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J Appl Physiol 93: 1959-1965, 2002. First published August 23, 2002; doi:10.1152/japplphysiol.00511.2002
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Vol. 93, Issue 6, 1959-1965, December 2002

Increased antioxidant capacity does not attenuate muscle atrophy caused by unweighting

T. J. Koesterer1, S. L. Dodd2, and Scott Powers2

1 Humboldt State University, Arcata, California 95521; and 2 Center for Exercise Science, College of Health and Human Performance, University of Florida, Gainesville, Florida 32611


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have increased antioxidant capacity in skeletal muscle to attenuate oxidative stress and muscle atrophy during limb immobilization (Appell HJ, Duarte JAR, and Soares JMC. Int J Sports Med 18: 157-160, 1997; Kondo H, Miura M, Nakagaki I, Sasaki S, and Itokawa Y. Am J Physiol Endocrinol Metab 262: E583-E590, 1992). The purpose of this study was to determine the level of oxidative stress in muscle during hindlimb unweighting (HLU) and whether antioxidant supplementation can attenuate the atrophy and changes in contractile properties resulting from 14 days of unweighting. Muscle unweighting caused a 44% decrease in soleus (Sol) and a 30% decrease in gastrocnemius (GS) mass, a 7% decrease in body weight, and 28% decrease in tetanic force in the GS. Protein carbonyls increased by 44% in the Sol with HLU. Antioxidant supplementation did not attenuate the GS or Sol atrophy or the decrease in GS force generation during HLU. Sol and GS protein concentration was not different between groups. The GS was also subjected to three different oxidative challenges to determine whether the supplement increased the antioxidant capacity of the muscle. In all cases, muscles exhibited an increased antioxidant capacity. These data indicate that antioxidant supplementation was not an effective countermeasure to the atrophy associated with HLU.

hindlimb unweighting; oxidative stress; muscle wasting


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE ATROPHY DUE TO UNWEIGHTING is a significant problem during periods of prolonged spaceflight, extended bed rest, and orthopedic injuries. The loss of muscle protein in these conditions has been attributed to both a decrease in protein synthesis (6) and an increase in protein degradation (39). Protein synthesis in the unweighted soleus (Sol) decreases within 5 h of unweighting and may decrease by 50-60% over 7-14 days (6). In contrast, protein degradation increases at a much slower rate, peaks after ~14 days, and then returns to normal levels after ~24 days (40). The mechanisms that initiate and promote the protein degradation have not been delineated.

Kondo et al. (18) have demonstrated an increase in oxidative stress during immobilization-induced muscle atrophy. They have shown that immobilization causes an increase in Cu-Zn-superoxide dismutase (SOD), an enzyme that dismutates superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) to hydrogen peroxide (H2O2). This increase in SOD indicates an increased level of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (a marker of oxidative stress) in the cytoplasm. They speculated that this increase in O<UP><SUB>2</SUB><SUP>−</SUP></UP>· was due to an increased activity of xanthine oxidase resulting from an increased nucleotide degradation. Also, it is possible that an increase in intracellular calcium would activate proteases, which would convert xanthine oxidase to the O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-producing isoform. Glutathione peroxidase (GPX) and catalase (Cat), which convert H2O2 to H2O, did not increase in the cell. Therefore, the cell is producing O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and converting it to H2O2, resulting in increased levels of both in the cell. In another study (19), they demonstrated an increase in Fe2+ in the cell, which can lead to production of the highly reactive hydroxyl radical (·OH), through either Fenton chemistry or the Haber-Weiss reaction. Production of these radicals can lead to oxidation of lipid, protein, and DNA. There are several mechanisms that could explain how oxidative stress accelerates muscle atrophy.

First, the plasma membrane and sarcoplasmic reticulum may be damaged by oxidative stress, causing an increase in cellular calcium (7, 24). Calcium has been shown to stimulate phospholipid hydrolysis and nonlysosomal proteolysis (26, 28). Second, free radical damage to the lysosomal membrane causes the leakage of lysosomal proteases into the cytoplasm, and these proteases have been shown to increase in muscle atrophy (23). Finally, radicals may damage proteins directly, thereby making them more susceptible to proteolysis (30, 37).

It is unknown which of these or other mechanisms may begin the cascade of oxidative events, but there is strong evidence that the process can be accelerated rapidly. Although evidence is mounting that free radical damage occurs during immobilization of muscle, it is unknown whether these mechanisms are activated in unweighted muscle.

Given the potential role of reactive oxygen species (ROS) in muscle atrophy, it is not surprising that muscle cells have extensive protective systems against damage from radicals. Endogenous antioxidant enzymes include SOD, GPX, and Cat. Nonenzymatic compounds that have potent antioxidant properties include vitamin E, vitamin C, carotenoids, glutathione, ubiquinones, and flavonoids (12, 44). Antioxidants work not only individually but also with additive and synergistic interactions to maintain redox homeostasis (8, 44).

Countermeasures designed to increase the cells' antioxidant defenses are a promising strategy for muscle atrophy associated with muscle unweighting. An effective method of increasing antioxidant defenses is through supplementation with nonenzymatic antioxidants that result in the elevation of cellular antioxidant concentration and increased protection against oxidative damage (8). Thus the purpose of this study was to determine whether 1) oxidative stress is increased during unweighting and 2) antioxidant supplementation will attenuate the muscle atrophy and functional changes in contractile properties resulting from unweighting.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted in conformance with the Guiding Principles for Research Involving Animals and Human Beings and received approval from the University of Florida Institutional Animal Care and Use Committee. Adult, female Sprague-Dawley rats (Harlan), weighing between 245 and 255 g, were given water ad libitum, maintained on a 12:12-h light-dark cycle, and handled daily to reduce contact stress with humans. Animals were provided a control diet for 5 days and then randomly assigned by body weight to one of two groups (control or antioxidant supplemented), and they were fed their respective diets for 7 days. Concurrently, the antioxidant-supplemented animals were injected once a day with vitamin E (30 mg/kg ip). Vitamin E injections were given in the form of alpha -tocopherol solubilized in corn oil. The control diet animals were sham injected with a corresponding volume of vitamin E-stripped corn oil (Purina). Control diet animals were fed a diet designed to meet the National Research Council's recommended requirements (AIN-93M) in the adult rat (29). The antioxidant-supplemented animals were fed the AIN-93M diet supplemented with the seven antioxidants listed in Table 1.

                              
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  		Table 1.   Antioxidants supplemented to the AIN-93M diet

Control and antioxidant animals were further randomly assigned by body weight to either weight-bearing or hindlimb unweighted (HLU) groups to achieve the four following treatment groups: 1) weight bearing with control diet (C), 2) weight bearing with antioxidant supplementation (AO), 3) HLU with control diet (HLU-C), and 4) HLU with antioxidant supplementation (HLU-AO).

All animals continued their respective diets during the 14-day experimental period. Vitamin E and sham injections were given every other day, at the same dosages as during preexperimental period. The HLU-C and HLU-AO animals were hindlimb unweighted, whereas the C and AO animals remained weight bearing for the same 14 days. The tails of the HLU-C and HLU-AO rats were wrapped and connected to a swivel for suspension similar to the procedure described by Roer and Dillaman (34) for 14 days. The rats were suspended from the top of a cage such that the hindlimbs were 1 cm above the cage floor and had complete (360°) range of motion by use of the forelimbs. This afforded the animals free range of motion around the floor of the cage but prevented climbing on the sides of the cage.

In situ protocol. On day 14, the animals were anesthetized with pentobarbital sodium (30 mg/kg ip) and ventilated through an endotracheal tube with room air. The Sol and gastrocnemius (GS) muscles of the right leg were removed, immediately placed in cold antioxidant buffer [100 µM EDTA, 50 mM Na2HPO4, and 1 mM butylated hydroxytoluene (BHT)], blotted dry, weighed, and frozen for biochemical analysis. Although the Sol muscle is more widely studied during HLU, the GS is similarly and significantly affected by unweighting and provides a better in situ model for determining contractile properties. In addition, the GS provides the much greater amount of muscle needed for the biochemical analyses.

The GS muscle of the left hindlimb was prepared in situ to determine contractile properties. The muscle was dissected free from overlying muscles and surrounding connective tissue, with care taken not to disrupt the blood supply. A bone pin was placed through the femur, and the foot was placed into a clamp to prevent movement. The GS tendon was attached to an isotonic force transducer (model 400, Cambridge Instruments, Boston, MA) with a lightweight chain. The transducer output was amplified and differentiated by operational amplifiers and underwent analog-to-digital conversion for analysis with a computer-based data acquisition system (Superscope II, GW Instruments, Somerville, MA). The sciatic nerve was carefully isolated, tied, cut, and placed in a bipolar electrode connected to a square-wave stimulator (model S48, Grass Instruments, West Warwick, RI). The preparation was kept moist with saline covered with saline-soaked gauze and plastic wrap.

Measurement of contractile properties. After a 15-min equilibration, the muscle was stimulated with a supramaximal square-wave pulse (6 V, 0.2-ms duration) delivered in 100-ms trains at 150 Hz. The muscle was repeatedly stretched and stimulated to determine optimal length. Maximal isometric tetanic tension was determined by stimulating the muscle three separate times with the above parameters (6 V, 0.2-ms duration, 100-ms trains, 150 Hz). Three independent twitch stimulations (6 V, 0.2-ms duration) were given to determine the maximal isometric twitch tension and one-half relaxation time (RT1/2).

The left Sol and GS were then removed, immediately placed in cold antioxidant buffer (100 µM EDTA, 50 mM Na2HPO4, and 1 mM BHT), blotted dry, weighed, and frozen for later analysis.

Biochemical analysis. Approximately 25 mg of tissue were removed from the frozen Sol and GS samples, and a wet weight was measured. The samples were then freeze-dried (Virtis Sentry Benchtop 3L), and dry weights were measured.

Myofibrillar protein was isolated in Sol and GS muscles by using a modification of the myofibril-extraction technique described by Solaro et al. (36); myofibrillar protein concentration was then determined with the biuret technique of Watters (42).

Oxidative damage to proteins is accompanied by increased levels of protein carbonyls (2, 43). Spectrophotometric detection and quantification of protein carbonyls is facilitated by the reaction of 2,4-dinitrophenylhydrazine with protein carbonyls to form protein hydrazones. To quantify the amount of protein oxidation that occurred during HLU, total protein carbonyl derivatives were measured spectrophotometrically as described by Reznick and Packer (32), with modifications reported by Yan et al. (43).

Tissue antioxidant capacity. To evaluate the ability of the supplement to increase the antioxidant capacity of the muscles, GS homogenates from all four groups were subjected to three different ROS-generating systems and then analyzed for lipid peroxidation with the thiobarbituric acid-reactive substance (TBARS) assay. A section of the right GS was homogenized at a concentration of 10:1 (wt/vol) in either 50 mM potassium phosphate buffer (for the aqueous generating systems) or in ethanol [for the 2,2'-axobis(2-4 dimethylvaleronitrile) (AMVN) system] according to the method of Haramaki et al. (13). Aliquots of the homogenates were incubated at a concentration of 10 mg protein/ml in the presences or absence of an ROS-generating system. The following is a brief description of the three systems that were used.

·OH were generated by an Fe2+-catalized system according to the method of Bernier et al. (5). O<UP><SUB>2</SUB><SUP>−</SUP></UP>· were generated by a hypoxanthine-xanthine oxidase system according to the method of Fridovich (11). Peroxyl radicals were generated in lipids in the GS homogenate by thermal decomposition of AMVN according to the method of Kagan et al. (17). To determine the amount of ROS-mediated oxidative damage in the muscle, malondialdehyde (MDA), a by-product of lipid peroxidation, was measured. MDA levels were determined spectrophotometrically by using the TBARS assay previously described by Uchiyama and Mihara (40) with 1,1,3,3-tetramethoxypropane used as the standard.

Statistical analysis. All dependent measures (i.e., maximum twitch and tetanic tension, RT1/2, wet muscle weight, wet-to-dry muscle ratio, and biochemical parameters) were subjected to a one-way analysis of variance with a Scheffé's test used post hoc. Significance was established at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary considerations. Animals tolerated the antioxidant supplementation, and no differences (P > 0.05) in food intake existed between the control diet and antioxidant-supplemented groups (Table 2). Body weights were not different between groups before treatments. However, after 14 days of HLU, there was a significant difference between weight-bearing and HLU groups (Table 3).

                              
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  		Table 2.   Food and antioxidant intake


                              
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  		Table 3.   Body weights, muscle weights, and protein carbonyls for all groups

Muscle weights. Muscle weights were taken as an indicator of muscle atrophy. Sol and GS wet weights of the HLU groups decreased significantly (P < 0.05) compared with the respective weight-bearing groups. Furthermore, no significant difference (P > 0.05) was demonstrated between C and AO groups for either the Sol or GS (Table 3). Because body weight decreased with HLU, we expressed muscle mass changes relative to body weight. Both Sol and GS muscle weight-to-body weight ratios decreased with HLU (~48% in Sol and 20% in GS), and antioxidant supplementation had no effect.

Protein carbonyls. Protein carbonyls were assayed as a measure of protein oxidation in the Sol muscle of all experimental groups. Sol protein carbonyl concentrations were significantly (P < 0.05) increased for both HLU groups compared with weight-bearing groups. Comparison of control diet groups to their respective antioxidant-supplemented group indicated no difference (P > 0.05) in carbonyl concentrations (Table 3).

Muscle protein concentration. Muscle protein concentration was measured in both muscles to determine alterations in myofibrillar and soluble protein. There were no differences between groups for water content or for total, myofibrillar, or soluble protein (Table 4).

                              
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  		Table 4.   Muscle protein concentration and water content

GS contractile function. GS contractile function was assessed in situ at the end of the experimental period. Absolute tetanic and twitch forces were significantly decreased (P < 0.05) in the HLU groups compared with the weight-bearing groups, whereas the antioxidant-supplemented groups were not significantly different (P > 0.05) from the control diet groups, respectively. No significant differences existed between groups for specific tension or RT1/2 (Table 5).

                              
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  		Table 5.   Contractile properties of gastrocnemius during all treatments

Oxidative challenges. In vitro oxidative challenges were employed to evaluate the ability of the supplement to increase the antioxidant capacity of the muscles. The GS from the supplemented groups was better protected (P < 0.05) against lipid peroxidation in the two aqueous radical-generating systems compared with the GS from control diet groups (Fig. 1, A and B). Similarly, compared with control, GS from antioxidant-supplemented animals experienced less (P < 0.05) lipid peroxidation when exposed to the lipid phase (AMVN) oxidative challenge (Fig. 1C).


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  		Fig. 1.   Lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay] in gastrocnemius homogenate subject to in vitro oxidative challenges. A: iron challenge. B: xanthine oxidase challenge. C: 2,2'-axobis(2-4 dimethylvaleronitrile) challenge. Values are means ± SE; n = 8 rats per group. C, weight bearing with control diet; AO, weight bearing with antioxidant supplementation; HLU-C, hindlimb unweighting with control diet; HLU-AO, hindlimb unweighting with antioxidant supplementation. * Significant decrease compared with coresponding control diet group, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Overview of experimental findings. This is the first study to examine the effects of an antioxidant supplementation on disuse atrophy associated with HLU. The major findings of this study were that protein oxidation increased significantly (P < 0.05) with HLU. Muscle weight, body weight, and absolute force all decreased (P < 0.05), which is consistent with other HLU studies (9, 14). Although in vitro experiments demonstrated antioxidant protection against lipid peroxidation induced by different radical-generating systems, the antioxidant supplementation did not attenuate the disuse atrophy associated with HLU. Together, these data indicate that the antioxidant supplementation was not an effective countermeasure to the disuse atrophy associated with HLU.

Antioxidant supplementation and muscle antioxidant capacity. The antioxidant supplementation used in these experiments was modeled after supplementation used by others who demonstrated protection of a variety of tissues against oxidative injury (8, 27). The supplementation protocol used in this study resulted in increased antioxidant protection. This was supported by the finding that, after exposure to three ROS-generating systems, lower levels of TBARS were detected in skeletal muscle homogenates of supplemented animals compared with the control diet groups. Kondo et al. (20) and Appell et al. (3) demonstrated attenuation of the disuse atrophy associated with immobilization by supplementation with vitamin E. However, no attempts were made to evaluate whether the treatment elevated vitamin E levels or antioxidant protection in the muscle. The findings of this study, compared with those of Kondo et al. (20) and Appell et al. (3), suggest that the mechanism governing muscle wasting in immobilization and unweighting may be fundamentally different.

Protein oxidation and lipid peroxidation. To our knowledge, this is the first study to evaluate the level of protein oxidation and lipid peroxidation post-HLU. In the Sol, protein carbonyls, a measure of protein oxidation, were elevated in the HLU groups, and antioxidant supplementation did not attenuate this increase. The increase in protein carbonyls with the increased antioxidant capacity of the muscle would indicate that disuse muscle atrophy is not related to the antioxidant capacity of the muscle.

TBARS assay, a measure of lipid peroxidation, in the GS was not different between groups. Although Kondo et al. (20) demonstrated an increase in TBARS with immoblization, the data in this study do not indicate an increase in lipid peroxidation with HLU. This implies that the mechanisms for degradation and/or oxidation may be different between immobilization and unweighting, as suggested by the data of others (15, 16). Several studies have also demonstrated different results between TBARS and protein carbonyls (1, 21, 22). These studies and the present data would indicate that different mechanisms may be involved in protein and lipid oxidation, and further research would be warranted to investigate the possible mechanisms of protein and lipid oxidation with HLU.

Muscle weight and body weight. Muscle weights decreased by 44.5 and 29.2% for Sol and GS, respectively. This finding is consistent with the theory that HLU causes atrophy, with the greatest change observed in antigravity muscles such as the Sol (10). Antioxidant supplementation did not affect the degree of atrophy for Sol (45.8%) or GS (28.4%). This finding is not consistent with studies that have demonstrated attenuation of atrophy due to immobilization with the administration of vitamin E (3, 20).

The body weight gain of AO and C groups were similar (17.1 ± 5.2 and 12.7 ± 4.0 g, respectively). Furthermore, the body weight loss of HLU-AO (-19.7 + 4.4 g) and HLU-C (-18.6 ± 2.9 g) groups were in the range seen in other studies (25, 31). Average daily food comsumption was consistant between all groups (C, 17.2 ± 0.5 g; AO, 16.7 ± 0.6 g; HLU-C, 17.0 ± 0.4 g; HLU-AO, 16.9 ± 0.5 g). These data indicate that the muscle mass and body weight loss observed in the HLU groups was mainly related to unweighting conditions and that the antioxidant supplementation did not effect the amount of atrophy due to HLU.

Contractile properties. The mixed-fiber type GS was used to determine variations of in situ contractile properties. The absolute twitch and tetanic force production decreased for the HLU-C group (24.4 and 30.8%, respectively). These data are consistent with decreases in muscle mass with other HLU studies (10). The antioxidant supplementation did not attenuate the decreases in twitch (24.2%) and tetanic (30.1%) forces. Specific tension was unaltered as a result of HLU, which is consistent with the findings of others (9, 10).

RT1/2 is an index of calcium handling properties of the sarcoplasmic reticulum. A decrease in RT1/2 has been shown in the Sol with HLU (35). However, it has not been well characterized in muscles other than the Sol (10). The fact that GS RT1/2 did not change with HLU may be due to differences in recruitment patterns between the two muscles. Thus, if the Sol is used more in weight-bearing activities, the GS may not experience the same degree of unweighting as demonstrated in the slow Sol (10).

Protein concentration and water content. The loss of muscle mass, and the decreased force production that accompanies it, may be due to a decrease in muscle protein and/or water. Our data indicate that there is no change in protein concentration or percent water content. This agrees with the findings of others (3, 15, 25) and suggests that all components of muscle are lost in equal proportions.

Antioxidant supplementation and HLU. The mechanisms of atrophy may be fundamentally different between immobilization and HLU. Immobilization does not permit for movement, but does allow the muscle to generate an isometric force, whereas HLU permits a free range of motion but does not offer any resistance against which the muscle can generate any force. Limb position during immobilization appears to have a significant effect on atrophy. Jaspers et al. (15) demonstrated that immbolization with the Sol in a stretched position attenuated atrophy. This mechanism is thought to be due to increased internal tension in the muscle from the stretching. Thus, because the degree of stretch during immobilization may alter the adaptive responses, it is difficult to compare the responses between HLU and immobilization.

A fundamental question of our methodology was the degree to which the antioxidant supplementation provided benefit to the muscle. In contrast to measuring the amount of antioxidant in the muscle, we chose to determine the antioxidant capacity of the muscle by posing three oxidative challenges to GS muscle from each group (Fe2+ to generate the ·OH, xanthine oxidase to generate the O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, and AMVN to generate the peroxyl radical). Exposure to these systems caused lower levels of TBARS in the skeletal muscle homogenates of the antioxidant supplemented animals compared with control diet groups. Thus it appears that the antioxidant supplementation offered significant protection against oxidative stress.

If oxidation of proteins is not initiating the protein degradation, protein degradation may have been due to activation of other degradation pathways not involving free radicals. Berlett and Stadtmann (4) state that the accumulation of oxidized protein reflects not only the rate of protein oxidation but also the rate of oxidized protein degradation, which is also dependent on many variables, including concentrations of proteases that preferentially degrade oxidized proteins and numerous factors (metal ions, inhibitors, activators, and regulatory proteins) that affect their proteolytic activity. For example, oxidized forms of some proteins (e.g., cross-linked proteins) and proteins modified by glycation or lipid peroxidation products are not only resistant to proteolysis but, in fact, can inhibit the ability of proteases to degrade the oxidized forms of other proteins (4).

Alterations in calcium homeostasis may have an effect on protein degradation. Ingalls et al. (14) demonstrated that unloading of the Sol increased the resting calcium concentration by 36% above control. One mechanism that may allow the influx of calcium is the L-type calcium channel, which opens during passive shortening (41). Increased calcium can activate calpains, which seem to degrade structural proteins (e.g., titin) during muscle disuse (38). Once released, it is likely that the major contractile proteins, actin and myosin, are further degraded by the ATP-dependent ubiquitin pathway (38). Determination of the time course of calcium changes resulting from HLU would offer further information toward the hypothesis that alterations in calcium homeostasis may have an effect on protein degradation.

Summary and conclusions. This is the first study to examine the effects of antioxidant supplementation on disuse atrophy associated with HLU. An increase in protein oxidation, indicated by an increased level of protein carbonyls, was demonstrated with HLU. Changes in muscle weight, body weight, and contractile properties were consistent with exposure to HLU. Antioxidant supplementation did not attenuate the disuse atrophy resulting from HLU. The use of oxidative challenges to the muscle illustrated that there was an increased antioxidant protection. These data indicate that the antioxidant supplementation was not an effective countermeasure to the disuse associated with HLU.


    FOOTNOTES

Address for reprint requests and other correspondence: S. L. Dodd, PO Box 118205, Univ. of Florida, Gainesville, FL 32611 (E-mail: sdodd{at}hhp.ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 23, 2002;10.1152/japplphysiol.00511.2002

Received 12 June 2002; accepted in final form 22 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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