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Biphasic Stress Response in the Soleus during Reloading after Hind Limb Unloading


Medicine & Science in Sports & Exercise: April 2012 - Volume 44 - Issue 4 - p 600–609
doi: 10.1249/MSS.0b013e31823ab37a
Basic Sciences

Introduction Extreme disuse and spaceflight elicit rapid skeletal muscle atrophy, accompanied by elevated proinflammatory signaling and impaired stress response proteins (e.g., heat shock proteins (HSP), insulin-like growth factor 1 (IGF-1)). Recovery of muscle mass is delayed during the early stage of reloading after prolonged unloading, with a concomitant impairment of HSP70 and IGF-1. We postulated that proinflammatory signaling and stress response alterations would characterize early and late phases of signaling during reloading.

Methods Twenty-four adult SD rats were divided into the following groups: controls, 28 d of hind limb unloading (HU), HU + early (7 d) reloading (HU-R7), and HU + late (28 d) reloading (HU-R28).

Results Soleus mass decreased (−55%) with HU and remained depressed (−41%) at HU-R7. Nuclear factor κB activation and oxidative stress were elevated with HU and remained high during reloading. HU elevated inducible nitric oxide synthase and returned to baseline during reloading, whereas 3-nitrotyrosine did not increase with HU and peaked at HU-R7. HU depressed levels of HSP25 phosphorylation at Ser82 and IGF-1. Although p-HSP25 and Akt phosphorylation (Ser473) recovered during early reloading, HSP70, heat shock factor 1, and IGF-1 remained depressed. HSP70, heat shock factor 1, and IGF-1 recovered, whereas p-Akt and 3-nitrotyrosine decreased to control levels at HU-R28.

Conclusions Reloading elicited an early phase characterized by elevated nuclear factor κB activation, 3-nitrotyrosine, p-HSP25, and p-Akt levels and a delayed phase with recovery of HSP70, IGF-1, and muscle mass. We conclude that the reloading phenotype in skeletal muscle is expressed in two distinct phases related to (a) pro-inflammatory signaling and (b) muscle mass recovery.

1Texas A&M University, Redox Biology and Cell Signaling Laboratory, College Station, TX; and 2Department of Mechanical Engineering, University of Houston, Houston, TX

Address for correspondence: John M. Lawler, Ph.D., Redox Biology & Cell Signaling Laboratory, Department of Health and Kinesiology, Texas A&M University, College Station, TX 77843-4243; E-mail:

Submitted for publication March 2011.

Accepted for publication September 2011.

Mechanical unloading during disuse (e.g., bed rest, spaceflight) elicits substantial muscle atrophy and weakness, as typical limb loading is removed (4). Unloading-induced muscle atrophy is a function of increased protein degradation, coupled with decreased protein synthesis (4,46). Potential signaling pathways leading to muscle atrophy include oxidative stress, proinflammatory signaling, and impaired stress response, including heat shock proteins (HSP) and insulin-like growth factor (IGF-1) (1,6,14,24,25,27,38). Suppression or insufficient HSP and antioxidant enzymes elicit oxidative damage of proteins and lipids (20,24). Indeed, mechanical unloading elevates oxidative stress while disrupting antioxidant capacity, HSP, and IGF-1 (24,25,27). Since Kondo et al. (20) first demonstrated that casting increased oxidative stress in limb muscles, elevated oxidative stress has also been found in hind limb unloading (1,24,45) and mechanical ventilation (46) models.

Elevated oxidative stress could drive muscle atrophy via protein degradation pathways (e.g., ubiquitin–proteasome, caspase-3), apoptosis of myonuclei and satellite cells, and/or through nuclear factor κB (NF-κB) activation and FoxO3a (1,28,31). NF-κB is a transcription factor that regulates the expression of numerous inflammatory genes that encode for inducible nitric oxide synthase (iNOS), cytokines (e.g., tumor necrosis factor α), apoptosis, and ubiquitin pathway ligases (e.g., MuRF, atrogin-1) (6,8,14). NF-κB can be activated through a myriad of signaling including oxidative stress, altered mechanical stress, and withdrawal of stress response (e.g., HSP25, HSP70, IGF-1/Akt pathway) (22,25). NF-κB activation is activated during unloading and contributes to muscle atrophy via elevating protein degradation (6,14), and inhibition of NF-κB or genetic ablation of the p50 subunit gene attenuates atrophy (6,14). Accumulating evidence indicates that prooxidant and proinflammatory signaling, including activation of NF-κB, contribute to unloading-induced atrophy particularly when combined with impaired stress protein levels (1,8,14,20,24,25,27,31,38,43).

Interestingly, NF-κB DNA-binding activity also responds to increased mechanical strain and exercise (20,43) as well. Because dynamic changes in mechanical stress are an integral challenge in maintaining of skeletal muscle homeostasis, either an increase or a decrease in mechanical loading may stimulate remodeling. Therefore, we hypothesize that reloading, in addition to mechanical unloading, will stimulate significant activation of NF-κB.

It is possible that muscle wasting in response to mechanical unloading is related to up-regulation of iNOS and NF-κB via oxidative stress. Previously, Vaziri et al. (44) demonstrated that hind limb unloading increased iNOS levels in the rat aorta, heart, kidney, and brain. iNOS may promote the inflammatory response and muscle fiber necrosis during early reloading after hind limb suspension (29). iNOS up-regulation has also been linked with oxidative stress, protein degradation, and muscle wasting observed with chronic heart failure, cancer, acquired immunodeficiency syndrome, and sepsis (5,11,27).

Withdrawal of HSP may permissively increase oxidative stress, upregulate NF-κB activation, and lead to muscle atrophy with unloading (16,27). HSP70, HSP25, and α,β-crystalline provide antioxidant protection, protect cytoskeletal integrity, and inhibit NF-κB activation (7,18,19,27). Soleus HSP70 and HSP25 levels indeed decreased with hind limb unloading (25,27). Moreover, HSP70 remained depressed in the soleus muscle during the early stages of reloading (25,27). Moreover, application of heat stress or transfection of HSP70 or HSP25 before hind limb unloading provided partial protection against unloading-induced muscle atrophy (27,37,38). HSP25 phosphorylation at Ser83 may also integrate with the IGF-1/Akt signaling to augment cell protection (4,26,32).

The early portion of reloading after prolonged mechanical unloading is characterized by muscle damage and inflammation (4,17,44). Although muscle mass recovery after short-term unloading (7–10 d) is rapid (7–9 d) (35), reloading when preceded by long-term hind limb unloading (>17 d) exhibits impaired recovery of muscle mass (7,17). We propose that recovery from prolonged unloading includes two distinct phases during reloading. First, an early reloading phase (7 d) will be characterized by elevated oxidative stress, NF-κB activation, iNOS, and HSP25 phosphorylation (p-HSP25) coupled with impairment of HSP70 and the IGF-1/Akt pathway. Second, delayed phase of reloading (28 d) will elicit recovery of HSP70 and IGF-1/Akt pathways, with normalization of oxidative stress, iNOS, and p-HSP25.

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Four-month-old male Sprague-Dawley adult rats (Harlan Sprague Dawley, Indianapolis, IN) were used in these experiments. Adult rats were chosen to avoid any confounding effects of development. Animals were housed and cared for in accordance with National Institutes of Health (NIH) policy (DHEW publication no. 85-23, revised 1985) and American College of Sports Medicine animal care standards and used with the approval of the University Laboratory Animal Care Committee. Rat chow and water were provided ad libitum, and the animals were maintained in a temperature-controlled room (23°C ± 2°C) with a 12-h light–12-h dark cycle.

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Hind limb unloading and reloading

The hind limb unloading model is the preferred ground model to mimic the mechanical and hydrostatic changes of spaceflight (4). Our unloading and loading periods as well as study design are based on the model used by Kasper et al. (17). Animals were randomly assigned to four groups: a normal weight bearing control group (CON, n = 6), 28 d of hind limb unloading (HU, n = 6), 28 d of hind limb unloading followed by 7 d of ambulatory reloading (HU-R7, n = 6), and 28 d of hind limb unloading followed by 28 d of ambulatory reloading (HU-R28, n = 6). Controls were euthanized at equivalent ages after the duration of the hind limb unloading period. HU procedure was performed as described previously (25). The hind limbs of the HU group were elevated to a spinal orientation of 45° above horizontal using flexible orthopedic tape wrapped around the proximal two-thirds of the tail. Hind limb elevation was adjusted so that the hind limb was suspended, whereas the forelimbs were in contact with the ground, and rats were free to ambulate around the entire range of the cage. Unloading for 28 d was chosen because it represents a prolonged period susceptible to impaired recovery of muscle mass upon reloading (17). Short-term reloading for 7 d after prolonged unloading (HU-R7) was chosen as a time point near peak skeletal muscle inflammation (24). The 28 d of long-term reloading (HU-R28) time point was selected to contrast with the short-term reloading period (HU-R7), and postinflammatory responses in postural muscles. Control rats were permitted to ambulate within their cages for 28 d.

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Tissue preparation and experimental design

Rats were anesthetized with sodium pentobarbital (50 mg·kg−1 i.p.) after the hind limb suspension period and weighed for all four experimental groups. Animals in the HU group were anesthetized while still suspended to avoid risk of reloading-induced injury. Preparation of hind limb muscles followed the procedure described by Lawler et al. (25). The soleus muscle was quickly extracted, weighed, and placed in ice-cold phosphate-buffered saline (PBS) at a pH of 7.4. Soleus muscles were then frozen in liquid nitrogen and transferred to a −80°C freezer for future analyses. The soleus was chosen because of its recruitment as a postural muscle, because of its high percentage of slow-twitch fibers, and because of its consistent atrophy with unloading during spaceflight and bed rest.

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Western immunoblot analysis

Soleus samples were minced and homogenized in a manner adapted from previous studies (25). Tissues were further minced into fine pieces, then homogenized (20:1 w/v) in ice-cold (4°C) lysis buffer solution (pH 7.4) containing the following: 20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% Igepal-CA630, 1 mM MgCl2, 0.1 mM dithiothreitol, 0.5 mM EDTA, 0.1 mM EGTA, and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). Minced muscle samples were homogenized (20:1 w/v) using a ground glass on a ground-glass homogenizer (Bellco Biotechnology, Vineland, NJ) at 4°C and then centrifuged at 3000g (4°C), and the supernatant was removed for soluble protein analysis. The pellet was resuspended (9:1 v/v) in buffer containing 20 mM HEPES-free acid, 20 mM HEPES Na salt, 350 mM NaCl, 10% glycerol, 1 mM MgCl2, and 0.5 mM EDTA and centrifuged at 12,000g for 30 min at 4°C. The supernatant was kept for nuclear assay analysis. Protein was determined via the Bradford technique per manufacturer’s specifications for a Pierce-Endogen kit.

Protein expression for 4-hydroxy-2-nonenal (4-HNE), iNOS, HSP70, heat shock factor-1 (HSF-1), HSP25 phosphorylation at Ser82 (p-HSP25), IGF-1, and Akt phosphorylation at Ser473 (p-Akt) was determined by Western immunoblot analysis. Separating gel (375 mM Tris-HCl, pH 8.8, 0.4% sodium dodecyl sulfate [SDS], 10% acrylamide) and stacking gel (125 mM Tris-HCl, pH 6.8, 0.4% SDS, 10% acrylamide monomer) solutions were made, and polymerization was then initiated by N,N,N′,N′-tetramethylethylene diamine and ammonium persulfate. Separating and stacking gels were then quickly poured into a Bio-Rad Protein III gel-box (Bio-Rad, Hercules, CA). Proteins (30 μg) from skeletal muscle homogenates in the sample buffer (Tris pH 6.8 with 2% SDS, 30 mM dithiothreitol, and 25% glycerol) were then loaded into the wells of the 10% polyacrylamide gels and electrophoresed at 150 V. The gels were then transferred (30 V) overnight onto a nitrocellulose membrane (Bio-Rad). Membranes were blocked in 5% nonfat milk with 0.1% Tween-20 in PBS for 6 h. After blocking, membranes were incubated at room temperature for 12 h in PBS with the appropriate primary antibodies: iNOS (1:2500; BD Transduction Laboratories, Lexington, KY), 4-HNE (1:1000; Calbiochem, La Jolla, CA), 3-nitrotyrosine (3-NT; Cayman Chemical, Ann Arbor, MI), HSP25 phosphorylation at Ser82 (1:1000; Cell Signaling Technology, Beverly, MA), HSP70 (1:1000; StressGen, Victoria, Canada), HSF-1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), IGF-1 (1:1000; Upstate, Lake Placid, NY), nNOS (1:200; Cayman Chemical), and Akt phosphorylation at Ser473 (1:1000; Calbiochem). After three washings using PBS with 0.4% Tween-20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies in PBS at room temperature for 90 min. After three washes in PBS with 0.4% Tween-20, an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ) was used for visualization. Densitometry and quantification were performed using a Kodak film cartridge (Sigma-Aldrich, St. Louis, MO), HP scanner interfaced with a microcomputer, and the NIH ImageJ software program. Equal amounts of protein were loaded in each well (30 μg) as determined via Bradford analysis. To confirm equal loading and transfer consistency of muscle protein, Ponceau S staining was performed for each membrane before the blocking step, and the membrane was used if lane stains were congruent in density of staining, demonstrating equal loading of protein among lanes. As an additional control, the lane background readings were subtracted from each lane’s protein blot density reading before quantifying protein blots. Blots were quantified as the pixel density (subtracted from background) × area and then equated per microgram of protein. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an additional loading control, where membranes were stripped, reprobed for GAPDH antibody (RGM2; Advanced Immunochemical, Long Beach, CA) and by stripping the blots from the membranes and reprobing membranes. GAPDH protein levels were quantified to ensure less than a 10% deviation among lanes and were not statistically different among groups.

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NF-κB DNA-binding activity

We used a sensitive high throughput ELISA technique that is specific for the activation of NF-κB (34). This ELISA kit (Active Motif, Carlsbad, CA) contains a 96-well plate with the immobilized oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′). Biotinylated double-strand probe containing the consensus NF-κB binding sequence for p65 were fixated on streptavidin-coated microplate wells. Cell lysate samples and nuclear fractions were prepared and assayed (in triplicate) in accordance with the manufacturer’s instructions, with incubation performed at 25°C for 60 min. Next, wells were incubated with rabbit anti–NF-κB antibodies for 60 min. After three washings with PBS, a horseradish peroxidase-conjugated anti-rabbit secondary antibody was incubated in the wells for 60 min. Colorimetric development was quantified via a microplate reader (Molecular Devices, Sunnyvale, CA) set at an absorbance wavelength of 450 nm with a reference wavelength of 655 nm.

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Statistical analysis

One-way ANOVA with Fisher LSD post hoc were conducted to determine the existence of mean differences among controls, hind limb unloaded groups, 7 d of reloaded groups, and 28 d of reloaded groups. The level of significance was set at P < 0.05.

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Mean ± SD body masses were as follows: CON = 445.5 ± 15.3 g, HU = 424.3 ± 28.9 g, HU-R7 = 478.2 ± 49.4 g, and HU-R28 = 492.8 ± 24.7 g. There was no significant loss in body mass during prolonged unloading, although body mass was higher at the end of the reloading period. Mean soleus mass was significantly lower after 28 d of hind limb unloading compared with controls (−55%; Fig. 1). However, 7 d of reloading did not result in a full recovery of soleus mass to control levels. The soleus mass after 7 d of short-term reloading (122.4 ± 12.8 mg) remained significantly depressed (−41%) when compared with CON (169.4 ± 9.5 mg) but was greater than hind limb unloaded (76.5 ± 7.9 mg) rats. In contrast, after 28 d of long-term reloading, the soleus mass (164.3 ± 9.7 mg) was significantly higher than short-term reloading (7 d) but not significantly different from CON. Soleus mass–body mass ratios followed a similar pattern. Muscle mass–body mass (mean ± SD) was significantly lower in HU (172.3 ± 14.7 mg·kg−1) than in CON (379.9 ± 39.2 mg·kg−1). Partial recovery of soleus mass–body mass was noted after 7 d of reloading (233 ± 26.5 mg·kg−1), with muscle mass–body mass ratios not significantly different from CON after 28 d of reloading (365.2 ± 21.0 mg·kg−1).



Oxidative stress, as detected via 4-HNE adducts, increased significantly (+105%) in the soleus exposed to 28 d of hind limb unloading (Fig. 2A). Soleus 4-HNE levels remained elevated after 7 d of reloading but were significantly lower compared with the HU group. After 28 d of reloading, soleus 4-HNE adducts remained significantly higher (+66%) versus the CON group. NF-κB DNA-binding activity also increased significantly (+77%) after 28 d of hind limb unloading when compared with CON (Fig. 2B). Interestingly, NF-κB DNA-binding activity did not decrease in the HU-R7 group but was greatly elevated compared with controls (+442%) and HU (+306%). HU-R28 NF-κB values were approximately 30% higher than CON and 70% lower than HU but not included in Figure 2 because of a small sample size.



iNOS protein abundance in the soleus was significantly greater with HU (+143%) compared with the CON group (Fig. 3A). In contrast with NF-κB activity, iNOS levels were sharply reduced by 7 d of reloading (HU-R7) to a level 90% below the CON group. Similarly, low levels of iNOS protein content were evident during 28 d of long-term reloading (HU-R28), 70% lower than controls. 3-NT, a marker of nitrosative stress, was not altered in the soleus by hind limb unloading (Fig. 3B). In contrast with iNOS, 3-NT levels were significantly elevated after 7 d of reloading (HU-R7) compared with CON (+375%) and hind limb unloading (HU). After 28 d of reloading (HU-R28), 3-NT levels in the soleus decreased significantly, returning to CON levels. Because nNOS may be a contributor to the spike in 3-NT during the early stage or reloading, we measured nNOS protein levels in CON, HU, and HU-R7 groups. Indeed, nNOS protein abundance decreased during hind limb unloading (−38%) but recovered remarkably during the early (+41%) and late (+62%) stages of reloading to levels significantly above HU (Fig. 3C).



Hind limb unloading significantly decreased (−92%) HSP25 phosphorylation at Ser82 in the soleus (Fig. 4A). In contrast, both HU-R7 (+260%) and HU-R28 (+93%) resulted in elevated p-HSP25 compared with the CON group. Finally, p-HSP25 values were significantly lower after 28 d of reloading (HU-R28) compared with 7 d of reloading. Soleus HSP70 protein expression decreased significantly with hind limb unloading (−38%) (Fig. 4B). Early stages of reloading (HU-R7) resulted in a further depression (−65%) of HSP70 protein expression compared with CON. However, HSP70 protein abundance returned to CON levels after 28 d of reloading. HSF-1, the upstream transcription factor that regulates HSP70 and HSP25 expression, displayed a pattern with unloading and reloading that was similar to HSP70 (Fig. 4C). HSF-1 protein expression was significantly lower (−32%) with hind limb unloading in the soleus. Furthermore, HSF-1 levels decreased even further (−68%) after 7 d of reloading (HU-R7) compared with controls. HSF-1 then returned to CON levels only after long-term (28 d) reloading.



Hind limb unloading significantly reduced IGF-1 protein expression (−62%) in the soleus as expected (Fig. 5A). Surprisingly, soleus IGF-1 protein expression did not increase in the early stages of reloading (HU-R7) but remained depressed at levels significantly lower (−67%) than those at CON. IGF-1 protein expression demonstrated marked recovery and elevation by 28 d of reloading, to levels that were significantly higher than HU (+208%) and early reloading (HU-R7) (+255%). Akt phosphorylation at Ser473 is thought to be a cell signaling event downstream of IGF-1 and triggered by mechanical stress and insulin signaling (31). In the current study, phosphorylation of Akt was not significantly altered by hind limb unloading in the soleus. However, a very large increase (+530%) in Akt phosphorylation at Ser473 was observed after 7 d of reloading (HU-R7; Fig. 5B), in contrast with IGF-1 expression. p-Akt returned to controls levels by 28 d of reloading.



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The major and novel findings of our study include the following observations. Both prolonged unloading and subsequent reloading resulted in a large up-regulation of soleus NF-κB DNA-binding activity. Similarly, oxidative stress peaked during prolonged hind limb unloading but remained elevated during short (7 d) and long-term (28 d) reloading. Although iNOS protein expression increased markedly in the soleus as a result of hind limb unloading, iNOS levels surprisingly returned to control levels with short- and long-term reloading. In contrast, 3-NT levels were reduced during hind limb unloading but were markedly elevated during early reloading. HSP25 phosphorylation (Ser82) decreased with prolonged unloading, while increasing during short-term reloading, then returning to control levels after 28 d of reloading. HSP70, transcription factor HSF-1, and IGF-1 remained depressed during short-term reloading but then recovered back to control levels with long-term reloading as muscle mass recovered back to preunloading levels. In contrast, Akt phosphorylation (Ser473) was greater with short-term reloading but decreased back to baseline after 28 d of reloading. A discussion of the principal findings follows.

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Oxidative stress and NF-κB signaling during unloading and reloading

Our data clearly indicate that both mechanical unloading and reloading of the rat soleus can trigger NF-κB activation. Indeed, NF-κB DNA-binding activity was substantially greater during reloading than unloading. To our knowledge, these are the first data to indicate that NF-κB activation increases with early reloading after prolonged unloading in skeletal muscle. Previously, NF-κB DNA-binding activity had been found to be elevated in response to hind limb unloading (14,38) and was linked with increased protein degradation via the ubiquitin–proteasome system (14). Furthermore, NF-κB inhibition via transgenic means attenuated elevation of protein degradation and muscle fiber atrophy as a result of hind limb unloading (6,14). Whereas these findings may seem paradoxical, stretch and acute exercise have also been found to stimulate NF-κB activation (15,21). Although the precise upstream mechanisms that increase NF-κB activation during reloading have not been elucidated, based on our findings and the literature, several possible explanations exist. First, oxidative stress, a potential upstream activator of NF-κB activation (30), is elevated during both unloading and reloading (1,25) (Fig. 2). In addition, elevated mechanical stress and strain via stretch may activate NF-κB activation (15,21). Muscle damage and inflammation upon reloading can also stimulate activation of NF-κB as well (25). Moreover, withdrawal of protective stress proteins in soleus during reloading observed here and previously (28) (Figs. 4 and 5) can also elevate NF-κB activation (25). It is possible that reloading-induced activation of NF-κB could promote remodeling and early growth by regulating protein turnover and satellite cell activation (14,43). Therefore, our data are consistent with the postulate that adaptations in response to either a decrease or an increase in mechanical loading, which require muscle remodeling, involve activation of NF-κB as a central regulator. More work is needed regarding the upstream regulation of NF-κB during muscle reloading by performing a detailed temporal analysis of oxidative stress marker expression and whether these markers correlate to the magnitude of reloading duration.

Higher levels of oxidative stress are a consistent finding for unloading and reloading (1,24,25) (Fig. 2). Recently, Xu et al. (45) reported that mitochondrial production of superoxide anions increases as hind limb unloading duration increases. Several physiological roles for oxidative stress have been suggested including activation of NF-κB and proteolytic systems including calpains, caspase-3, serine protease, and the ubiquitin–proteasome system, all exquisitely redox sensitive (1,6,14,24,25,28,30,33,44). Lipid peroxidation products, specifically 4-HNE, may also suppress the cell cycle and satellite cell proliferation (2). Our current and previous findings (24,25) are consistent with the hypothesis that elevated oxidative stress guides remodeling and triggers NF-κB activation during both hind limb unloading and reloading. Sources of elevated oxidative stress with muscle unloading and reloading are not fully elucidated but may include up-regulation of xanthine oxidase, NAD(P)H oxidase, uncoupling of NOS, nonheme iron, and impaired stress protection (antioxidant enzymes, HSP, IGF-1) (1,20,24,25,46).

An important source of ROS during mechanical unloading could be the mitochondria, particularly with prolonged periods of disuse. Mitochondrial dysfunction is believed to play a role in many muscle myopathies, including sarcopenia. Chronic or long-term inactivity, coupled with aging, may lead to insufficient MnSOD activity in skeletal muscle and heart (22,36). Exercise, chronic, or lifelong physical activity may upregulate MnSOD and provide protection (12,36). Recently, Xu et al. (45) demonstrated that mitochondrial superoxide anion production increases with both disuse (in a time-dependent fashion) and aging in skeletal muscle. Further, Powers et al. (30) demonstrated that an increase in mitochondrial ROS in the diaphragm during mechanical ventilation contributed to weakness. Additional studies are warranted to determine the identity and importance of ROS sources during mechanical unloading, including mitochondria.

Given that oxidative stress and NF-κB activity was elevated with reloading, in addition to unloading (24,25), we expected that iNOS protein levels would also be elevated in a similar manner in the soleus after reloading as well as HU. Indeed, iNOS protein expression was significantly upregulated during unloading (Fig. 3). This is consistent with elevated iNOS levels after hind limb unloading in the heart, kidney, and brain (44) and may reflect a proposed role in proteolysis and muscle atrophy (33). In contrast with our hypothesis, iNOS levels decreased during early unloading, indicating that iNOS was not coupled to or downstream of NF-κB activation. Further, iNOS was an unlikely candidate as a prooxidant during reloading of skeletal muscle. Although up-regulation of iNOS and oxidative stress have been associated with muscle wasting pathologies such as cachexia with acquired immunodeficiency syndrome and cancer (5), it is possible iNOS may provide a protective role, even limiting wasting and apoptosis (9).

Surprisingly, the pattern of 3-NT adducts did not follow iNOS protein expression in response to either hind limb unloading or reloading. Hind limb unloading resulted in a decrease in 3-NT, whereas iNOS levels increased. In contrast, 7 d of reloading increased 3-NT levels, whereas iNOS levels decreased. These findings suggest that tyrosine nitration by reactive nitrogen species could be related to unloading and reloading patterns for other NOS isoforms, such as nNOS. Indeed, our data indicated that up through the early stage of reloading, 3-NT nNOS abundance in the soleus, suggesting nitrosative stress is highest then. Given that 3-NT adducts returned to control levels, tyrosine nitration may reflect a reversible growth or repair signaling mechanism in response to elevated muscle loads and/or damage rather than simply pathology (3). Criswell et al. demonstrated that NOS may be a key regulators of muscle hypertrophy and fiber-type shift when a dynamic increase in muscle loading is imparted, using the muscle ablation model of compensatory hypertrophy (39,41). Recent data indicate that nNOS levels and location may play a role in nitrosative stress and muscle atrophy during hind limb unloading (43). Clearly, further research is needed to test the hypotheses that nNOS translocation is more important than iNOS up-regulation in eliciting muscle atrophy.

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Stress response signaling during unloading and reloading

As expected stress response markers including (p-HSP25, IGF-1, p-Akt, HSP70) decreased or trended lower in the soleus with hind limb unloading in a manner similar to that seen previously (25,27). Moreover, Seo et al. (40) used proteomic analysis to demonstrate that HSP are suppressed with 3-wk of hind limb unloading and yet recover after 2 wk. Selsby and Dodd (37) showed that heat stress provides partial protection against oxidative stress and reduced muscle mass. Recently, Senf et al. (38) demonstrated that HSP70 transfection provided significant protection against atrogin-1 and MuRF-1 up-regulation and atrophy. Together, these findings are consistent with notion that withdrawal of HSP and IGF-1 play permissive roles in elevating oxidative stress and NF-κB activation, while promoting muscle atrophy (17,25,27,37).

Stress response signaling in the soleus with reloading clearly revealed a biphasic pattern with distinct early (3-NT, p-HSP25, p-Akt) and late responders (HSF-1, HSP70, IGF-1). Increased phosphorylation of HSP25 at Ser82 during the early (7 d) phase of reloading seems to be a function of total HSP25 levels, which also increase early during reloading (25), whereas p-HSP25 returned to control levels during the later phase of reloading. Huey (13) reported that overloading, using the synergistic ablation model, caused an up-regulation of soluble HSP25 and p-HSP25 (Ser82) at 1 and 7 d after overloading. It has been postulated that phosphorylation of HSP25 may play a role in modulating oxidative stress, stabilizing the cytoskeletal–extracellular matrix interface, and regulating growth and remodeling during early phases of reloading or hyperloading (13,19,25). Our current and recent data (Fig. 4 [25]) suggest that HSP25 phosphorylation at Ser82 may be independent of HSF-1 signaling, whereas HSP70 seems to be tightly linked with HSF-1 protein expression.

Contrary to our expectations, Akt phosphorylation at Ser473 was not reflective of biphasic changes in IGF-1 during hind limb unloading or reloading, but in fact, changes in Akt phosphorylation were inversely related to IGF-1 (Fig. 5). Akt phosphorylation at Ser473 increased more than fivefold during the early phase of reloading, while returning to control levels during the late (28 d) reloading phase. These data are thus inconsistent with the canonical view whereby alterations in Akt phosphorylation in response to changes in loading are downstream of IGF-1/PI-3K signaling (19). Interestingly, reloading patterns for Akt phosphorylation observed in this study were similar to those observed for HSP25 phosphorylation at Ser82 (Figs. 4 and 5), suggesting a potential cooperative relationship between phosphorylation of both Akt and HSP25 or altered sensitivity of IGF-1 binding proteins, such as IGFBP-3 (31). Additional research is warranted to focus on the potential crosstalk between HSP25 and Akt signaling.

Because changes in HSP70 and HSF-1 protein levels mirrored each other tightly during unloading and reloading, this is consistent with the role of HSF-1 as a robust upstream regulator of HSP70 protein expression during reloading as well as unloading of skeletal muscle. Our data indicate a potential role for HSP70 and IGF-1 in contributing to the later phase of muscle mass recovery (28 d), after prolonged hind limb unloading, but not the initial phase of reloading.

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Potential limitations of the study include potential fiber-type shifts in the soleus muscle with hind limb unloading and reloading. Although some fiber-type shifts from slow to fast myosin isoforms in the soleus occur with unloading, the soleus remains primarily a slow-twitch muscle, with these fibers seeing the greatest reduction in cross-sectional area. Fast-twitch fibers may exhibit higher levels of oxidative stress because they have lower levels of antioxidant enzymes (23). In addition, HSP reduction could in part be related to fiber-type differences (10) as a result of unloading. Thus, it cannot be discounted that a portion of the impairment of stress protection during hind limb unloading and early reloading could be related to fiber-type shifts. Although there is no evidence in the literature that a shift from slow- to fast-twitch fibers contributes to muscle atrophy, fiber-type shift could clearly affect fatigue. The addition of other locomotor muscles such as the gastrocnemius and vastus lateralis in future experiments will expand our understanding of regulation of stress response during reloading in mixed and fast-twitch fibers.

In addition, future studies should focus on potential interventions to promote faster recovery of from damage and return of mass and contractile after prolonged periods of disuse or unloading. Our data from the early stage of reloading suggest that HSP70, IGF-1, and oxidative stress are prospective targets of countermeasure development to promote recovery of mass and force production.

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Summary and perspectives

In summary, prolonged hind limb unloading was characterized by decreases in soleus mass, elevated oxidative stress, increased NF-κB activation, and increased iNOS concomitant with reduced levels of HSP and IGF-1. However, signaling patterns with reloading were clearly biphasic with the early phase (7 d) of reloading characterized by partial recovery of soleus mass, elevation of oxidative stress, NF-κB activation, 3-NT, HSP25 phosphorylation at Ser82, and Akt phosphorylation at Ser473, whereas protein expression of HSP70 and IGF-1 remained depressed. The later phase (28 d) of reloading showed that soleus mass recovered close to control levels in concert with HSP70 and IGF-1 levels, whereas 3-NT, HSP25 phosphorylation, and Akt phosphorylation returned to control levels. These data indicate that countermeasure development targeting oxidative stress, HSP70, and IGF-1 may prove efficacious in promoting the recovery of muscle mass and strength and limiting injury in astronauts during reloading back to a gravitational environment.

Dr. Lawler received a grant from the NIH (AR054084) as part of support. Dr. Kwak received a 2007 Environmental and Exercise Physiology Section Predoctoral Gravitational Physiology Award (American Physiological Society).

Hyo-Bum Kwak, Jong-Hee Kim, Yang Lee, Jeffrey M. Hord, and Daniel A. Martinez have no conflicts of interest.

The results of the present study do not constitute an endorsement by the American College of Sports Medicine.

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