDH across the time course. We focused on GAPDH due to its importance in glycolysis, reports of altered GAPDH function with posttranslational modification i.e., nitrosylation [23], and a recent report that overexpression of GAPDH can rescue cells in culture from caspase-independent cell death [27]. These reports suggest that GAPDH has multiple functions and may integrate different cellular activities with the metabolic state of the cell [28].

After normalizing the nitration of the 37 kD band to GAPDH protein pool levels as measured by Western blotting, it was found that the nitration of GAPDH in the young was significantly increased at the one and three day time points (Figure 8; p = 0.001 and 0.043 respectively). The highest level of nitration in the samples from young mice occurs at the 3 day time point.

Figure 8. Tyrosine nitration of GAPDH in plantaris/FHL lysate from young mice. (A) Tyrosine nitration was assessed by Western blot of one dimensional gel electrophoresis. (B) Densitometric analysis shows nitration was significantly increased for GAPDH at 1 and 3 days of reperfusion, with the greatest increase at 3 days reperfusion. C = contralateral control; Is = I/R; Group I = no reperfusion; II = 1 day reperfusion; Group III = 3 days reperfusion; Group IV = 5 days reperfusion, and Group V = 7 days reperfusion. * = p < 0.05.

After normalization to GAPDH pool level, the nitration of the 37 kD band in the samples from old mice was significantly increased at 1, 3, and 5 days reperfusion (Figure 9; p = 0.019, 0.048, and 0.011, respectively). In contrast to the young, the highest levels of nitration measured were at the 5 and 7 day time points. However, the increase in nitration was not statistically significant at 7 days due to the variability within the I/R group (Figure 9).

Figure 9. Tyrosine nitration of GAPDH in plantaris/FHL lysate from old mice. (A) Tyrosine nitration was assessed by Western blot of one dimensional gel electrophoresis. (B) Densitometric analysis shows nitration was significantly increased for GAPDH at 1, 3, and 5 days of reperfusion. C = contralateral control Is = I/R; Group I = 1 day reperfusion; Group II = 3 days reperfusion; Group III = 5 days reperfusion, and Group IV = 7 days reperfusion. * = p < 0.05.

Comparing the percent increase in nitration (control to I/R) of the 37 kD band, a significant difference (p = 0.02) was noted between the young and old skeletal muscle at the 5 day time point corresponding to the peak of GAPDH nitration in the old. The level of nitration in the young had decreased by the 5^th day with the peak of the increase in nitration appearing at the 3^rd day of reperfusion. In summary, the greatest difference between control and I/R in the young occurs at 3 days of reperfusion, while the greatest difference in the old occurs at 5 days of reperfusion.

Animals and tissues

Young (6-7 months) and old (24-27 months) C57Bl/6 mice were used in these experiments. Prior to their use, the mice were acclimatized to their new surroundings for one week. Upon completion of the experiments, animals were euthanized using sodium pentobarbital. For one-dimensional gel electrophoresis and Western blots, 3 animals were used per time point, with the exception of the young at 1 day, which had 2 animals. Plantaris and flexor hallicus longus (FHL) muscles were collected for the one dimensional gels. Time points examined for young and old mice were 2 hours of ischemia followed by either 1, 3, 5, or 7 days of reperfusion. For the young mice, an additional time point with 2 hours of ischemia and no reperfusion was also examined. For two dimensional gel electrophoresis, a representative sample from the 5 day time point was selected from the young and the old samples. For RNA extraction and PCR analysis, plantaris muscles from 6 young and 3 old animals were collected after 2 hours of ischemia followed by 5 days of reperfusion. All experimental procedures were approved by and conducted in accordance with guidelines set by the University of Texas at Austin IACUC.

Ischemia/Reperfusion protocol

Tourniquet-induced I/R was performed as previously described [2]. Mice were anesthetized using 2% isofluorane prior to and for the duration of the tourniquet application. A heat lamp was used to maintain the animal's body temperature at 32-35 ± 1°C during the tourniquet procedure. After 2 hours, the tourniquet was removed, and the animal returned to its cage to recover. After the appropriate length of reperfusion, the muscles were taken, snap frozen in liquid nitrogen, and the samples were stored at −80°C until analysis.

Protein extraction

Flexor halicus longus and plantaris muscles were homogenized together in 2D lysis buffer (8 M urea, 4% CHAPS, 40 mM DTT, protease inhibitors) using the Ettan Sample grinding kit (Amersham BioSciences), according to manufacturer's instructions. Briefly, the samples were homogenized in the sample grinding tube with abrasive resin by using the provided plastic pestles. The samples were centrifuged for 5 minutes at 10,000g to pellet the resin and debris. Supernatant was removed to a fresh tube. Additional 2D lysis buffer was added and the previous steps repeated to maximize yield. In some experiments supernatants were combined.

Protein assays

Bradford protein assays (BioRad) of muscle homogenates were performed in triplicate using bovine serum albumin as a protein standard. The Bradford assays were performed in triplicate after the samples were homogenized, and the average value used. The samples to an equal concentration. The samples were then diluted, aliquoted and frozen at −80°C. until further use.

One dimensional gel electrophoresis

Muscle samples (30 μg) were mixed with 5X Laemli loading buffer (10% SDS, 500 mM DTT, 300 mM Tris, 50% glycerol, 0.05% bromophenol blue, pH 6.8) and brought up to 25 μL with 2D lysis buffer. Samples were separated on a 4-20% tris-glycine gradient gel (Bio-Rad) at 135 volts for 2 hours in SDS running buffer (25 mM Tris base, 0.1% SDS (w/v), 192 mM glycine). Immobilon PVDF membranes (Millipore) were prepared by wetting the membrane for 15 seconds in methanol, equilibrating the membrane in distilled water for 2 minutes, and then equilibrating the membrane in transfer buffer (25 mM Tris base, 10% methanol (v/v), 192 mM glycine) for 5 minutes. Gel transfers were performed in a Hoeffer wire transfer tank at 50 volts for 2 hours at 4°C.

Two dimensional gel electrophoresis

Muscle samples (180 μg) isolated in 2D lysis buffer were mixed with Destreak reagent (GE -BioSciences) plus 0.75% IPG 3-10 pH IPG buffer. Samples were made up to 200 μl with 2D lysis buffer. The sample was pipetted into an 11 cm Ettan IPGphor strip holder (Amersham Biosciences) and overlaid with an 11 cm Immobiline Drystrip 4-7 pH gel strip (GE Biosciences) with the backing strip of the gel toward the top. Immobiline DryStrip cover fluid (Amersham BioSciences) was pipetted over the strip and sample to prevent evaporation, the cover placed on the holder, and the assembly rehydrated on an Ettan IPGphor isoelectric focusing system (Amersham Biosciences). Conditions used were as follows: 12 hours in-gel rehydration at 20°C with a constant current of 50 mA/strip followed by 3 focusing steps – 500V for 50 Vh, 1000V for 1000Vh and 8000V for 16000Vh.

Strips were equilibrated for 15 minutes in SDS equilibration buffer (6 M urea, 29.3% glycerol (v/v), 2% SDS (w/v), 0.002% bromophenol blue (w/v), and 75 mM Tris-HCl, pH 8.8. Prior to equilibration, DTT (1% w/v) was added to the SDS equilibration buffer. The gel strip was then placed in the sample lane of a 10-20% Tris-HCl IPG +1 well, 1 mm Criterion gel (BioRad) and sealed with agarose sealing solution (25 mM tris, 192 mM glycine, 0.1% SDS (w/v), 0.5% agarose, and 0.002% (w/v) bromophenol blue. After the agarose solidified the gels were placed in a Criterion running tank (BioRad), filled with running buffer (25 mM Tris base, 0.1% SDS (w/v), and 192 mM glycine). The sample was separated at 135 volts for approximately 2 hours. The gel was transferred using a Criterion blotter system (BioRad) with conditions as described for transfer of 1D electrophoresis gels.

Western blotting

Membranes were blocked for one hour with 5% non-fat dried milk TBST (50.0 mM tris, 200 mM NaCl, 0.05% Tween-20, pH 7.4) and probed overnight with anti-nitrotyrosine antibody 1:2000 5% milk, TBS-T. Membranes were washed 3X for 5 minutes in TBS-T and probed with anti-mouse secondary antibody at RT for 1 hour. Prior to blotting membranes were washed 2X 5 minutes and 1X 15 minutes in TBS-T. The membrane was then incubated with Immobilon blot substrate (Millipore) for 5 minutes and exposed to film (Biomax MR, Kodak). Bands were assessed by densitometry (FluorChem 8900, AlphaInnotech). Membranes were stripped (Restore Western Blot Stripping Buffer, Pierce), reprobed with anti-mouse antibody to insure original primary anti-nitrotyrosine removal and then probed with GAPDH antibody (clone CA6, Santa Cruz) 1:5000 in 5% milk, TBST for 1 hour.

Protein normalization

Total protein per lane was assessed by densitometry (FluorChem 8900, AlphaInnotech) with automatic background subtraction and a value of 100 was assigned to the lane with the highest density. The remaining lanes were expressed as a decimal fraction of the 100 lane. The reciprocal of the decimal was multiplied by the values generated from the Western data. For nitration of the 37 kD band, a ratio computed from the GAPDH western blot was used to normalize the amount of GAPDH present rather than total protein. A correction factor was also used between gels of the same age group by rerunning samples in separate gels and taking a ratio of the 37 kD bands in the first and second gels. The average of the ratios was used as the correction factor. For nitrotyrosine blots the ratio was based on total nitration of repeated samples.

Sample preparation for mass spectrometry

Preparation of samples for mass spectrometry were performed by the Mass Spectrometry Core Facility at University of Texas Medical Branch, Galveston. Gel samples were cut into 1 mm size pieces or smaller and placed into separate 0.5 mL polypropylene tubes. Ammonium bicarbonate buffer (50 mM; 100 μl) was added to each tube and the samples were then incubated at 37°C for 30 minutes. After incubation the buffer was removed and 100 μl of water was added to each tube. The samples were then incubated again at 37°C for 30 minutes. After incubation the water was removed and 100 μl of acetonitrile was added to each tube to dehydrate the gel pieces. The samples were vortexed and after 5 minutes the acetonitrile was removed. Acetonitrile (100 μl) was again added to each of the sample tubes, vortexed and removed after 5 minutes. The samples were then placed in a speedvac for 45 minutes to remove excess solvent. Lyophilized trypsin (20 μg; Promega Corp.) was dissolved by adding 2 mL of 25 mM ammonium bicarbonate (pH 8.0). The trypsin solution was then vortexed and added to each sample tube in a minimal amount (~10 μL), i.e., just enough to cover the dried gel. The samples were then incubated at 37°C for 6 hrs. After digestion the samples were removed from the 37° C oven and 1 μL of sample solution was spotted directly onto a MALDI target plate and allowed to dry. Alpha-cyano-4-hydroxycinnamic acid (1 μL; Aldrich Chemical Co.) matrix solution (50:50 acetonitrile/water at 5 mg/mL) was then applied on the sample spot and allowed to dry. The dried MALDI spot was further dried with compressed air (Decon Laboratories, Inc.) before inserting into the mass spectrometer.

Mass spectrometry

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI TOF-MS) was used to analyze the samples and determine protein identity. Data were acquired with an Applied Biosystems 4800 MALDI TOF/TOF Proteomics Analyzer. Applied Biosystems software package included 4000 Series Explorer (v. 3.6 RC1) with Oracle Database Schema Version (v. 3.19.0) and Data Version (3.80.0). The software was used to acquire both MS and MS/MS spectral data. The instrument was operated in positive ion reflectron mode with a mass range of 850 – 3000 Da, and the focus mass was set at 1700 Da. For MS data, 1000-2000 laser shots were acquired and averaged from each sample spot. Automatic external calibration was performed using a peptide mixture with reference masses 904.468, 1296.685, 1570.677, and 2465.199.

Following MALDI-MS analysis, MALDI-MS/MS was performed on several (5-10) abundant ions from each sample spot. A 1kV positive ion MS/MS method was used to acquire data under post-source decay (PSD) conditions. The instrument precursor selection window was ±3 Da. For MS/MS data, 2000 laser shots were acquired and averaged from each sample spot. Automatic external calibration was performed using reference fragment masses 175.120, 480.257, 684.347, 1056.475, and 1441.635 (from precursor mass 1570.700).

Applied Biosystems GPS Explorer^TM (v. 3.6) software was used in conjunction with MASCOT to search the respective protein database using both MS and MS/MS spectral data for protein identification. Protein match probabilities were determined using expectation values and/or MASCOT protein scores. MS peak filtering included the following parameters: mass range 800 Da to 4000 Da, minimum S/N filter = 10, mass exclusion list tolerance = 0.5 Da, and mass exclusion list (for some trypsin and keratin-containing compounds) included masses 842.51, 870.45, 1045.56, 1179.60, 1277.71, 1475.79, and 2211.1. For MS/MS peak filtering, the minimum S/N filter = 10.

For protein identification, the murine taxonomy was searched in the SwissProtein database. Other parameters included the following: selecting the enzyme as trypsin; maximum missed cleavages = 1; fixed modifications included carbamidomethyl (C) for 2-D gel analyses only; variable modifications included oxidation (M); precursor tolerance was set at 0.2 Da; MS/MS fragment tolerance was set at 0.3 Da; mass = monoisotopic; and peptide charges were only considered as +1. The significance of a protein match based on both the peptide mass fingerprint (PMF) in the first MS and the MS/MS data from several precursor ions, is based on expectation values; each protein match is accompanied by an expectation value. The expectation value is the number of matches with equal or better scores that are expected to occur by chance alone. The default significance threshold is p < 0.05, so an expectation value of 0.05 is considered to be on this threshold. We used a more stringent threshold of 10⁻³ for protein identification (a lower expectation value represents a more significant score).

RNA extraction

Total RNA extraction was accomplished using the RNAqueous-4PCR kit (Applied Biosystems, U.S.) according to manufacturer's instructions. Plantaris muscle was collected from young (n = 6) and old (n = 3) mice subjected to 2 hours of ischemia and 5 days of reperfusion. The muscle samples were snap-frozen in liquid nitrogen and stored at −80° C until use. The plantaris was cut in half on dry ice. One half of the muscle was transferred to a microfuge tube and homogenized by hand in the lysis/binding solution supplied with the kit using a plastic pestle. An equal volume of 64% ethanol was added to the solution and mixed. This mixture was pipetted into a tube containing a filter cartridge supplied with the kit. The cartridge and solution were centrifuged at 15,000g for 1 minute. Wash solution #1 was added to the retentate, and the tube was centrifuged as in the previous step. The previous wash and centrifugation was repeated twice using wash solution #2/3. The filter cartridge was placed into a new tube, and elution buffer pre-warmed to 70-80°C was added. The cartridge and tube were centrifuged at 15,000g for 1 minute. Another volume of heated elution buffer was added, and the cartridge was centrifuged again. Finally, the cartridge was removed and the total filtrate was centrifuged again. The supernatant was removed to a fresh tube, leaving any glass fibers from the cartridge in the pellet. Residual amounts of DNA were removed by treating the samples with DNase 1 provided in the RNAqueous kit. The samples were incubated at 37°C for 20 minutes, and DNase inactivation reagent was added. The samples were then gently vortexed, incubated with the inactivation reagent for 2 minutes and then centrifuged at 10,000g for 1 minute. The supernatant was then transferred to fresh tubes.

To ascertain the quality of the extracted mRNA, the rRNA 6000 Nano Assay kit (Agilent Technologies, Germany) was used according to manufacturer's instructions, in conjunction with the Agilent 2100 bioanalyzer. This chip-based capillary gel electrophoresis unit utilizes a proprietary dye-based reagent that binds to nucleic acids for detection and quantitation of rRNA.

cDNA synthesis

Synthesis of cDNA was done using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. For each sample, an equal volume (8 μl) of DNAse-treated total RNA, 1 μl of 10 mM dNTP mix, and 1 μl of 0.5 μg/μl oligo(dT)_12-18 was added to one well of a PCR reaction plate. A 2X reaction mixture was prepared which contained the following: 2 μl of 10X reaction buffer, 4 μl of 25 mM MgCl₂, 2 μl of 0.1M DTT, and 1 μl RNaseOUT (provided in the kit). The RNA/primer mix was incubated at 65°C for 5 minutes using an Applied Biosystems GeneAmp 9700. The mixture was then placed on ice for 1 minute. Nine μl of the 2X reaction mixture was added to each reaction and mixed by pipetting. The reaction was incubated at 42°C for 2 minutes in the Applied Biosystems GeneAmp 9700 prior to the addition of 1μl of SuperScript II reverse transcriptase to each reaction (with the exception that no reverse transcriptase was added to the negative control). The reaction plate was incubated at 42°C for 50 minutes in the GeneAmp 9700. Subsequently, the temperature was increased to 70°C for 15 minutes to terminate the reaction. The reaction plate was chilled on ice for 5 minutes. RNase H (1μl) was added to each well and the plate was incubated at 37 °C for 20 minutes.

PCR

PCR was performed using a Taq polymerase-based fluorogenic assay and primers specific for GAPDH mRNA (Primetime qPCR, Integrated DNA Technologies). Primer sequences used were: 5'-AATCTCCACTTTGCCACTG-3' and 5'CCTCGTCCC GTAGA CAAAA-3'. Reactions were performed using the iCycler (Biorad) as follows: One period of 1.5 minutes at 95°C, followed by 50 cycles of 0.5 minutes at 95°C, 0.5 minutes at 55°C, and 0.5 minutes at 70°C.

Statistical analysis

The developed Western blot films were photographed using the FluorChem 8900 system (AlphaInnotech). Using the FluorChem 8900 spot densitometry analysis software, a rectangular area was selected which encompassed the region of interest. Selections for each lane were of equal area. The imaging software's automatic background subtraction was used for examining the density of immunostaining for each band. After normalization, statistical analysis was performed as previously described [49, 50]. Welch's modified version of the paired Student's t-test was used to compare means between control and ischemic muscle of each time point, or between the difference in average response between young and old (within each time point). Welch's modified version of the student t-test does not require that the sample variances be equal. Graphs represent the average of individual values (for example, the average GAPDH immunoreactivity of young controls versus the average of young I/R samples, at one day reperfusion). Error bars represent the standard deviation.