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 × Brown Norway rats after 14 days of hindlimb suspension. Medial gastrocnemius muscle weight was decreased by ∼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. H2O2 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, H2O2, 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.
- muscle disuse
- reactive oxygen species
generation of free radicals and reactive oxygen and nitrogen species (ROS/RNS) is a normal continuous process in the life of aerobic living organisms. When production of ROS/RNS exceeds the endogenous antioxidant buffering capacity, oxidative stress is provoked. There have been extensive data indicating that oxidative stress plays a central role in regulating a variety of cellular events (e.g., cell division, adhesion, differentiation, and death) (6, 16, 37, 47, 55). Moreover, substantial investigations have shown that the level of oxidative stress is elevated with diseases as well as aging, and accordingly the pathogeneses of cardiovascular diseases, diabetes mellitus, cancer, neurodegenerative diseases, and other chronic diseases are thought to be attributed to the deleterious effects of the increased oxidative damage (7, 11, 12, 18, 64). Aging is associated with an accretion of oxidative stress and increased incidence of oxidative injury in respiratory and locomotive skeletal muscles (4, 5, 9, 20, 26, 44, 45). Consequently, elevated oxidative stress has been thought to have a role in the development of sarcopenia, which comprises considerable age-related decline in muscle mass (10, 19).
Free radicals together with ROS/RNS are generated in both contracting and noncontracting skeletal muscles, although the magnitude and mechanisms of the oxidative stress generation may not be the same in muscles under contracting and inactive states (38, 42, 48, 49, 51, 52). In particular, it has been proposed that oxidative stress may have an important role in mediating the process of muscle atrophy during disuse (48, 49). In support of this hypothesis, several studies have demonstrated the incidence of oxidative injury in skeletal muscle under atrophic situations during limb immobilization or hindlimb suspension (27–32, 34). Although it has been demonstrated that unloading increases oxidative stress and disrupts antioxidant capacity in young adult skeletal muscle (34), the hypothesis that oxidative stress is also elevated in aged muscle during unloading has not been investigated. Moreover, it is unknown if aging may exacerbate the oxidative challenge in skeletal muscle when exposed to disuse (10, 19). Thus, in this study we examined the markers of oxidative stress (MDA/4-HAE, H2O2, and nitrotyrosine) and antioxidant enzyme [Cu-Zn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx)] gene and protein expressions or activities in medial gastrocnemius muscles of young adult and aged rats after 14 days of hindlimb suspension. We tested the hypothesis that oxidative stress is increased in both young adult and aged muscles in response to hindlimb unloading. Furthermore, we examined whether the hypothesized increase in oxidative stress markers with unloading would be exacerbated by aging.
Experiments were conducted on 6-mo-old young adult and 30-mo-old aged Fischer 344 × Brown Norway (FBN) rats (Harlan, Indianapolis, IN). It is noted that rats become sexually mature at ∼1.5 mo of age and reach maturity several months later at approximately 5–6 mo of age (1). The age of 28–30 mo to 36 mo of this strain of rat represents the late middle to senescent age stage based on their survival rate of 75% to 35% compared with the survival curves for humans (65). The rats were housed in pathogen-free conditions at ∼20°C and were exposed to a reverse light condition of 12:12 h of light:darkness each day. They were fed rat chow and water ad libitum throughout the study period.
Hindlimb suspension procedure.
The young adult and aged animals were randomly assigned to suspension group or control group, in which 10 animals per group were included in young control, young suspension, and aged suspension groups, whereas 8 animals were included in the aged control group. The procedure of hindlimb suspension described by Morey-Holton and Globus (41) was adopted in the present study. In brief, an adhesive (tincture of benzoin) was applied to the tail, air-dried, and an orthopedic tape was put along the proximal one-third of the tail. This practice distributed the load evenly and avoided excessive tension on a small area to the tail. The tape was then placed through a wire harness that was attached to a fish line swivel at the top of a specially designed NASA-approved hindlimb suspension cage. This provided the rats with 360° of movement around the cage and the forelimbs maintained contact with a grid floor, which allowed the animals to move and access food and water freely. Sterile gauze was wrapped around the orthopedic tape and was subsequently covered with a thermoplastic material, which formed a hardened cast (Vet-Lite, Veterinary Specialty Products, Boca Raton, FL). The distal tip of the tail was examined to verify that the procedure did not occlude the blood flow to the tail (i.e., tail remained pink). The suspension height was adjusted to prevent the animal's hindlimb from touching any supportive surface, with care taken to maintain a suspension angle of ∼30° (23). The suspension height and animal behavior were monitored daily. Control animals were allowed to move unconstrained around the cages. Following 14 days of suspension, animals were euthanized with an overdose of xylazine-ketamine. Medial gastrocnemius muscles from the hindlimbs were excised, weighed, and frozen in isopentane cooled to the temperature of liquid nitrogen and stored at −80°C until used for analyses.
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 following the policies and procedures detailed in the Guide for the Care and Use of Laboratory Animals as published by the U.S. Department of Health and Human Services and proclaimed in the Animals Welfare Act (PL89-544, PL91-979, and PL94-279).
RT-PCR analyses of CuZnSOD, MnSOD, and catalase.
Total RNA was extracted from the medial gastrocnemius muscle of both suspended and control animals with TriReagent (Molecular Research Center, Cincinnati, OH), which is based on the guanidine thiocyanate method. Frozen muscle was mechanically homogenized on ice in 1 ml of ice-cold TriReagent. Total RNA were solubilized in RNase-free H2O and quantified in duplicate by measuring the optical density (OD) at 260 nm. Purity of RNA was assured by examining OD260/OD280 ratio. RNA was reverse transcribed with decamer primers and Superscript II reverse transcriptase (RT) according to standard methods (Invitrogen Life Technologies, Bethesda, MD). Control RT reaction was done in which the RT enzyme was omitted. The control RT reaction was PCR amplified to ensure that DNA did not contaminate the RNA. One microliter of complementary DNA (cDNA) was then amplified by PCR using 100 ng of forward and reverse primers, ribosomal 18S primer pairs (Ambion, TX), 250 μM deoxyribonucleotide triphosphates (dNTPs), 1× PCR buffer, and 2.5 units Taq DNA polymerase (USB, Cleveland, OH) in a final volume of 50 μl. PCR was performed using a programmed thermocycler (Biometra, Göttingen, Germany). The primer pairs were designed from sequences published in GenBank (Table 1), and PCR products were verified by restriction digestions. Preliminary experiments were conducted with each gene to ensure that the number of cycles represented a linear portion for the PCR optical density curve for the muscle samples. The cDNA from all muscle samples was amplified simultaneously using aliquots from the same PCR mixture. After the PCR amplification, 30 μl of each reaction was electrophoresed on 1.5% agarose gels, stained with ethidium bromide. Images were captured and the signals were quantified in arbitrary units as OD × band area using Kodak image analysis system (Eastman Kodak, Rochester, NY). The size (number of base pairs) of each of the bands corresponded to the size of the processed mRNA. Ribosomal 18S primers were used as internal controls while all RT-PCR signals were normalized to the 18S signal of the corresponding RT product to eliminate the measurement error from uneven sample loading and provide a semiquantitative measure of the relative changes in gene expression.
The fractionation method described by Rothermel et al. (53) was used with minor modification to extract the cytosolic protein fraction from the medial gastrocnemius muscle. We have previously obtained the total cytosolic proteins including mitochondrial contents and nuclear proteins from skeletal and heart muscles using this modified protocol (56–59, 61). In brief, after removal of connective tissues, muscle was homogenized on ice in lysis buffer [10 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, pH 7.4, 20% glycerol, 0.1% Triton X-100, 1 mM dithiothreitol (DTT)] supplemented with a protease inhibitor cocktail containing 104 mM AEBSF, 0.08 mM aprotinin, 2 mM leupeptin, 4 mM bestatin, 1.5 mM pepstatin A, and 1.4 mM E-64 (Sigma-Aldrich, St. Louis, MO). Following centrifuging at 5,000 rpm for 5 min at 4°C to pellet the nuclei and cell debris, the supernatants were collected, and these supernatants were further centrifuged three times at 6,000 rpm for 5 min at 4°C to remove residual nuclei. The final collected supernatants were stored as total cytosolic protein fraction. The above described protein fractionation procedure has been routinely used in our laboratory to obtain high-purity cytosolic fraction protein as assessed by immunoblotting the fractions with an anti-histone H2B (a nuclear protein), an anti-CuZnSOD (a cytosolic isoform of SOD), and an anti-MnSOD (a mitochondrial isoform of SOD) antibody (56–58, 61). The protein content of the extract was then measured in duplicate by DC Protein Assay (Bio-Rad, Hercules, CA) based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent, which was similar to Lowry assay (36). As a further measure to ensure the protein content, all the protein samples were quantified in duplicate on a different occasion by BCA protein assay (Pierce, Rockford, IL) based on the biuret reaction and the bicinchoninic acid detection of cuprous cation (62).
Western immunoblot analyses of CuZnSOD and MnSOD.
Forty micrograms of cytosolic protein was boiled for 5 min at 95°C in Laemmli buffer and was loaded on each lane of a 12% polyacrylamide gel and separated by SDS-PAGE. The gels were blotted to nitrocellulose membranes (VWR, West Chester, PA) and stained with Ponceau S red (Sigma Chemical, St. Louis, MO) to verify equal loading and transferring of proteins to the membrane in each lane. As another approach to validate similar loading between the lanes, gels were loaded in duplicate with one gel stained with Coomassie blue. The membranes were blocked in 5% nonfat milk in PBS with 0.05% Tween 20 (PBS-T) at room temperature for 1 h and then probed with an anti-SOD-1 rabbit polyclonal antibody (1:500 dilution, sc-11407, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-MnSOD goat antibody (1:2,000 dilution, A300449A, Bethyl Lab, Montgomery, TX) diluted in PBS-T with 2% BSA incubated at 4°C for overnight. Secondary antibodies were conjugated to horseradish peroxidase (HRP) (Chemicon International, Temecula, CA), and signals were developed by chemiluminescence (Pierce Biotechnology, Rockford, IL). The signals were then visualized by exposing the membranes to X-ray films (BioMax MS-1, Eastman Kodak), and digital records of the films were captured with a Kodak 290 camera. Resulting bands were quantified as OD × band area by a one-dimensional (1-D) image analysis system (Eastman Kodak) and recorded in arbitrary units. The molecular sizes of the immunodetected proteins were verified by using prestained standard (LC5925, Invitrogen Life Technologies).
MnSOD activity assay.
The total cytosolic fraction of the muscle homogenate was used to assess the enzyme activities of MnSOD, catalase, and GPx using spectrophotometric assay kits (Calbiochem, San Diego, CA) according to the manufacturer's instructions. For the measurement of MnSOD activity, it was assayed by the use of tetrazolium salt for detection of superoxide generated by xanthine oxidase and hypoxanthine, in the presence of potassium cyanide. In brief, samples or known standards were incubated with the provided radical detector reagent followed by the addition of xanthine oxidase to initiate the reaction. Potassium cyanide (3 mM) was included to inhibit both CuZnSOD and extracellular SOD and resulted in the detection of only MnSOD activity. After 20 min of incubation with shaking at room temperature, the absorbance at 450 nm was read by using a Dynex MRX plate reader controlled through PC software (Revelation, Dynatech Laboratories). The activity of MnSOD was estimated according to the generated standard curve with known SOD activity in the same setting. Negative experiments were performed without inclusion of sample and known standard. Measurements were accomplished with control and suspended samples analyzed on the same microtiter plate. The results of MnSOD activity were expressed per milligram of protein.
Catalase activity assay.
In the catalase assay, samples were incubated in the presence of a known concentration of H2O2. After incubation for 60 s, the reaction was quenched with sodium azide. The amount of H2O2 remaining in the reaction mixture was then determined by the oxidative coupling reaction of 4-aminophenazone (4-aminoantipyrene or AAP) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) and catalyzed by HRP. The resulting quinoneimine dye, N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine, was measured at wavelength of 520 nm by using a Dynex MRX plate reader controlled through PC software (Revelation, Dynatech Laboratories), as an indicator of catalase activity. The enzymatic activity was calculated by using a catalase standard curve. Negative experiments were performed without inclusion of sample and known standard. Measurements were accomplished with control and suspended samples analyzed on the same microtiter plate. The results of catalase activity were expressed per milligram of protein.
GPx activity assay.
The analysis of the samples for GPx activity was based on oxidized glutathione produced on reduction of organic peroxide by GPx, which is recycled to its reduced state by the enzyme glutathione reductase. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance, which provides the spectrophotometric means of monitoring GPx activity. Briefly, samples were put to NADPH reagent in the provided assay buffer followed by the addition of the diluted tert-butyl hydroperoxide. Absorbance at 340 nm was monitored for 3 min by using a Dynex MRX plate reader controlled through PC software (Revelation, Dynatech Laboratories). GPx measurements were performed with control and suspended samples analyzed on the same microtiter plate. The results of GPx activity were expressed per milligram of protein.
Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAE) on decomposition (17). In the present study, the content of MDA/4-HAE was estimated in the cytosolic fraction as an indicator of lipid peroxidation using a commercial colorimetric assay kit (21012, Oxis International, Portland, OR). Briefly, 50 μl of the cytosolic protein fraction or standards with known concentrations was incubated with a chromogenic reagent, N-methyl-2-phenylindole, at 45°C for 1 h, and the generated chromophore after incubation was detected by spectrophotometry at 586 nm. The MDA/4-HAE concentration of samples was determined according to the standard curve generated in the same setting. Results were presented as MDA/4-HAE (μM) normalized to milligram protein used in the assay. Control and suspended samples were run in the same setting to eliminate any assay-by-assay variations. Although spectrophotometric analysis of MDA/4-HAE has been widely employed for the assessment of lipid peroxidation in living systems, the assay has some limitations because aldehydes other than MDA/4-HAE could possibly contribute to the signal measured by the spectrophotometer. Other approaches including mass spectrometry would have provided a more definitive measure of lipid peroxidation; however, the technical limitations of the small muscle sizes in this study prevented us from further analysis following the MDA/4-HAE evaluations. Nevertheless, we view the MDA/4-HAE spectrophotometric assay as a reasonable estimation of lipid peroxidation.
The content of H2O2 in the muscle homogenate was measured in the cytosolic fraction using a fluorometric H2O2 detection kit (FLOH 100–3, Cell Technology, Mountain View, CA). This assay is based on the static detection of H2O2 using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP). ADHP is a highly sensitive and stable probe for H2O2 (40, 66) and is a nonfluorescent substrate that becomes fluorescent after it is oxidized by H2O2. By following the manufacturer's instruction, 50 μl of the total cytosolic fraction protein of the medial gastrocnemius muscle was incubated in 50 μl of reaction cocktail containing HRP and ADHP in sodium phosphate buffer at room temperature in the dark for 10 min. The fluorescence was measured on a spectrofluorometer with an excitation wavelength of 530/25 nm and an emission wavelength of 590/35 nm (CytoFluor, Applied Biosystems, Foster City, CA) after the incubation. H2O2 content was estimated as the arbitrary fluorescence units normalized to milligram protein used in the assay. Measurements were performed with the suspended and control samples run on the same microplate in the same setting. Negative inhibitory control experiments were performed with the inclusion of catalase (219261, Calbiochem, San Diego, CA) in the reaction buffer.
Nitrotyrosine dot blot.
As an index of the production of RNS, an immuno-dot blot was carried out to estimate the total nitrotyrosine content in the cytosolic fraction of the medial gastrocnemius muscles from suspended and control animals. In brief, 4 μg of cytosolic protein was dotted on a nitrocellulose membrane (VWR). After air dry, the membrane was blocked in 5% nonfat milk in PBS-T at room temperature for 1 h and then probed with an anti-nitrotyrosine mouse monoclonal antibody (1:1,000 dilution, MAB5404, Chemicon International) diluted in PBS-T with 2% BSA incubated at 4°C for overnight. The membrane was incubated with a HRP-conjugated anti-mouse IgG secondary antibody (AP124P, Chemicon International) at room temperature for 1 h, and then signals were developed by chemiluminescence (Pierce Biotechnology). Negative control experiments were performed by omitting either the anti-nitrotyrosine or the secondary antibodies. The signals were then visualized by exposing the membranes to X-ray films (BioMax MS-1, Eastman Kodak), and digital records of the films were captured with a Kodak 290 camera. Resulting dot signals were quantified in arbitrary units as OD × dot area using Kodak 1-D image analysis system (Eastman Kodak).
Statistical analyses were performed using the SPSS 10.0 software package. ANOVA was performed to examine the main effects of suspension, age, and interaction (suspension × age) on the measured variables. ANOVA followed by Tukey HSD post hoc analysis was used to examine differences between groups. Relationships between given variables were examined by linear regression analysis and computing the Pearson product-moment correlation coefficient, r. All data are given as means ± standard error of mean (SE). Statistical significance was accepted at P < 0.05.
Following 14 days of hindlimb suspension, the whole medial gastrocnemius absolute muscle weight decreased by ∼30% in young adult and aged rats (Fig. 1A). When the muscle mass was expressed as relative muscle weight normalized with animal body weight, the relative muscle weight of the suspended muscle was reduced by 12% in young adult rats and by 22% in aged rats compared with the muscles of their corresponding age-matched control animals (Fig. 1B). Comparisons between young adult and aged control animals demonstrated a sarcopenic decline in muscle mass. Medial gastrocnemius wet weight was ∼25% greater in young adult rats than aged rats (Fig. 1A), whereas muscle weight normalized to the animal body weight was 51% greater in young adult rats compared with the aged rats (Fig. 1B).
As an indicator of the level of lipid peroxidation, suspended muscles had a greater content of MDA/4-HAE with a 29% and 58% increase compared with the control muscle in young adult and aged animals, respectively (Fig. 2). The interaction effect of suspension × age in the ANOVA indicated that the response of MDA/4-HAE content to hindlimb suspension in these animals followed an age-dependent manner. The increase in MDA/4-HAE with suspension in the aged animals was greater than that in young adult animals (Fig. 2).
According to our fluorometric assay, no difference was found in H2O2 content between the suspended and control muscles in young adult animals. However, there was a 43% increase in the H2O2 content in aged suspended muscle compared with the aged control muscle (Fig. 3). The main effect of age in the ANOVA suggested that aged muscle had a higher content of H2O2 relative to the young adult muscle (Fig. 3). Moreover, an interaction (suspension × age) effect was found in the change of H2O2 content in the muscles of these animals, indicating that the change of H2O2 content during hindlimb suspension was age dependent (Fig. 3).
As a marker of the level of RNS, we found that the content of nitrotyrosine in the cytosolic fraction of suspended muscle was elevated by 76% and 65% relative to the control muscle in young adult and aged animals, respectively (Fig. 4). Moreover, a main effect of age in the ANOVA suggested that the aged muscle had a higher level of nitrotyrosine content compared with young adult muscle (Fig. 4).
mRNA content of antioxidant enzymes MnSOD, CuZnSOD, and catalase.
As indicated by RT-PCR analyses, the mRNA abundance of MnSOD was not different between the suspended and control muscles in both young adult and aged animals (Fig. 5). The main effect of age in the ANOVA suggested that there was a lower content of MnSOD mRNA in the aged muscle relative to the young adult muscle (Fig. 5). CuZnSOD mRNA in the suspended muscle was not different from the control muscle in both young adult and aged animals (Fig. 5). Moreover, no difference was found in the mRNA content of CuZnSOD between the young adult and aged control muscles (Fig. 5). Although no difference was found in the mRNA content of catalase between the suspended and control muscles in both young adult and aged animals, the main effect of age in the ANOVA suggested that there was a lower mRNA content of catalase in the aged muscle compared with the young adult muscle (Fig. 5).
Protein content of antioxidant enzymes MnSOD and CuZnSOD.
Although MnSOD protein content of the suspended muscle was not different from the control muscle in young adult animals, in the aged animals, MnSOD protein content in the suspended muscle was 28% lower than the control muscle (Fig. 6). An interaction effect of suspension × age in the ANOVA indicated that the response of MnSOD protein content to hindlimb suspension is dependent on the age of the animals (Fig. 6). As indicated by immunoblot analyses, CuZnSOD protein content in the suspended muscle was not different from the control muscle in both young adult and aged animals (Fig. 6). Moreover, no difference was found in protein content of CuZnSOD between the young adult and aged control muscles (Fig. 6).
Enzymatic activity of antioxidant enzymes MnSOD, catalase, and GPx.
No significant difference was found in the enzymatic activity of MnSOD between the suspended and control muscles in both young adult and aged animals (Fig. 7). In contrast, catalase activity in the total cytosolic fraction of medial gastrocnemius muscle was elevated by 69% and 43% in response to hindlimb suspension in young adult and aged animals, respectively (Fig. 7). Furthermore, there were significant main effects of age and suspension in the ANOVA of catalase activity measurement (Fig. 7). There was no difference in the enzyme activity of GPx between the suspended and control muscles in both young adult and aged animals (Fig. 7).
Relationship between medial gastrocnemius muscle mass and oxidative stress markers, MnSOD, and catalase.
The young adult, aged, control and suspended animals were pooled to perform the regression analyses (n = 38). We found that the relative muscle mass of medial gastrocnemius was negatively correlated with the contents of MDA/4-HAE (r = −0.364), H2O2 (r = −0.648), and nitrotyrosine (r = −0.616) (Fig. 8). Positive significant correlation coefficients were found between the relative muscle mass of medial gastrocnemius and the contents of MnSOD mRNA (r = 0.354) and protein (r = 0.894) (Fig. 9). A positive correlation coefficient was established between the relative muscle mass of medial gastrocnemius and the catalase mRNA (r = 0.480) whereas a negative significant correlation coefficient was found between the relative muscle mass and the catalase activity (r = −0.848) (Fig. 10).
Although considerable muscle wasting has been exhibited during muscle disuse (14, 15, 60), the underlying mechanisms responsible for the disuse-induced muscle loss are still largely unknown. It has been proposed that oxidative stress may play an important role in regulating the process of muscle loss during disuse (48, 49). This hypothesis is strongly supported by data showing signaling links between oxidative stress and the proteolytic processes mediating muscle loss (caspase-3, calpain-mediated and proteasome-mediated proteolyses) (48, 49). In addition, reactive oxidants are produced in inactive skeletal muscle through different sources such as xanthine oxidase, nitric oxide/peroxynitrite, reactive iron, NAD(P)H oxidase, and/or mitochondrial ROS (28–32). With muscle atrophy, as induced by immobilizing one ankle joint of rats in a fully extended position for days, Kondo and colleagues (28–32) demonstrated increases in iron concentration (especially in microsomal fraction), lipid peroxidation marker TBAR, H2O2 content, glutathione disulfide, hydroxyl radical, activities of CuZnSOD, glutathione-S-transferase, and glutathione reductase but decreases in total glutathione and MnSOD activity. In addition, administration of antioxidant vitamin E attenuates the rate of muscle atrophy during immobilization (2, 30). In particular, oxidative stress has been investigated in atrophied skeletal muscle in response to hindlimb suspension-mediated muscle disuse (27, 34, 63). Stevenson et al. (63) reported the activation of the oxidative stress-related genes including glutathione-S-transferase, glutathione peroxidase, selenoprotein P and W, peroxiredoxin 5, SOD-1 and thioredoxin interacting factor in rat soleus muscle after 1, 4, 7, or 14 days of hindlimb suspension by using the microarray technique. Koesterer and coworkers (27) also demonstrated that protein carbonyl content, a marker for protein oxidation, is increased, although they did not find change in the content of TBARS in the young hindlimb-suspended muscle. Oxidative stress increases in the slow-twitch soleus muscle (muscle that undergoes severe atrophy during disuse) from young adult rats following hindlimb unloading, as indicated by increases in dichlorohydrofluorescein and total hydroperoxides and disruption of antioxidant capacity including reduction of glutathione peroxidase, catalase and MnSOD activities, and nonenzymatic antioxidant scavenging capacity and increase in CuZnSOD activity (34). In this study, our results extend the knowledge by demonstrating that mixed muscle such as the medial gastrocnemius (which is predominantly but not exclusively composed of fast fibers) also increases oxidative stress in response to unloading. This observation is important because unlike the soleus that achieves marked atrophy during unloading, the gastrocnemius muscle only undergoes moderate muscle loss in response to hindlimb unloading (43% and 55% reduction in soleus absolute muscle mass following 14 and 28 days of unloading, respectively, whereas there was a 30% reduction in gastrocnemius after 14 days of unloading as shown in the present study) (34, 46). Consistent with the documented differential atrophic responses that slow extensor responds greater than fast extensor in hindlimb during suspension unloading (43), we have previously showed that the effect of unloading on the decline in muscle mass was the least in fast-twitch plantaris muscle (20% reduction in absolute muscle mass after 14 days of unloading) compared with gastrocnemius and soleus muscles (46). As this fast-twitch plantaris muscle has also been demonstrated to be affected by aging (8, 21, 46), it would be intriguing to examine whether oxidative damage is induced in plantaris muscle during unloading and the effect of age in a future study.
Based on the findings that oxidative stress is elevated and antioxidant capability is impaired in skeletal muscle with aging (5, 9, 10, 19, 20, 26, 44, 45), it is reasonable to hypothesize that aging may influence redox events and increase the muscle's susceptibility to oxidative injury during muscle disuse. Indeed, the effects of age on antioxidant systems in atrophied slow-twitch skeletal muscle following hindlimb unloading have been lately reported. Chen and colleagues (13) examined the influence of age in the ability of soleus muscle antioxidants to adapt to 3, 7, or 14 days of hindlimb unloading in adult and old Fischer 344 rats of 13 and 26 mo of age. They found that unloading was related to a compensatory increase in catalase activity and aging was associated with an elevation of glutathione peroxidase activity, whereas neither disuse nor aging changed the enzymatic activity of CuZnSOD (13). Age-related increases in the reduced and total glutathione with muscle unloading were shown, and it was concluded that the aged soleus muscle was more susceptible to oxidative damage during hindlimb unloading (13). In the present study, medial gastrocnemius muscle was investigated. We also demonstrated the age-specific changes in oxidative stress markers in rat gastrocnemius muscle during hindlimb suspension. Consistent with the soleus data reported by Chen and colleagues (13), our gastrocnemius findings of age-dependent increases in MDA/4-HAE and H2O2 and decrease in MnSOD protein abundance in response to hindlimb suspension are in agreement with the general hypothesis that aging elevates oxidative stress and exacerbates the disturbance of redox balance during muscle unloading. However, it is noteworthy to mention that our data failed to demonstrate the age-induced exacerbation of muscle loss with unloading. The exact reasons in explaining the observation that changes in muscle mass with unloading did not go parallel with the age-related changes of oxidative stress markers are unknown. However, the fact that our data were collected at a single time point (i.e., 14 days after hindlimb suspension) may have limited the interpretation as there might be temporal differences in the responses of the change of muscle mass and the oxidative stress markers. This speculation is supported by the previous findings demonstrating the temporal gene expression changes of oxidative stress markers in rat soleus muscle following 1, 4, 7, and 14 days of hindlimb unloading (63). Although a general parallel change of muscle mass/size and oxidative stress parameters has been established with muscle disuse in young adult muscle (3, 54), our data indicate that this parallel change may be more complicated in aging muscle. Nonetheless, the differential age-related responses of muscle mass decline and alteration of oxidative stress markers suggest a complex interaction in the link between oxidative stress and muscle atrophy with aging. Further studies are warranted in addressing the temporal issues of oxidative signaling with muscle disuse and aging.
In this study, we found that the activity of MnSOD remained unchanged while the protein and/or mRNA contents were decreased following unloading and in aged muscles. This finding on the differential responses of mRNA, protein, and activity is not entirely unexpected as it has been shown that the regulation of the antioxidant enzyme activity can be influenced at translational and/or posttranslational level (22). Hollander and coworkers have examined the gene and protein expressions of MnSOD and CuZnSOD in soleus, gastrocnemius, and superficial vastus lateralis muscles of young and aged Fischer 344 rats that were 8 and 25 mo of age. Similar to our observations, they reported differential responses of mRNA, protein and enzymatic activity of SOD to aging and they concluded that the translational and/or posttranslational modifications were involved in the modulation of SOD with aging (22). Nevertheless, our observed increase in nitrotyrosine content with unloading and aging was expected to result in inactivation of MnSOD as peroxynitrite has been shown to negatively modulate this antioxidant enzyme (39). Indeed, our findings of decreased MnSOD mRNA and/or protein agreed with the result of elevated nitrotyrosine, but it is unknown why we failed to demonstrate the reduction of MnSOD activity. Nonetheless, our data support that the adaptation mechanism of antioxidant enzyme to unloading and aging possibly involves posttranscriptional, translation, and/or posttranslational regulation. Although our MDA/4-HAE, H2O2, and MnSOD protein data clearly showed that the unloading-induced oxidative stress was exacerbated by aging, it is noted that our interpretation on the regulatory mechanism of antioxidant enzymes are limited by the lack of the full spectrum measurements in mRNA, protein content, and enzymatic activity of all the examined antioxidant enzymes. Additional research is needed to reveal the precise regulatory mechanisms of the mRNA, protein, and activity of the antioxidant enzymes.
When comparing the medial gastrocnemius muscles of control young adult and control aged animals in this study, the contents of MDA/4-HAE and H2O2 were not found to be different, and these results appear to be inconsistent with the revealed elevation of oxidative stress in aging muscle (24, 33). However, our regression analyses indicated that moderate but significant negative correlation existed between muscle mass and MDA/4-HAE (r = −0.364) and H2O2 (r = −0.648) when the young adult, aged, unloaded and control animals were pooled for analysis. These results suggest that the observed age-associated decline in muscle mass was, at least moderately, related to increased MDA/4-HAE and H2O2. Nonetheless, our data did not show greater contents of MDA/4-HAE and H2O2 in the examined aged muscle relative to young adult muscle. We interpret that our findings generally agreed with the demonstrated increase in oxidative stress in aging muscle. The discrepancies in the age-related change of some oxidants (e.g., MDA/4-HAE and H2O2) observed between our data and previous findings might be attributed to the differences in strain and age of the animals and/or the differences in sensitivity of the assays adopted for measurements. Regarding the response of antioxidant enzyme to age, it is surprising but not completely unexpected to observe that catalase activity was increased but mRNA content was reduced in the aged muscle compared with young adult muscle in the present study. Hollander and colleagues (22) have reported differential alterations of MnSOD activity, protein, and mRNA contents in different type of muscles with aging. We interpret our catalase findings that the regulation by posttranscriptional and posttranslational process was possibly involved in the age-related adaptation of catalase. It is worth noting that our observed age-related compensatory elevation of catalase activity in medial gastrocnemius muscle was consistent with previous reports examining aged soleus (33, 35), vastus lateralis (22, 25, 35), and gastrocnemius muscles (50). However, our reported age-related decrease in catalase mRNA in medial gastrocnemius muscle was contradictory to the shown age-associated increase in catalase mRNA in soleus muscle reported by Lambertucci et al. (33). The divergent results are probably due to the difference in the examined muscle type (i.e., medial gastrocnemius vs. soleus), as well as different strain (FBN rat vs. Wistar-Kyoto rat) and age (young and aged: 6 and 30 mo vs. 2 and 21 mo) of the studied animals. With regard to the aged rats that were examined in this study, it is noted that their age (i.e., 30 mo) represents a late middle age rather than a senescent age based on the comparison of their survival curves with humans (65). Therefore, the effect of aging in our shown results should be interpreted as the aging process in the late middle age. As the sarcopenic muscle mass loss of gastrocnemius muscle has been demonstrated to be much greater in 36-mo-old senescent FBN rats relative to 28- to 30-mo-old late middle aged FBN rats (36 mo vs. 28–30 mo: 60% vs. 19% muscle mass loss compared with 8- to 9-mo-old young adult FBN rats) (21), it is unclear whether our findings still apply when sarcopenia becomes more severe at later age (e.g., 36 mo of age). Nevertheless, additional research is warranted to answer this question.
In conclusion, the novel results reported in this paper demonstrate that increases in oxidative stress markers are also evident in mixed medial gastrocnemius muscles of both young adult and aged FBN rats after 14 days of hindlimb suspension. We report that catalase activity and MDA/4-HAE and nitrotyrosine contents are increased in the suspended muscle compared with the control muscle in both young adult and aged animals, whereas H2O2 content was elevated and MnSOD protein content was reduced relative to the control muscle exclusively in the aged suspended muscle. We also found that the unloading-induced changes of the contents of MDA/4-HAE, H2O2, and MnSOD protein were age dependent. The magnitude of unloading-induced increase in MDA/4-HAE was greater in aged than young adult animals. The unloading-induced increased H2O2 and decreased MnSOD protein contents were exclusively found in aged muscles. Moreover, in addition to a sarcopenic muscle mass decline, aged muscle exhibited greater catalase activity and contents of H2O2 and nitrotyrosine but lower abundances of MnSOD mRNA and protein and catalase mRNA. When young adult, aged, suspended, and control animals were pooled for analysis, negative significant relationships were found between muscle mass and oxidative stress indicators, including MDA/4-HAE, H2O2, and nitrotyrosine. Positive significant relationships existed between muscle mass and MnSOD mRNA and protein contents and catalase mRNA, whereas catalase activity was found to be negatively correlated to muscle mass. In general, our data are consistent with the hypothesis that oxidative stress is increased in both young adult and aged muscles in response to hindlimb unloading, and the changes of oxidative stress markers during unloading are age dependent. These findings are in agreement with the proposition that aging may predispose skeletal muscle to an advanced level of oxidative stress at rest and under unloading or decreased loading conditions.
This study was supported by National Institute on Aging Grant R01-AG-021530 (S. E. Alway) and The Hong Kong Polytechnic University Research Funds A-PH69 and A-PA7N (P. M. Siu).
We thank Dr. William Wonderlin for providing access to the CytoFluor spectrofluorometer.
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