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J Appl Physiol 96: 1341-1348, 2004; doi:10.1152/japplphysiol.01015.2003
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RhoA expression during recovery from skeletal muscle disuse

J. M. McClung,1 R. W. Thompson,1 L. L. Lowe,2 and J. A. Carson1

1Department of Exercise Science, Integrative Muscle Biology Laboratory, School of Public Health, University of South Carolina, Columbia 29208; and 2Department of Biology and Physical Sciences, Benedict College, Columbia, South Carolina 29204

Submitted 22 September 2003 ; accepted in final form 21 November 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Functional overload and anabolic steroid administration induce signaling pathways that regulate skeletal muscle RhoA expression. The purpose of this study was to determine RhoA and associated protein expression at the onset of disuse and after a brief period of reloading. Male Sprague-Dawley rats were randomly assigned to cage control (Con), 3 days of hindlimb suspension (Sus), or 3 days of hindlimb suspension with 12 h of reloading (12-h Reload). The reloading stimuli consisted of 12 h of resumed normal locomotion after 3 days of hindlimb suspension. Plantaris muscle-to-body weight (mg/g) ratio decreased 17% from Con with Sus but returned to Con with 12-h Reload, increasing 13% from Sus. Sus decreased RhoA protein concentration 46%, whereas 12-h Reload induced a 24% increase compared with Sus. The ratio of cytosolic- to membrane-associated RhoA protein was not changed with either Sus or 12-h Reload. RhoA mRNA concentration was decreased 48% by Sus, and 12-h Reload induced a 170% increase from Sus. {beta}1-Integrin protein, a transmembrane protein associated with RhoA activation, was not altered by Sus but increased 155% with 12-h Reload. Although {beta}1-integrin mRNA was not altered by Sus, it increased 70% from Con with 12-h Reload. Rho family member Cdc42 protein associated with the muscle membrane was decreased 60% with Sus, and 12-h Reload induced a 172% increase compared with Sus. In conclusion, decreased RhoA protein expression and mRNA abundance are early adaptations to disuse but recover rapidly after normal locomotion is resumed.

hypertrophic signaling; muscle growth; muscle wasting; mechanotransduction; muscle atrophy

THE LOADING STATE PLACED ON skeletal muscle dramatically influences gene expression related to phenotype and growth regulation (4, 23). Weight-bearing activity is an essential prerequisite for maintaining skeletal muscle phenotype and physiological function. Skeletal muscle disuse induces mass loss, isomyosin profile shifts, and altered metabolic enzyme expression. The extent of these changes is related to the length of time the muscle is unloaded (14, 16, 44). Extended periods of disuse induce the specific loss of proteins associated with contraction and cytoskeletal structure (12, 43, 44). Skeletal muscle's sensing of the reduced load is likely responsible for at least some of the cellular signaling that alters muscle protein expression during disuse (20).

Transmembrane integrin proteins can integrate mechanical stimuli into intracellular signaling that regulates skeletal muscle gene expression (26). Membrane-associated integrin receptors exist as {alpha}- and {beta}-subunit heterodimers, which mediate extracellular matrix cell adhesion and focal adhesion complex formation. Proteins involved in focal adhesion mechanotransduction include focal adhesion kinase, paxillin, and RhoA (17, 20). Rho is a member of a low-molecular-weight phosphoprotein family of small GTPases, including Rac and Cdc42. RhoA association with the muscle membrane coincides with its activation (38). Cdc42 associates with the dystrophin-glycoprotein signaling complex (DGC) at the sarcolemma. Cdc42 expression and activity are decreased in atrophied muscle (11). RhoA- and Cdc42 GTPase-mediated signaling are involved in a variety of cellular processes, including cell migration (13), differentiation (40, 47), survival (2), actin cytoskeletal rearrangement (1), and DNA transcription factor activity (5, 41). Rho-GTPase expression is not solely regulated by mechanical stimuli. RhoA and Cdc42 are also regulated by growth factors and cytokines (29, 30). Ten days of tenotomy-induced disuse decreases RhoA activity in rat gastrocnemius muscle (11). Alterations in muscle loading can change both RhoA expression and localization (30). However, RhoA expression and activity in muscle recovering from disuse have not clearly been defined.

During skeletal muscle disuse and subsequent recovery, several stimuli must be integrated to provide a cellular response. Stimuli that can influence skeletal muscle gene expression include the muscle's loading state, circulating growth factors, circulating hormones, and inflammatory processes due to damage and injury. RhoA is either a primary effector or a potential modifier of many of these signaling pathways (30, 36), suggesting that RhoA signaling may be important for muscle gene regulation during recovery from disuse. The purpose of this study was to determine RhoA and associated signaling protein expression during the early onset of muscle disuse and after a brief period of recovery in rat plantaris muscle. It was hypothesized that RhoA, {beta}1-integrin, and Rho-family member Cdc42 expression would be decreased at the onset of disuse and that brief periods of reloading would induce their expression. Male rats were subjected to hindlimb suspension-induced disuse for 3 days and then a 12-h reloading stimulus. Plantaris muscle RhoA protein, mRNA, and activity were determined. In addition, {beta}1-integrin protein and mRNA, as well as the expression and activity of Cdc42 and p70s6k, were analyzed.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals and housing. Five-week-old male Sprague-Dawley rats (n = 20, ~125 g) were acquired from Harlan rodent colony (Indianapolis, IN). Animals were divided into three groups, which consisted of ground-based controls (n = 10), disuse induced by 3 days of hindlimb suspension (n = 5), and disuse induced by 3 days of hindlimb suspension plus 12 h of subsequent recovery (n = 5). Animals were housed individually, kept on a 12:12-h light-dark cycle, and given ad libitum access to normal rodent chow and water for the duration of the study at the fully accredited animal care facilities at the University of South Carolina, Columbia. All procedures were approved by the University of South Carolina Animal Care and Use Committee.

Hindlimb suspension-induced disuse. Disuse of the muscles of the hindlimb was performed as previously described (43, 44). Briefly, unanesthetized animals' tails were cleaned with rubbing alcohol and air dried, covered with a light coat of benzoin tincture, and dried with a hair dryer until tacky. Strips of elastoplast (Biersdorf, Norwalk, CT) adhesive bandage were applied to the proximal two-thirds of all sides of the tail and looped through a swivel attachment mounted above the cage and designed to allow the animal to move rotationally 360° with only the forelimbs able to come into contact with the cage floor. The animals were provided food and water ad libitum and were monitored daily for signs of lethargy or illness.

Reloading after hindlimb suspension. The recovery stimulus consisted of the removal of all suspension apparatus and the resumption of normal ambulation about the cage for 12 h after 3 days of hindlimb suspension-induced disuse.

Total RNA, total protein, and total DNA. Total muscle RNA, protein, and DNA were quantified by using the method of Fleck and Munro as previously described (18, 30). Total RNA was quantified by ultraviolet absorbance at 260 nm. Protein content was determined by standard Bradford assay (Bio-Rad, Hercules, CA), and total muscle DNA was quantified by a standard flourimetric assay (27). RNA, protein, and DNA are expressed as the concentration per milligram of muscle and total per whole muscle.

Crude protein extracts. Crude protein extracts were made as previously described (17, 20). Tissue was homogenized in Mueller buffer on ice with a Polytron homogenizer (Kinematica) using 3 x 15-s pulses at a low setting. Homogenates were fractionated into soluble and insoluble fractions by centrifugation, and the protein concentration was determined by Bradford assay (Bio-Rad) and aliquoted at -80°C until use for Western blotting.

Western blot analysis. Western blot analysis was performed as previously reported (6, 7, 30). Dose-response analysis for {beta}1-integrin (1:1,000), RhoA (1:1,000; M-106 and -119, respectively), and Cdc42 (1:200; BD Transduction Laboratories, 610928) demonstrated that 40 µg of crude protein extract gave signals in a linear range for quantification (data not shown). Secondary antibodies were visualized by chemiluminescence (Amersham Life Sciences) as per manufacturer instructions and quantified by densitometry scanning (Scion Technologies, Frederick, MD).

p70s6k Phosphorylation quantification. p70s6k Protein and phosphorylation levels were determined as previously described (3). Dose-response analysis for p70s6k (1:6, 500; C-18) demonstrated that 10 µg of crude protein extract gave signals in a linear range for quantification (data not shown). Relative phosphorylation amounts were determined as previously described (3) by summing the total amount of phosphorylated p70s6k and dividing that by the total p70s6k.

RhoA and Cdc42 localization. RhoA and Cdc42 protein localization was determined as previously described (30, 38). Briefly, muscle was homogenized in a complete lysis buffer on ice with a Polytron homogenizer (Kinematica). Crude soluble and insoluble fractions were separated by centrifugation, and the supernatant was transferred to a new tube and centrifuged to generate membrane and cytosolic fractions. The membrane pellet was resuspended in lysis buffer. Protein concentrations were determined by Bradford assay (Bio-Rad) and aliquoted at -80°C until use for Western blotting.

Total RNA isolation and complementary DNA synthesis. Total RNA was isolated by using TRIzol reagent (Life Technologies, Grand Island, NY) as per manufacturer's instructions. RNA concentration and purity were determined by ultraviolet spectrophotometry. Five micrograms of purified total RNA were used as a substrate for reverse transcription. Complementary DNA was reverse transcribed from RNA by using the superscript II kit (GIBCO-BRL, Rockville, MD) and priming with random hexamer, following the manufacturer's recommendations.

Northern blotting. Northern blot analysis for {beta}1-integrin mRNA abundance was performed as previously described (7, 8). Briefly, 10-15 µg of total RNA were fractionated on a denaturing 1% agarose gels and then transferred to a nylon membrane by capillary action. The rat {beta}1-integrin probe was a kind gift of Dr. R. J. Schwartz (Baylor College of Medicine, Houston, TX). Northern analysis probes were made by random priming and radiolabeled with 32P as previously described (7). Hybridization of the labeled probes with the RNA-containing membrane was performed as previously described (7, 8). Membranes were visualized by autoradiography (-80°C, 12 h) and quantified by densitometry scanning (Scion), obtaining an integrated optical density (IOD), which was used to calculate mRNA IOD per microgram of total RNA.

Competitive polymerase chain reaction. Competitive polymerase chain reaction (PCR) for RhoA mRNA abundance was performed as previously described (30, 32). Primers for RhoA used in reverse transcription PCR were as follows: forward primer, 5'-ACCAGTTCCCAGAGGTTTATGT-3'; reverse primer, 5'-TTTGGTCTTTGCTGAACACT-3'; composite, 5'-CCAGTTCCCAGAGGTTTATGTGAAGTCAAGCATTTCTGTCCAAA-3' (32). RhoA target (411 base pairs) and competitor (211 base pairs) were amplified for different cycle numbers (Fig. 3, A-C). The linear portion of the amplification curve for each transcript was defined by coamplification of duplicate tubes containing 10 pg of wild-type plasmid and 10 ng of competitor plasmid for different cycles. Wild-type plasmid (50 pg) was coamplified with decreasing amounts of competitor plasmid to establish linearity over a different range of concentrations. PCR reactions were fractioned by SDS-PAGE, stained with ethidium bromide, and quantified by densitometry.



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  		Fig. 3. Linear and equal efficiency of the amplification of RhoA target and competitor (comp). A: equal efficiency: RhoA target (411 bp) and competitor (211 bp) were amplified for different cycle numbers. PCR reactions were fractioned by SDS-PAGE, stained with ethidium bromide and quantified by densitometry. The integrated optical density (IOD) of each pair of bands was plotted against the cycle numbers. B: linearity: RhoA target was coamplified with a dilution series of RhoA competitor. C: representative gel electrophoresis of a competitive RhoA RT-PCR demonstrating the effects of RhoA competitor dilutions (50, 10, 5, and 1 pg).

 

Data analysis. Results are reported as means ± SE. All variables were analyzed by one-way ANOVA to determine significant main effects of treatment (P < 0.05). Post hoc analysis between treatments was performed by using independent t-tests (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Body weights, muscle wet weights, muscle wet weight-to-body weight ratio, protein concentration, and total protein. Body weight (g) did not change from control (Con; 161 ± 5 g), with 3-days of suspension disuse (Sus; 156 ± 1 g), or after 12 h of reloading (12-h Reload; 154 ± 4 g). Plantaris muscle weight decreased 20% (P < 0.001) from Con (148 ± 5 mg) with Sus (119 ± 7 mg) and remained decreased (P < 0.035) after 12-h Reload (132 ± 6 mg). Plantaris muscle-to-body weight ratio decreased 17% (P < 0.015) from Con with Sus but returned to Con with 12-h Reload, increasing 13% from Sus (P < 0.04; Table 1). Total protein (mg) did not change from Con with Sus or 12-h Reload treatments (Table 1). In addition, muscle protein concentration (µg/mg) did not change with Sus or 12-h Reload treatments (Table 1).


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  		Table 1. Plantaris muscle weight-to-body weight ratio, total protein, and protein, RNA, and DNA concentrations after 3 days of disuse and disuse with reloading

 

Total RNA and DNA abundance. Plantaris muscle RNA concentration (µg/mg) was significantly decreased by 3 days of disuse due to Sus (P < 0.006; Table 1) and returned to Con values after normal locomotion was resumed for 12 h (P = 0.092; Table 1). Total muscle RNA (µg) was significantly decreased (P < 0.001) from Con (297 ± 7 µg) by Sus (202 ± 9 µg) and remained decreased after 12-h Reload (251 ± 18 µg; P < 0.006). However, total RNA increased 24% with 12-h Reload compared with Sus (P < 0.01), demonstrating the sensitivity of total RNA to resumed activity in the plantaris muscle. Plantaris muscle DNA concentration (µg/mg) was not changed from Con with Sus or 12-h Reload (Table 1). In addition, total DNA was not changed from Con (78 ± 7 µg) with Sus (75 ± 11 µg) or 12-h Reload (63 ± 13 µg).

p70s6k Protein and relative phosphorylation. Western blot analysis revealed that neither Sus (1.2 ± 0.2 IOD) nor 12-h Reload (0.9 ± 0.1 IOD) resulted in significant changes in total p70s6k protein expression from Con (1.0 ± 0.3 IOD). The relative phosphorylation of p70s6k (expressed as the percentage of total p70s6k in the phosphorylated form; Fig. 1) decreased 52% (P < 0.001) with Sus and returned to Con values, increasing 123% (P < 0.001) with 12-h Reload.



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  		Fig. 1. p70s6k Protein expression and activation in disuse and recovering plantaris skeletal muscle. A: representative Western immunoblots of phosphorylated (pi) and nonphosphorylated p70s6k protein (pan) from suspended (Sus), 3-day suspended, 12-h reloaded (12-h Reload), and cage control (Con) treatments were separated by SDS-PAGE, transferred to nitrocellulose, and Western blotted. B: p70s6k activation is determined by the proportion of the hyperphosphorylated fraction of total p70s6k protein expression. Bars show the values of 3-day plantaris relative hyperphosphorylated protein fraction with Con (n = 10), Sus (n = 5), and 12-h Reload (n = 5) plantaris skeletal muscle. Values are means ± SE. *Significantly different from Con (P < 0.05). {dagger}Significantly different from Sus (P < 0.05).

 

RhoA expression and localization. Western blot analysis revealed that RhoA protein expression decreased 46% (P < 0.001) from Con with Sus (Fig. 2B). RhoA expression remained decreased (P < 0.002) from Con with 12-h Reload but increased 24% (P < 0.05) from Sus. There was considerable variation in the results for the determination of RhoA localization in the plantaris muscle. Due to this variation, RhoA was not altered (P < 0.087) from Con (1.0 ± 0.1 IOD) with Sus (0.79 ± 0.1 IOD). There was also no significant effect of 12-h Reload (0.90 ± 0.12 IOD) on membrane-associated fractions of RhoA protein compared with Con. There was also no effect (P < 0.089) of Sus (0.83 ± 0.1 IOD) on cytosolic fractions of RhoA protein compared with Con (1.0 ± 0.1 IOD). There was no effect of 12-h Reload (0.89 ± 0.1 IOD) on cytosolic fractions of RhoA protein. RhoA activity, expressed as a membrane-to-cytosolic ratio (38), displayed no significant differences from Con with Sus or with 12-h Reload (Fig. 2C). Competitive quantitative PCR (Fig. 3) revealed that RhoA mRNA abundance significantly (P < 0.01) decreased 48% with Sus (Fig. 4). The 12-h Reload resulted in a 170% increase in RhoA mRNA from Sus levels (P < 0.03; Fig. 4).



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  		Fig. 2. RhoA protein expression and localization in disuse and recovering rat plantaris muscle. A: fold induction of RhoA protein expression and representative Western immunoblot and Ponceau stained nitrocellulose membrane (right) of total (pan) RhoA expression. B: fold induction RhoA localization. RhoA activity is determined by the portion of the total fraction associated with the muscle membrane (38). Values are expressed as the membrane-to-cytosol ratio (Memb:Cyt) and representative Western immunoblot (right) of fractionated membrane and cytosolic RhoA. Values are means ± SE and normalized to Con. *Significantly different from Con (P < 0.05). {dagger}Significantly different from Sus (P < 0.05).

 


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  		Fig. 4. RhoA mRNA abundance decreases at the onset of hindlimb suspension-induced disuse in rat plantaris muscle and recovers at the onset of recovery due to reloading. RhoA mRNA abundance was quantified by quantitative PCR (see Fig. 3). Sus (n = 5), 12-h Reload (n = 5), and Con (n = 10) treatments are presented. Values are means ± SE, normalized to cage (Con). *Significantly different (P < 0.05) from Con. {dagger}Significantly different from Sus (P < 0.05).

 

{beta}1-Integrin expression. Western blot analysis revealed that {beta}1-integrin protein concentration was not different from Con values with Sus (Fig. 5A) in the plantaris muscle. The 12-h Reload, however, resulted in a 155% increase in {beta}1-integrin protein expression from Sus (P < 0.001; Fig. 5A). Northern blot analysis revealed no change in {beta}1-integrin mRNA abundance with Sus in the plantaris muscle (Fig. 5B). The 12-h Reload resulted in a 70% increase from Con (P < 0.001) and a 52% increase in {beta}1-integrin mRNA abundance from Sus (P < 0.001).



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  		Fig. 5. {beta}1-Integrin protein and mRNA abundance with hindlimb suspension-induced atrophy and reloading after atrophy. A: representative Western blot and Ponceau stained nitrocellulose membrane (right) and fold induction of {beta}1-integrin protein expression in Sus, 12-h Reload, and Con rat plantaris muscle. B: fold induction {beta}1-integrin mRNA abundance in treated plantaris skeletal muscle. Representative Northern blot (right) of plantaris {beta}1-integrin and 18S mRNA abundance. {beta}1-Integrin mRNA abundance was quantified by Northern blot analysis. Ten to fifteen micrograms of total RNA were fractionated on a denaturing 1% agarose gels (1 x 3 MOPS, 6.7% formaldehyde) and then transferred to a nylon membrane by capillary action. The 18S and 28S mRNA were visualized by ethidium bromide staining of the agarose gel. Visualization of {beta}1-integrin mRNA was performed by autoradiography (-80°C, 12-h), and {beta}1-integrin and 18S were quantified by densitometry scanning. Values are means ± SE, corrected for 18S (Northern) and normalized to Con values. *Significantly different (P < 0.05) from Con. {dagger}Significantly different from Sus (P < 0.05).

 

Cdc42 protein expression and localization. Western blot analysis revealed that total Cdc42 protein expression was not altered from Con by Sus but increased 52% from Sus with 12-h Reload (Fig. 6A). Cdc42 protein localized to the membrane fraction of plantaris skeletal muscle after Sus was decreased 60% from Con (P < 0.004; Fig. 6B). The 12-h Reload returned the fraction of Cdc42 protein localized to the plasma membrane to Con values, resulting in a 172% increase in its localization to the plasma membrane compared with Sus (P < 0.005; Fig. 6B).



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  		Fig. 6. Cdc42 protein expression and localization in disuse and recovering rat plantaris muscle. A: fold induction of Cdc42 protein expression and representative Western immunoblot and Ponceau stained nitrocellulose membrane (right) of total Cdc42 expression. B: fold induction of Cdc42 membrane localization and representative Western immunoblot (right) of fractionated membrane localized Cdc42. Values are means ± SE and normalized to Con. *Significantly different from Con (P < 0.05). {dagger}Significantly different from Sus (P < 0.05).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Although RhoA expression is altered in skeletal muscle subjected to functional overload and tenotomy, its expression during the early onset of hindlimb suspension disuse and after a brief return to normal locomotion is not known. Therefore, we investigated RhoA expression after Sus-induced muscle disuse and a subsequent 12-h period of normal locomotion. This study's primary finding is that decreased mechanical loading reduces rat plantaris muscle RhoA expression, and a brief return to normal loading attenuates this decrease. Repressed RhoA expression during disuse was regulated, in part, by pretranslational mechanisms. The pattern of RhoA expression at the early onset of disuse could be related to several signaling pathways that are not associated with muscle mass regulation. RhoA expression was decreased before changes in total muscle protein content. The loss of muscle mass without a corresponding loss of muscle protein implies that muscle fluid shifts occurring with hindlimb suspension may be responsible for initial muscle weight fluctuations (33). Longer periods of muscle disuse have also demonstrated repression of RhoA and associated signaling. Ten days of tenotomy-induced disuse decreases gastrocnemius muscle RhoA, H-Ras, and Cdc42 activity (11).

Decreased RhoA expression occurs concurrently with the downregulation of protein synthetic capacity at the onset of muscle disuse. Rho-associated signaling can activate protein synthesis through phosphatidylinositol 3-kinase and phospholipase D1 activation, which can act on the mammalian target of rapamycin (mTOR) (9, 42). The p70s6k is involved in the regulation of protein synthesis initiation and is activated by mTOR-associated signaling (9). p70s6k Phosphorylation state decreases with muscle disuse and increases after a 3-day recovery period (10, 25). However, the present study has extended these findings by demonstrating that the decreased p70s6k is rapidly reversed by the resumption of normal locomotion for 12 h. The plantaris muscle total RNA pool decrease with disuse also points to a reduction in ribosomal synthetic capacity. Decreased ribosomal RNA combined with altered translation initiation and decreased mRNA availability are important regulatory points for producing muscle atrophy at later time points of muscle disuse (21, 22). Repressed Rho signaling through phospholipase D1 and phosphatidylinositol 3-kinase signaling to mTOR could contribute to this decrease (9, 42).

Focal adhesion complex-associated transmembrane receptors appear to have differentially regulated expression compared with associated cytosolic proteins. The brief period of plantaris muscle disuse in the present study did not alter {beta}1-integrin expression. However, {beta}1-integrin expression induction during muscle recovery from disuse demonstrates the importance of cytoskeletal reorganization for these processes (45). In atrophying skeletal muscle, focal adhesion-related protein expression is decreased, which has been interpreted to demonstrate their load-dependent expression (20). Signaling linkage between the extracellular matrix and the cytoskeleton occurs at sites of DGC-Cdc42 association (11). Cdc42 localization at the sarcolemma decreases after 3 days of disuse. However, Cdc42 membrane association increases rapidly during recovery. Cdc42 regulates cell adhesion activation of Akt- and extracellular-regulated kinases (13) through membrane localization at DGC. Cellular hypertrophy occurs through Akt-mediated mTOR induction of protein synthesis and insulin signaling-mediated Akt inhibition of protein degradation (15). In addition, mechanical tension is critical for Akt induction in fast-twitch rat skeletal muscle (35). Androgen receptor interactions with the p85{alpha} catalytic subunit of phosphatidylinositol 3-kinase and Src regulates androgen responsive Akt activation at the skeletal muscle membrane (39). This demonstrates the potential of plasma membrane signaling to mediate non-genomic pathways associated with cellular growth and survival. Akt induction by Cdc42 could regulate protein synthesis with altered mechanical tension during disuse and subsequent reloading.

Although members of the same GTPase family, RhoA and Cdc42 regulate different cellular functions. Rho GTPases have been linked to mechanical signaling and cytoskeletal organization through integrin and DGC receptors at the sarcolemma (11, 46). However, they also regulate other signaling pathways related to muscle regeneration or regrowth processes. Satellite cell activation is critical for postnatal skeletal muscle growth (34). Satellite cell activity is reduced during hindlimb disuse (37). Differential expression of RhoA and Cdc42 during disuse may be related to their regulatory role in myoblast differentiation. RhoA signaling can induce myoblast differentiation. This regulation involves nuclear transcription factor, serum response factor (SRF), and the myogenic regulatory factor myoD (5, 24). Cdc42 activation is associated with depressed myoblast differentiation (31). Activated Cdc42 inhibits the expression of troponin T, myosin heavy chains, and the myogenic differentiation factor myogenin (31). In contrast, activated RhoA potentiates troponin T and myogenin expression through SRF-dependent mechanisms (31). RhoA and Cdc42 expression can be regulated by muscle loading, cytokines, and growth factor stimuli (29, 30, 41). These facts make RhoA and Cdc42 proteins excellent candidate molecules for integrating multiple stimuli during skeletal muscle recovery from disuse.

Other than roles in protein synthesis and satellite cell activity, RhoA signaling can also modulate gene transcription. RhoA is involved in the transduction of mechanical signals to the nucleus (11, 13). Regulation of skeletal {alpha}-actin promoter activity occurs through RhoA-mediated interaction with the focal adhesion complex at the sarcolemma (46). Activated RhoA can phosphorylate focal adhesion kinase and stimulate the formation of focal adhesion complexes at the sarcolemma, modulating integrin-mediated signaling (13). This regulation is dependent on SRF DNA binding (46, 47). SRF signaling has been shown to be necessary for skeletal {alpha}-actin promoter activation in stretch overload chicken skeletal muscle (7). RhoA activation of SRF is also linked to actin cytoskeletal reorganization due to transcriptional regulation of cytoskeletal-related vinculin, actin, and SRF genes (2). RhoA induction during muscle recovery from disuse may be involved in load-sensitive gene transcription, which is important for muscle regrowth.

Functional overload can regulate RhoA expression (30). The present study demonstrates that RhoA expression is also regulated by muscle disuse and subsequent recovery. The rapid and pretranslational regulation of RhoA expression by increased or decreased loading point to it as a potential modulator of muscle adaptation due to these stimuli. Additionally, transmembrane-associated {beta}1-integrin expression was responsive to increased loading after disuse but not to disuse alone. Localization of Rho family member Cdc42 was altered during skeletal muscle disuse and recovery and appears to have differential expression compared with family member RhoA. In conclusion, the expression pattern of RhoA during disuse and recovery further establishes it as a potential modulator of load-induced changes in gene expression in rat plantaris skeletal muscle.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by National Science Foundation/EPSCoR Grant EPS-0132573, National Center for Research Resouces Grant 8-PO RR-13461A, the South Carolina Space Grant Consortium Grant (to J. A. Carson), and South Carolina Space Grant Consortium and American College of Sports Medicine/National Aeronautics and Space Administration Student grants (awarded to J. M. McClung).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Won Jun Lee for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Carson, Univ. of South Carolina, Dept. of Exercise Science, 1300 Wheat St., Columbia, SC 29208 (E-mail: carsonj{at}gwm.sc.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.


   REFERENCES
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 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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