Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle

Free Radical Biology & Medicine, Vol. 35, No. 1, pp. 9 –16, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00186-2

Original Contribution HINDLIMB UNLOADING INCREASES OXIDATIVE STRESS AND DISRUPTS ANTIOXIDANT CAPACITY IN SKELETAL MUSCLE JOHN M. LAWLER, WOOK SONG,

and

SCOTT R. DEMAREE

Redox Biology and Cell Signaling Laboratory, Department of Health and Kinesiology, Texas A&M University, College Station, TX, USA (Received 26 November 2002; Revised 7 March 2003; Accepted 14 March 2003)

Abstract—Skeletal muscle disuse with space-flight and ground-based models (e.g., hindlimb unloading) results in dramatic skeletal muscle atrophy and weakness. Pathological conditions that cause muscle wasting (i.e., heart failure, muscular dystrophy, sepsis, COPD, cancer) are characterized by elevated “oxidative stress,” where antioxidant defenses are overwhelmed by oxidant production. However, the existence, cellular mechanisms, and ramifications of oxidative stress in skeletal muscle subjected to hindlimb unloading are poorly understood. Thus we examined the effects of hindlimb unloading on hindlimb muscle antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase), nonenzymatic antioxidant scavenging capacity (ASC), total hydroperoxides, and dichlorohydrofluorescein diacetate (DCFH-DA) oxidation, a direct indicator of oxidative stress. Twelve 6 month old Sprague Dawley rats were divided into two groups: 28 d of hindlimb unloading (n ⫽ 6) and controls (n ⫽ 6). Hindlimb unloading resulted in a small decrease in Mn-superoxide dismutase activity (10.1%) in the soleus muscle, while Cu,Zn-superoxide dismutase increased 71.2%. In contrast, catalase and glutathione peroxidase, antioxidant enzymes that remove hydroperoxides, were significantly reduced in the soleus with hindlimb unloading by 54.5 and 16.1%, respectively. Hindlimb unloading also significantly reduced ASC. Hindlimb unloading increased soleus lipid hydroperoxide levels by 21.6% and hindlimb muscle DCFH-DA oxidation by 162.1%. These results indicate that hindlimb unloading results in a disruption of antioxidant status, elevation of hydroperoxides, and an increase in oxidative stress. © 2003 Elsevier Inc. Keywords—Hindlimb unloading, Superoxide dismutase, Catalase, Glutathione peroxidase, Skeletal muscle, Soleus, Dichlorohydrofluorescein diacetate, Free radicals

INTRODUCTION

and molecular triggers that lead to muscle wasting with hindlimb unloading are not well understood. A potential mechanism that would trigger increased protein degradation and atrophy in skeletal muscle is “oxidative stress,” where antioxidant protein and scavenger protection are overwhelmed by oxidant production [6]. However, there are limited data in the literature concerning oxidative stress and skeletal muscle in disuse models. Girten et al. [7] first described a reduction in the antioxidant enzyme activities for catalase and total superoxide dismutase following 14 d of hindlimb unloading. However, the cytosolic Cu,Zn- and mitochondrial Mn-isoforms of superoxide dismutase were not distinguished, and glutathione peroxidase was not measured. In contrast, Kondo et al. [8,9] reported a more complex response to 8 d of cast immobilization. Glutathione peroxidase and catalase did not change with immobilization, while the Cu,Zn isoform of superoxide dismutase in-

Loss of skeletal muscle mass occurs with disuse or “hypokinesia” [1] as well as muscle wasting pathologies including sepsis, muscular dystrophy, heart failure, COPD, cancer, and AIDS [2– 4]. Extreme disuse and muscle wasting occur with spaceflight, chronic bed rest, hindlimb unloading, and immobilization. Hindlimb unloading is the preferred animal model for extreme disuse and spaceflight [1]. There is agreement that muscle wasting with hindlimb unloading, and other disuse models, is a product of atrophy of skeletal muscle fibers and increased protein degradation [1,5]. However, the cellular Address correspondence to: John M. Lawler, Ph.D., Redox Biology and Cell Signaling Laboratory, Department of Health and Kinesiology, Texas A&M University, College Station, TX 77843-4243, USA; Tel: (979) 862-2038; Fax: (979) 847-8987; E-Mail: jml2621@neo. tamu.edu. 9

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creased by 41%. Immobilization also increased contributors to oxidative stress in skeletal muscle including lipid peroxidation, oxidized glutathione/reduced glutathione ratio, free iron, and xanthine oxidase [9 –12]. In addition, Kondo et al. [13] and Appell et al. [14] reported that vitamin E supplementation blunted the amount of immobilization-induced muscle fiber atrophy ranging from 23 to 66%, suggesting that oxidative stress may play a role in muscle atrophy. Thus the responses of the full spectrum of antioxidant enzymes to hindlimb unloading have not been fully delineated. In addition, there are disparate reported responses of the antioxidant enzyme system among limited disuse studies. It is very possible that differences between hypokinesia models are a factor. Moreover, there is lack of information regarding the effects of disuse on nonenzymatic antioxidant scavenging capacity. Finally, a direct marker of oxidant production and oxidative stress, such as dichlorohydrofluorescein diacetate (DCFH-DA), has not been used to confirm the existence of oxidative stress in hindlimb unloading. Thus our purpose was (i) to identify changes in direct and indirect indicators of oxidative stress in skeletal muscle with 28 d of hindlimb unloading, and (ii) to identify changes in muscle antioxidant enzyme and scavenger capacity with hindlimb unloading. We hypothesized that hindlimb unloading would increase direct and indirect indicators of oxidative stress, and oxidative stress would be linked to an imbalance in the antioxidant system and increased hydroperoxides. MATERIALS AND METHODS

Animals Twelve 6 month old male Sprague Dawley rats were housed and cared for in accordance with NIH policy (DHEW Publication No. 85-23, revised 1985). An animal use protocol had been approved previously by the University Laboratory Animal Care Committee. Rat chow and water were provided ad libitum, and the animals maintained in a temperature-controlled room (23 ⫾ 2°C) with a 12 h light/12 h dark cycle. Hindlimb unloading protocol The hindlimb unloading model is the preferred ground model for spaceflight with similar changes in mechanical and hydrostatic loading, unlike immobilization [1]. Animals were randomly assigned to either 28 d of hindlimb unloading (HU: n ⫽ 6) or to a normal weight-bearing control group (n ⫽ 6). Hindlimb unloading was performed as described previously [15]. Briefly, the hindlimbs of the HU group were elevated to a spinal orientation of 40 – 45° above horizontal using orthopedic

traction tape wrapped around the proximal two-thirds of the tail. Hindlimb elevation was adjusted so that the hindlimb paws were suspended, while the forelimbs were free to ambulate around the entire range of the cage. Tissue preparation Rats were anesthetized with sodium pentobarbital (50 mg · kg⫺1 i.p.) following the hindlimb suspension period in both the HU and control groups. Animals in the HU groups were anesthetized immediately upon completion of unloading to avoid any risk of reloading-induced injury. Preparation of hindlimb muscles followed the procedure described by Lawler and Demaree [16]. Hindlimb muscles were quickly extracted and weighed. Strips were dissected in ice-cold phosphate-buffered saline (PBS) (pH ⫽ 7.4) from fresh whole hindlimb muscles (soleus ⫹ gastrocnemius), where large aggregate mass is needed, using the fluorochrome probe dichlorohydrofluorescein-diacetate (DCFH-DA) as a direct oxidative stress marker. Strips were placed in PBS at 4°C until the DCFH-DA oxidation procedure began. Soleus muscle samples, from the contralateral limb, to be used in the measurement of antioxidant enzyme activities, nonenzymatic antioxidant scavenging capacity (ASC), and total hydroperoxides were frozen in liquid nitrogen and then transferred to a ⫺80°C freezer until biochemical analysis. Homogenization procedure Soleus samples were minced and homogenized as described previously [16] for the measurement of antioxidant enzyme activities, ASC, and total hydroperoxides. Briefly, the tissues were further minced into fine pieces and homogenized (20:1 w/v) in ice-cold potassium phosphate buffer (100 mM; pH ⫽ 7.40; temp. ⫽ 5°C). Samples were then homogenized using 10 passes of a ground glass on ground glass pestle system, found to be superior to blade homogenizer and sonication techniques [17]. Ten mM EDTA, 0.13 mM butylated hydroxytoluene (BHT), and 0.13 mM desferrioxamine added to minimize oxidation of lipids and proteins during the homogenization procedure, as detailed previously [18]. Total protein was measured using the Bradford technique. Antioxidant enzyme activities Antioxidant enzyme activities were conducted on muscle homogenates as described previously [16]. Catalase assays were conducted in the above homogenization buffer, except without EDTA. Homogenization was followed by centrifugation for 10 min at 300 ⫻ g (3°C) to rid the homogenate of cellular debris and the supernatant decanted.

Hindlimb unloading impairs skeletal muscle antioxidant capacity

Superoxide dismutase (SOD) activity was measured using an adaptation of the technique of Vanneste and Vanneste [19,20]. This assay utilized the reduction of cytochrome c by superoxide anions produced via the xanthine oxidase reaction; 0.06 U/ml xanthine oxidase, 6 mM xanthine, and 60 ␮M cytochrome c were mixed with 300 ␮l of homogenate to a final volume of 1.6 ml. The cytosolic Cu,Zn-SOD isoform was distinguished from the mitochondrial Mn-SOD isoform by the addition of 1 mM KCN. The change in absorbance at 550 nm was subtracted from controls, with 1 U of SOD activity defined as a 50% reduction in ⌬ absorbance. Soleus muscle catalase activity was determined using the technique of Aebi [21] adapted by Lawler & Song [20]. Briefly, ethanol was added 1:10 v/v to muscle homogenates and incubated for 30 min in an ice bath. Then 1% Triton X-100 was added and the homogenates incubated in ice for 15 min. One ml of 100 mM K⫹ phosphate buffer (pH ⫽ 7.4) was added to 500 ␮l of the homogenate in a glass cuvette and the reaction commenced with the addition of 1 ml of 50 mM H2O2. Absorbance was read at 240 nm for 90 s. Activity units for catalase were calculated from the rate constant (k) and expressed as U · mg protein⫺1. Analysis of muscle glutathione peroxidase activity was performed using the technique described previously [16,22]. Briefly, 800 ␮l of 0.30 U/ml glutathione peroxidase, 1.25 mM reduced glutathione, and 0.19 mM NADPH in potassium phosphate buffer (pH ⫽ 7.4) were introduced to each cuvette. Then 100 ␮l of homogenate was added. Finally, 100 ␮l of t-butyl hydroperoxide was added to commence the reaction, and the absorbance was then read for 4 min at 340 nm. Total antioxidant scavenging capacity (ASC) We used a modification of the technique described by Pellegrini et al. [23] that determines the ability of muscle homogenates to scavenge the blue/green chromophore (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulABTS⫹ fonic acid) (Sigma Chemical, St. Louis, MO, USA). The ASC technique is a robust measure of the nonenzymatic antioxidant scavenging ability of a tissue or chemical. The assay was based on the principle that the introduction of antioxidants reduces the ABTS•⫹ in solution to ABTS with a concomitant reduction in absorption at 734 nm. The nonenzymatic antioxidant scavenging capacity of a sample is then the rate of decolorization expressed as a percentage inhibition of ABTS•⫹, and quantified relative to the reactivity of Trolox, used as a standard curve under identical conditions. Thus this assay is a functional marker of a composite of skeletal muscle antioxidant scavengers, including reduced glutathione, tocopherols,

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ascorbate, urate, thioredoxin, etc. ABTS•⫹ was activated by reaction of ABTS stock (7 mM) with 2.5 mM potassium persulfate (Aldrich, Milwaukee, WI, USA) in 100 mM potassium phosphate buffer (pH ⫽ 7.4). The resulting radical cation was found to be stable in the dark at 4°C for a week. We made ABTS⫹ 24 h before use in the ASC assay. Immediately prior to assay, the ABTS⫹ solution was diluted (⬃ 82:1) to an absorbance of 0.70 ⫾ .02 at 734 nm, the most interference-free absorption maxima. Following dilution, 1000 ␮l of ABTS⫹ solution was added to each cuvette. The initial absorbance was read and recorded. Then 70 ␮l of sample or Trolox standard was added to each cuvette, the contents mixed by inversion, and the absorbance recorded for 4 min. Parallel blanks were run with the buffer alone. The decolorization of ABTS•⫹, as determined through a reduction in absorbance, was a function of antioxidant concentration and equated relative to the reactivity of Trolox (used as a standard under the same conditions). ASC for each sample was then calculated as ␮mol Trolox equivalent per gram of tissue. Total hydroperoxides We measured total hydroperoxides using the technique of Hermes-Lima et al. [24] that mixes tissue lipid hydroperoxides with FeSO4 (250 ␮M) in sulfuric acid (25 mM), and a reactive dye (xylenol orange: 100 ␮M). The principle relies on the oxidization Fe2⫹ to Fe3⫹ when hydroperoxides are reduced. The ionized sulfuric acid assists in the reduction and prevents spontaneous reaction of Fe2⫹ with xylenol orange, unless reacting with the hydroperoxides first. The Fe3⫹ then reacts with the xylenol orange to form a Fe3⫹-xylenol complex and changes to a purplish color. Absorbance was then measured at 580 nm. Concentrations of hydroperoxides were quantified against standard curve using t-butyl hydroperoxide as a substrate. DCFH-DA oxidation DCFH-DA, a redox-sensitive fluorescent probe that emits light in the green spectrum when oxidized, was purchased from Molecular Probes (D-399, Eugene, OR, USA). DCFH-DA passes through cell membranes where it is cleaved by esterases to DCFH and becomes activated by oxidation. We isolated hindlimb muscle strips as described above and loaded one-half of the samples loaded with DCFH-DA (50 ␮M) in PBS, and placed the other half of the samples in PBS alone as a control. Samples were then incubated on a shaker at 37°C for 30 min. Following incubation, the muscle strips were homogenized (20:1 w/v) in potassium phosphate buffer (pH ⫽ 7.4). Fluorescence of the samples was measured using a Farland Optical fluorometer. Peak excitation wave-

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Fig. 2. Effect of hindlimb unloading on mean soleus mass. * indicates significantly different than controls (p ⬍ .05).

Statistics Data was analyzed with one-way ANOVAs. Where appropriate, Student-Newman-Keuls was used for post hoc comparisons. Statistical significance was established at p ⬍ .05. RESULTS

Fig. 1. (a) Fluorescein standard (nM) for dichlorohydrofluorescein diacetate (DCFH-DA) with Farland Optical fluorometer at an excitation wavelength of 488 nm and emission of 525 nm. (b) Hydrogen peroxide (H2O2) standard (␮M) for DCFH oxidation Farland Optical fluorometer at an excitation wavelength of 488 nm and emission of 525 nm. Serial standards were preloaded with DCFH-DA and 20 U/ml esterase and incubated for 30 min.

length for oxidized DCFH was 488 nm and emission was 525 nm. Calibration of the fluorometry procedure was accomplished by running standard curves for serial dilutions of fluorescein (Molecular Probes) (Fig. 1a). Standards for the samples were conducted using serial dilutions of metal contaminant-free hydrogen peroxide (Merck, Darmstadt, Germany) incubated in DCFH-DA with esterase (20 U/ml) for 30 min at 37°C (Fig. 1b). DCFH oxidation was then calculated per ␮M H2O2. While all samples were run concurrently, we found that data from standard curves were reproducible on a dayto-day basis as well. Samples loaded with DCFH-DA were subtracted by their respective controls to determine the true DCFH oxidation levels in the muscle samples.

Body weights of the hindlimb-unloaded and control groups were not significantly different (data not shown). However, mean soleus mass decreased to 45% of controls (90.6 ⫾ 7.3 g vs. 202 ⫾ 7.1 g), reflecting significant anti-gravity muscle wasting over the 28 d unloading period (Fig. 2) while total protein expressed per gram muscle mass was unchanged. Hindlimb unloading resulted in significantly lower (⫺10.2%) soleus Mn-SOD activity when compared with controls (331.12 ⫾ 9.05 vs. 368.3 ⫾ 6.44 U/g.w.w.). Results for Mn-SOD are displayed in Fig. 3a. In stark contrast, Cu,Zn-SOD activity was dramatically elevated by hindlimb unloading (⫹71.2%) when compared with controls (436.10 ⫾ 56.23 vs. 254.80 ⫾ 44.58 U/g.w.w.). Results for hindlimb unloading of Cu,Zn-SOD activity are found in Fig. 3b. Thus, total SOD was also higher in soleus muscle from the hindlimb-unloaded group (767.2 ⫾ 29.67 U/g.w.w.) than from controls (623.1 ⫾ 25.49 U/g.w.w.) (data not displayed). Superoxide dismutase removes superoxide anions while producing hydrogen peroxide that is removed enzymatically by catalase and glutathione peroxidase primarily. Mean catalase activities were significantly lower in soleus muscles from the hindlimb-unloaded rats when compared with controls (Fig. 4a). Catalase activity was depressed by 54.5% with hindlimb unloading (1.60 ⫾ 0.18 vs. 3.52 ⫾ 0.63 U/g.w.w.). Similarly, hindlimb

Hindlimb unloading impairs skeletal muscle antioxidant capacity

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Fig. 3. (a) Effect of hindlimb unloading on manganese-superoxide dismutase (Mn-SOD) activity in the rat soleus. * indicates significantly different than controls (p ⬍ .05). (b) Effect of hindlimb unloading on copper, zinc-superoxide dismutase (Cu,Zn-SOD) activity in the rat soleus. * indicates significantly different than controls (p ⬍ .05).

unloading also resulted in a significant downregulation of glutathione peroxidase activity (Fig. 4b). Glutathione

peroxidase activity was 16.1% lower with hindlimb unloading than in control soleus muscles (55.3 ⫾ 3.0 vs.

Fig. 4. (a) Effect of hindlimb unloading on catalase activity in the rat soleus. * indicates significantly different than controls. (b) Effect of hindlimb unloading on glutathione peroxidase activity in the rat soleus. * indicates significantly different than controls (p ⬍ .05). (c) Effect of hindlimb unloading on nonenzymatic antioxidant scavenging capacity (ASC) in the rat soleus. ASC measures the integrated effects of endogenous scavengers such reduced glutathione, Vitamin E, etc. on the ability to remove ABTS⫹ cation in vitro. * indicates significantly different than controls (p ⬍ .05).

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J. M. LAWLER et al. DISCUSSION

Fig. 5. (a) Effect of hindlimb unloading on total hydroperoxides in the rat soleus. * indicates significantly different than controls (p ⬍ .05). (b) Effect of hindlimb unloading on difluorochlorofluoroscein diacetatte (DCFH-DA) oxidation activity in the rat hindlimb muscle. * indicates significantly different than controls (p ⬍ .05).

65.93 ⫾ 1.67 ␮mol/g.w.w./min). Moreover, nonenzymatic antioxidant scavenging capacity (ASC) of the soleus was also significantly reduced with hindlimb unloading. ASC was 12.8% lower in HU than controls (1722 ⫾ 58 vs. 1975 ⫾ 45 ␮mol Trolox Eq/g.w.w.) (Fig. 4c). Depression of enzymatic and nonenzymatic protection against hydroperoxides with hindlimb unloading was manifested in significantly higher total hydroperoxides in the soleus muscle following unloading when compared with controls (Fig. 5a). Total hydroperoxides were 21.6% higher in the hindlimb-unloading group than controls (2152.3 ⫾ 138.2 vs. 1770.1 ⫾ 101.4 nmol/ g.w.w.). Finally, hindlimb unloading significantly increased DCFH-DA oxidation, a direct measure of oxidant content and production, in mixed hindlimb muscle (Fig. 5b). In fact, DCFH-DA oxidation was 162.1% higher in HU when compared with controls (238.0 ⫾ 58.3 vs. 90.8 ⫾ 32.5 ␮M H2O2 equivalents).

The unique findings of our study include the following: (i) Hindlimb unloading resulted in an imbalance of antioxidant status, characterized by a large increase in Cu,Zn-superoxide dismutase activity while catalase, glutathione peroxidase, and nonenzymatic antioxidant scavenging capacity decreased. (ii) Hindlimb unloading indeed resulted in an increase in oxidative stress, as indicated directly by DCFH-DA oxidation, as well as elevated total hydroperoxides when compared with controls. To our knowledge, this is the first report that definitively demonstrates that oxidative stress increases in hindlimb muscle with hindlimb unloading, and that a disrupted antioxidant status and accumulation of hydroperoxides may be critical contributory factors. The physiological significance of altered antioxidant status and elevated oxidative stress in skeletal muscle resulting from disuse is discussed below. The current study is the first to demonstrate that hindlimb unloading increases both DCFH-DA oxidation and hydroperoxide content in skeletal muscle. These data are consistent with the hypothesis that hindlimb unloading would increase direct and indirect indicators of oxidative stress. Our study is also the first to fully characterize alterations in major skeletal muscle antioxidant enzymes as a result of unloading. The results were consistent with the hypothesis that hindlimb unloading would be linked to an imbalance in the antioxidant system and increased hydroperoxides. These data clearly indicate that hindlimb unloading increases oxidative stress with unloading. Moreover, unloading-induced disruption of the antioxidant enzyme and scavenger profile could predispose skeletal muscle to inflammation and muscle damage upon reloading [25]. Fourteen years ago, Girten et al. [7] reported a decrease in total superoxide dismutase and catalase activities of the rat soleus with 14 d of hindlimb unloading which was not prevented by dobutamine, but partially prevented by treadmill exercise training, prior to unloading. In contrast, Kondo et al. [9] found that 7–14 d of immobilization did not alter catalase or glutathione peroxidase, and in fact increased Cu,Zn-SOD activity. However, significant increases in soleus lipid peroxidation and oxidized glutathione/reduced glutathione ratio, indirect markers of oxidative stress, occurred with immobilization [9,10,12]. The current data appear to rectify some of the apparently contradictory findings of previous investigations. Similar to Kondo’s data, Cu,Zn-SOD isoform activity in the current study was dramatically upregulated (⫹71.2%) in the soleus by hindlimb unloading (Fig. 3b). In contrast, hindlimb unloading resulted in a decrease of Mn-SOD (Fig. 3a), indicating differential

Hindlimb unloading impairs skeletal muscle antioxidant capacity

regulation of superoxide dismutase isoforms in skeletal muscle as a result of unloading. Cu,Zn-SOD upregulation would increase elimination of superoxide anions while catalyzing greater production of hydrogen peroxide. The mechanisms causing the differential response of Mn-SOD and Cu,Zn-SOD in skeletal muscle from hindlimb unloading are unknown. As Mn-SOD is located in the mitochondria and Cu,Zn-SOD in the sarcoplasm [6], it is logical that the divergent responses could be related to different micro-environments between the mitochondria and sarcoplasm as a result of unloading. Specifically, one would expect that hindlimb unloading would induce in skeletal muscle different changes in oxidant levels, species of oxidants, other stimulatory and inhibitory molecular signals between the mitochondria and sarcoplasm. For example, elevation in Cu,Zn-SOD may also suggest a response to upregulation of cytosolic sources of superoxide anions in skeletal muscle with unloading. In addition, responsiveness of the Mn-SOD and Cu,Zn-SOD isoforms to changes in the mechanical in redox stresses, such as differences in the influence of specific growth factors and transcription factors on the promoter regions, could be different [26]. While an upregulation of an antioxidant protein such as Cu,Zn-SOD might at first appear to be beneficial or protective of muscle mass and function against oxidative stress, the interaction of increased product and location could be important factors leading to increased oxidative stress. Other skeletal muscle models have previously identified that elevated Cu,Zn-SOD, under certain circumstances, can promote oxidative stress, damage, and fatigue in skeletal muscle. Our laboratory previously demonstrated that the addition of Cu,Zn-SOD greatly enhanced fatigue in intact muscle fiber bundles that had been exposed to a reactive oxygen species donor (i.e., xanthine oxidase) [27]. In contrast, catalase produced a protective effect against muscle fatigue, indicating the direct or indirect contribution of hydrogen peroxide. Paled-Kamar et al. [28] found that transgenic overexpression of Cu,Zn-SOD, without increasing other antioxidant enzymes, induced overt signs of muscle damage associated with an increase in hydroxyl radicals (•OH). Rando et al. [29] observed that overexpression of Cu,ZnSOD increased muscle creatine kinase release, atrophy, oxidative stress, and necrosis in a pattern similar to muscular dystrophy and ALS (amyotrophic lateral sclerosis). Elevation of Cu,Zn-SOD may lead to increased hydroperoxide and oxidative stress (i.e., DCFH oxidation) due to unloading-induced suppression of two key antioxidant enzymes that remove hydroperoxides, catalase and glutathione peroxidase (Fig. 4a, b). Hollandier et al.

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[30] previously reported that spaceflight downregulated catalase and glutathione peroxidase in the liver, raising the possibility of a systemic effect on antioxidant enzymes protective against hydroperoxides in spaceflight models. In addition, nonenzymatic antioxidant scavenging capacity, which is a composite of the scavenging ability of reduced glutathione, vitamin E, vitamin C, etc., was also depressed by hindlimb unloading (Fig. 4c). Other antioxidant or stress proteins, such as HSP70, may be reduced in muscle by hindlimb unloading as well [31]. These data indicate an imbalance in the antioxidant profile in muscle that promoted hydroperoxide and accumulation and oxidative stress (Fig. 5a, b). Elevations in oxidative stress and subsequent effect on protein oxidation could also be mediated indirectly by hydroperoxides through the production of hydroxyl radicals (•OH) catalyzed by Haber-Weiss or Fenton reactions [32]. Increased oxidative stress has the potential to promote muscle atrophy through increased proteolysis via the redox-sensitive ATP-ubiquitin system and calpains [1,33,34]. Whether oxidative stress is a cause or effect of unloading-induced atrophy is still uncertain. Additional research investigating changes in oxidant and antioxidant protein levels over time will be essential. Supplementation of the lipid-soluble antioxidant vitamin E resulted in a significant attenuation of lipid peroxidation and muscle atrophy due to immobilization [13,14], and suggest that oxidative stress resulting from hypokinesia could contribute to muscle atrophy. Changes in pro-oxidant and antioxidant pathways are likely to be complex and should be the subject of future investigations. Important questions still remain. (i) How does unloading alter antioxidant protein status and increase pro-oxidant signaling? (ii) Do changes in redox status with unloading regulate ubiquitin- and calpain-driven proteolysis? (iii) Does oxidative stress play a regulatory role in unloadinginduced skeletal muscle atrophy, or is it simply a consequence of unloading and atrophy? In conclusion, we demonstrated that hindlimb unloading results in an increase in oxidative stress in skeletal muscle. Elevated oxidative stress in skeletal muscle was related to an imbalance in the antioxidant enzyme system and depression of antioxidant scavenger status that promoted production of hydroperoxides. These data are consistent with the hypothesis that decreased mechanical loading leads to an increased oxidant state. Acknowledgements — This study was supported by grants from NASA (National Space and Biomedical Research Institute Grant NCC9-58-H) and the American College of Sports Medicine (NASA Space Physiology Research Grant). The authors would like to thank Matt Allen and Dr. Susan A. Bloomfield for their technical assistance.

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J. M. LAWLER et al. REFERENCES

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