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Am J Physiol Cell Physiol 292: C1636-C1644, 2007. First published December 13, 2006; doi:10.1152/ajpcell.00504.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3beta

Jos L. J. van der Velden,1 Ramon C. J. Langen,1 Marco C. J. M. Kelders,1 Jodil Willems,1 Emiel F. M. Wouters,1 Yvonne M. W. Janssen-Heininger,2 and Annemie M. W. J. Schols1

1Department of Respiratory Medicine, Maastricht University, Maastricht, The Netherlands; and 2Department of Pathology, University of Vermont, Burlington, Vermont

Submitted 27 September 2006 ; accepted in final form 6 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3beta signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (–40%) and plantaris (–27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3beta were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3beta and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3beta.

glycogen synthase kinase-3beta; Akt; muscle growth; muscle atrophy

MUSCLE ATROPHY COMMONLY OCCURS in the elderly and in chronic disease, partly because of an adaptive sedentary lifestyle. Muscle atrophy not only compromises physical functioning through adverse effects on muscle strength but has also been associated with increased mortality independent of body mass index. Therefore, understanding the mechanisms involved in muscle atrophy and muscle (re)growth is imperative for the development of approaches to maintain or improve muscle mass in an increasing susceptible population. Skeletal muscle is a highly plastic tissue, which is reflected in its ability to adapt to changes in functional demand. For example, muscle inactivity caused by a leg cast or prolonged bed rest in a disease condition results in muscle atrophy.

Muscle atrophy can be studied by unloading the weight-bearing skeletal muscles of mice with hindlimb suspension (HS). HS induces a rapid decline in weight and cross-sectional area (CSA) of skeletal muscle (atrophy), which results in muscle weakness. HS affects various muscles of the hindlimb musculature, including soleus and plantaris muscle, but the extent of atrophy varies between these muscles (9). Under physiological conditions, reloading of inactivity-atrophied skeletal muscle will restore muscle mass by induction of muscle regrowth, through hypertrophy and regeneration (36). Muscle growth can occur without myonuclear accretion (i.e., the addition of new myonuclei), as is the case during hypertrophy signaling (6). Muscle hypertrophy relies on increased mRNA translation and subsequent protein synthesis, resulting from signaling through the Akt/mammalian target of rapamycin (mTOR) pathway (44). However, after certain atrophic conditions, myonuclear accretion is a prerequisite for complete regrowth of skeletal muscle (11).

The number of myonuclei contained in a muscle fiber appears to be flexible, allowing muscle growth or atrophy beyond the margins defined by the myonuclear domain (38), which is the volume of cytoplasm within the myofiber regulated by the gene products of a single myonucleus (28, 43). Satellite cells have been shown to serve as a source of new myonuclei during muscle regeneration (48, 50) and muscle growth (17). Satellite cells proliferate upon activation, and the subsequent daughter cells, myoblasts (MB), differentiate into new fibers or fuse with existing fibers (5). MB differentiation and fusion are dependent on two members of the family of muscle regulatory factors (MRFs), MyoD and myogenin (41), and the expression of these MRFs is induced in some models of muscle growth (3) and regrowth (38). Insulin-like growth factor (IGF)-I plays a major role in skeletal muscle (re)growth in an autocrine/paracrine fashion (1). Downstream of IGF-I, the Akt pathway has been shown to be important in muscle regrowth following atrophy (44).

Nearly all studies investigating mechanisms of muscle (re)growth to date have only addressed IGF-I/Akt signaling in the context of hypertrophy, e.g., the regulation of mRNA translation. Indeed, substantial evidence suggests that increased protein synthesis during muscle hypertrophy relies on stimulation of mRNA translation via the Akt/mTOR pathway. (40). A downstream target of IGF-I/Akt signaling is glycogen synthase kinase (GSK)-3beta, which is phosphorylated and inhibited by activated Akt (16). GSK-3beta suppresses protein synthesis by inhibiting eukaryotic transcription factor (eIF)2B-dependent translation (31). Conversely, IGF-I stimulates eIF2B-dependent translation through inactivation of GSK-3beta (44). In addition to increasing mRNA translation during muscle growth, IGF-I has been shown to stimulate myogenic differentiation (21, 55) and muscle-specific gene transcription in an Akt-dependent fashion (55). Interestingly, inhibition of GSK-3beta was sufficient to reproduce the stimulatory effect of IGF-I on myogenic differentiation (55).

As it remains unclear whether the requirement for myonuclear accretion during muscle regrowth is stimulus- or muscle specific, we hypothesized that the magnitude of the regrowth response determines whether skeletal muscle requires myonuclear accretion and myogenic differentiation. Moreover, considering that Akt/GSK-3beta signaling during muscle regrowth has only been studied in relation to hypertrophy, we investigated whether Akt/GSK-3beta signaling differs if muscle regrowth also requires myonuclear accretion for complete recovery. To address these hypotheses we compared soleus and plantaris muscle regrowth during recovery from HS-induced muscle atrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and hindlimb suspension. All protocols and procedures involving mice were approved by the University of Vermont Institutional Animal Care and Use Committee. Animals were housed in a temperature-controlled room on a 12:12-h light-dark cycle with food pellets and water provided ad libitum.

Forty-eight C57/BL6 mice (4–5 mo) were randomly assigned to one of six groups: 1) baseline, 2) HS for 14 days, and 3–6) HS followed by reloading (RL) for 3, 5, 7, or 14 days. The animals were subjected to HS as described previously (34). This model causes atrophy of the postural muscles, and subsequent RL of the hindlimbs evokes muscle regeneration, resulting in the restoration of muscle mass (38). After the experimental procedures, mice were euthanized by halothane overdose and body weight and length were determined. Soleus and plantaris muscles were collected with standardized dissection methods, weighed, frozen in liquid nitrogen, and stored at –80°C.

Cell culture. The murine skeletal muscle cell line C2C12 was obtained from the American Type Culture Collection (ATCC no. CRL1772, Manassas, VA). These cells are able to undergo differentiation into spontaneously contracting myotubes (MT) after growth factor withdrawal (60). MB were cultured in growth medium (GM), composed of low-glucose Dulbecco's modified Eagle's medium (DMEM) containing antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and 9% (vol/vol) fetal bovine serum (FBS) (all from GIBCO, Rockville, MD). Cells were plated at 104/cm2 on Matrigel (BD Biosciences, Bedford, MA)-coated (1:50 in DMEM) dishes as described previously (33) and cultured in GM for 24 h. To induce differentiation, GM was replaced with differentiation medium, which contained DMEM with 0.5% heat-inactivated FBS and antibiotics. Murine IGF-I (Calbiochem, La Jolla, CA) was added 24 h after induction of differentiation or 5 days after induction of differentiation for 15 or 30 min or 24 h.

Western blotting. Phosphorylated (Ser473) Akt (p-Akt), Akt, phosphorylated (Ser9) GSK-3beta (p-GSK-3beta), and GSK-3beta protein abundance was evaluated by Western blotting. Protein lysates were prepared by mincing muscle tissue in cold lysis buffer [mM: 50 Tris, 150 NaCl, 1 EDTA, 1 DTT, 1 Na3VO4, 1 PMSF, 1 Na-pyro-PO4, and 1 beta-glycerophosphate, with 10% (vol/vol) glycerol, 0.5% (vol/vol) Nonidet P-40, 10 µg/ml leupeptin, and 1% (vol/vol) aprotinin] immediately followed by homogenization (Polytron, Kinematica). Alternatively, adherent cultured muscle cells were washed in PBS, and protein lysates were prepared by the addition of lysis buffer [mM: 20 Tris, 150 NaCl, 1 DTT, 1 Na3VO4, and 1 PMSF, with 1% (vol/vol) Nonidet P-40, 10 µg/ml leupeptin, and 1% (vol/vol) aprotinin].

After homogenization of muscle tissue or lysis of cells, lysates were incubated on ice for 30 min, followed by 30-min centrifugation at 16,000 g. A portion of the supernatant was saved for protein determination, before the addition of 4x Laemmli sample buffer [0.25 M Tris·HCl pH 6.8, 8% (wt/vol) SDS, 40% (vol/vol) glycerol, 0.4 M DTT, and 0.04% (wt/vol) bromophenol blue]. Next, samples were boiled for 5 min and stored at –20°C. Total protein was assessed with the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. For SDS-PAGE, 10–40 µg of protein was loaded per lane and separated on a 10% polyacrylamide gel (Mini Protean 3 System, Bio-Rad), followed by transfer to a 0.45-µm nitrocellulose membrane (Bio-Rad) by electroblotting. The membrane was blocked for 1 h at room temperature in 5% (wt/vol) nonfat dried milk diluted in Tris-buffered saline (TBS)-Tween 20 (0.05%). Nitrocellulose blots were washed in TBS-Tween 20 (0.05%), followed by overnight incubation (4°C) with a polyclonal antibody specific for p-Akt (no. 9271), Akt (no. 9272), p-GSK-3beta (no. 9336), or GSK-3beta (no. 9332), all at 1:1,000 dilutions (Cell Signaling, Beverly, MA), or a monoclonal antibody (1:2,500 dilution) specific for GSK-3beta (no. 610201, BD Biosciences, Lexington, KY) diluted in TBS-Tween 20 (0.05%). After three wash steps of 20 min each, the blots were probed with a peroxidase-conjugated secondary antibody (nos. PI1000 and PI200, Vector Laboratories, Burlingame, CA) and visualized by chemiluminescence with Supersignal WestPico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) according to manufacturers' instructions and exposed to film (Biomax light film, Kodak).

GSK-3beta immunoprecipitation and kinase activity assay. Muscle homogenates were prepared as described under Western blotting. An input of 100 µg of total protein was precleared with protein G-Sepharose (Invitrogen, Carlsbad, CA) and subsequently incubated with 1 µg of monoclonal anti-GSK-3beta under gentle agitation for 1 h at 4°C. The immune complexes were isolated by the addition of 25 µl of a suspension of protein G-Sepharose in lysis buffer [50% (vol/vol)] and incubation under gentle agitation overnight at 4°C. Immunoprecipitates were washed twice in lysis buffer and divided in two. To assess equal GSK-3beta content, one half of the immunoprecipitate was used for Western blotting as described above and the other half was used for GSK-3beta activity measurement. For this purpose, the immunoprecipitate was washed twice in kinase reaction buffer [mM: 8 MOPS, pH 7.4, 0.2 EDTA, 10 magnesium acetate, 1 Na3VO4, 1 DTT, and 2.5 beta-glycerophosphate, with 10 µg/ml leupeptin and 1% (vol/vol) aprotinin]. The kinase activity assays were performed in 40 µl of total reaction buffer containing 62.5 µM GSK-3beta peptide substrate that corresponds to the {epsilon}-subunit of eIF2B (BioMol, Plymouth, PA), 20 mM MgCl2, 125 µM ATP, and 10 µCi of [{gamma}-32P]ATP. The reaction mixture was incubated for 30 min at 30°C, and 25 µl of the supernatant was spotted onto Whatman P81 phosphocellulose paper. The filter squares were washed five times for 5 min each in 0.75% phosphoric acid. Next, the filters were briefly rinsed in acetone, dried at room temperature, and subjected to liquid scintillation counting.

RNA isolation and assessment of mRNA abundance for ribonuclease protection assay. Muscle tissue was homogenized (Polytron, Kinematica) in 0.75 ml of solution D. RNA was extracted by adding 0.1 vol of 2 M sodium acetate pH 5.0, 1 vol of water-saturated phenol, and 0.2 vol of chloroform-isoamyl alcohol (50:1). After 15-min incubation and centrifugation, RNA was precipitated with an equal volume of isopropanol. The pellet was resuspended in 0.5 ml of solution D and precipitated again with isopropanol. After a wash step in 70% ethanol, the RNA was dissolved in nuclease-free water and stored at –80°C.

Ribonuclease protection assay (RPA) was performed to determine MyoD, myogenin, Myf-5, MRF4, and proliferation marker histone 3.2 (H3.2) mRNA levels as previously described (34) with templates developed in our laboratory. Briefly, a radiolabeled multiprobe panel was prepared by a transcription reaction with the MaxiScript Kit (Ambion, Houston, TX) with [{alpha}-32P]UTP (800 Ci/mmol) followed by hybridization with 10 or 2 µg of RNA (8 x 104 cpm per probe) per sample at 42°C for 18 h. After incubation, unhybridized RNA was digested (1:100 diluted RNase A/RNase T1 cocktail) with the RPA III kit (Ambion), and protected fragments were resolved on a 5% denaturing polyacrylamide gel. Gels were dried and exposed to film (Biomax MR-1, Kodak) or an imaging screen for quantification in a Personal Molecular Imager FX (Bio-Rad), and band intensity was analyzed with Quantity One software (Bio-Rad).

RNA isolation and assessment of mRNA abundance for quantitative PCR. Total RNA from muscle tissue or C2C12 cells was isolated with the Totally RNA kit (Ambion) according to the manufacturer's instructions. After isolation RNA was dissolved in Tris-EDTA buffer and stored at –80°C. One microgram of RNA was reverse transcribed to cDNA with the Reverse iT First Strand Synthesis kit (ABgene, Epsom, UK) with oligo(dT) primers.

Myosin heavy chain-perinatal (MyHC-p) and beta-actin mRNA were determined by quantitative RT-PCR (Q-PCR). Q-PCR primers were designed with Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and obtained from Sigma Genosys (Haverhill, UK). Skeletal muscle IGF-IEa and MyHC-p were amplified with the following primers: IGF-IEa FP 5'-CAAGACTCAGAAGGAAGTACATTTGAA-3', RP 5'-GCGGTGATGTGGCATTTTC; MyHC-p FP 5'-gaggagcgggctgacatc-3', RP 5'-acccagagaggcaagtgacc-3'. PCR reactions (25-µl total volume) contained 1x qPCR MasterMix Plus for SYBR Green I (Eurogentec, Seraing, Belgium) and primers (300 nM). Standard curves were made in duplicate by performing serial dilutions of pooled cDNA aliquots. Real-time PCR reactions were performed in an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Cycle threshold values were obtained for each sample, and the relative DNA concentrations were derived from the standard curve. The expression of the genes of interest was normalized to beta-actin (primers obtained from Ambion).

Statistical analysis. Raw data were entered into SPSS (version 11.0) for statistical analysis. Values as expressed in Figs. 1–5 are means ± SE. To test for statistically significant differences (P < 0.05), data were subjected to Mann-Whitney test, one-way ANOVA analysis, or a paired Student's t-test as applicable.


Figure 1
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  		Fig. 1. Changes in soleus and plantaris muscle mass after hindlimb suspension (HS) and reloading (RL). Paired muscle weight of soleus and plantaris muscle was determined and expressed relative (%) to their respective baseline values (means ± SE). Statistically significant differences within soleus or plantaris muscles compared with baseline (n = 4–8, *P < 0.005) or compared with HS (n = 4–8, #P < 0.001) were determined by 1-way ANOVA. Statistically significant differences between soleus and plantaris muscle (n = 8, {dagger}P < 0.01) were determined for each time point by a Mann-Whitney test. RL3, RL5, RL7, RL14, reloading for 3, 5, 7, and 14 days.

 

Figure 5
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  		Fig. 5. Induction of GSK-3beta phosphorylation corresponds with increased MRF expression. C2C12 myoblasts were induced to differentiate for 24 h (MB) or 5 days (myotubes, MT) before the addition of IGF-I (5 nM) for 30 min (A) or 24 h (B). A: cell lysates were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blot analysis. To assess p-GSK-3beta (10 µg) and total GSK-3beta (5 µg) in soleus or plantaris muscle band intensity was determined as a ratio of p-GSK-3beta over total GSK-3beta. Representative blots for p-GSK-3beta and total GSK-3beta are depicted at bottom. B: total RNA was isolated and subjected to RPA to assess mRNA abundance of MyoD, Myf-5, and myogenin (Myog). Signal intensity was determined with PhosphorImager analysis, normalized to GAPDH, and expressed as relative (%) to control. Statistically significant differences between control and IGF-I treated cells (*P < 0.05) or between MB and MT ({dagger}P < 0.05) were determined by 1-way ANOVA.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in muscle mass after HS and RL. Soleus and plantaris muscle weights after HS and during the course of RL-induced regrowth are shown in Fig. 1. Muscle weight of soleus was more reduced than that of plantaris after HS (60 ± 3.7% vs. 73 ± 3.9% of baseline; P = 0.01) (Fig. 1). After 14 days of RL soleus muscle recovered to 92 ± 2.9% and plantaris muscle to 91 ± 3.1% of baseline muscle mass. Body weight showed a 15% decrease compared with control animals after 14 days of HS and was recovered completely after 7 days of RL (data not shown).

IGF-IEa mRNA expression after HS and RL. IGF-IEa mRNA expression was assessed after HS and RL in soleus and plantaris muscle. Under basal conditions there was no difference in IGF-IEa expression between the two muscles (data not shown). IGF-IEa mRNA expression was not affected in soleus and plantaris muscle after HS (Fig. 2A). However, after 3 days of RL, IGF-IEa mRNA expression was significantly increased in both soleus (620 ± 40% of baseline) and plantaris (202 ± 13% of baseline) muscle, with the strongest increase observed in the soleus muscle. After 7 days of RL IGF-IEa expression in soleus returned to baseline, whereas at that point in time IGF-IEa expression peaked (247 ± 10% of baseline) in plantaris muscle, to return to baseline after 14 days of RL.


Figure 2
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  		Fig. 2. Insulin-like growth factor (IGF)-I mRNA expression, Akt or glycogen synthase kinase (GSK)-3beta phosphorylation status, and GSK-3beta activity after HS and RL. A: total RNA was isolated and subjected to quantitative PCR (Q-PCR) analysis to assess mRNA abundance of IGF-IEa, which was normalized to histone 3.3 mRNA abundance and expressed as relative (%) to control. BL, baseline. B: muscle lysates were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blot analysis. To assess phosphorylated (p-)Akt, or total Akt, 40 and 10 µg were loaded, respectively. C: for p-GSK-3beta or total GSK-3beta, 20 or 10 µg protein was separated. For both kinases band intensity was determined, shown as the ratio of p-band over total, and expressed relative (%) to BL. D: muscle lysates were prepared for determination of GSK-3beta kinase activity in disintegrations per minute (DPM). Statistically significant differences compared with BL within soleus or plantaris muscles (*P < 0.05) or between soleus and plantaris muscles for each time point ({dagger}P < 0.05) were determined by 1-way ANOVA or an independent Student's t-test, respectively. Representative blots for p-Akt, Akt, p-GSK-3beta, and GSK-3beta are depicted below each respective graph.

 
Akt phosphorylation after HS and RL. p-Akt relative to total Akt was assessed after HS and RL in soleus and plantaris muscle. Under basal conditions there was no difference in p-Akt relative to total Akt (data not shown). Akt phosphorylation was not affected in soleus and plantaris muscle after HS (Fig. 2B). A marked and significant induction of Akt phosphorylation was observed in both muscles after RL. After 3 days of RL a maximal increase in Akt phosphorylation (540 ± 73% of baseline) was observed in soleus muscle, whereas the maximal phosphorylation of Akt in plantaris muscle (382 ± 28% of baseline) was apparent after 5 days of RL. Despite the differing kinetics of induction, p-Akt returned to baseline in both muscles after 7 days of RL.

GSK-3beta phosphorylation and activity after HS and RL. GSK-3beta is a downstream target of Akt (16). Therefore we investigated p-GSK-3beta relative to total GSK-3beta protein abundance and GSK-3beta kinase activity in soleus and plantaris after HS and RL. Under basal conditions p-GSK-3beta relative to total GSK-3beta was ~50% more abundant in plantaris muscle compared with soleus muscle (data not shown). After HS, the ratio of p-GSK-3beta to total GSK-3beta was not affected in either soleus or plantaris (Fig. 2C). During RL GSK-3beta phosphorylation strongly increased in soleus muscle (250 ± 38% of baseline) (Fig. 2C, top and bottom), whereas GSK-3beta phosphorylation was not affected in plantaris muscle compared with control (Fig. 2C, top and middle). Subsequently, GSK-3beta kinase activity was assessed in soleus and plantaris muscle. HS did not significantly affect GSK-3beta activity in soleus or plantaris (Fig. 2D). Consistent with increased GSK-3beta phosphorylation during RL in soleus muscle, GSK-3beta activity was significantly decreased after 5 days of RL (67 ± 2% of baseline) and returned to baseline levels at the later time points of RL. In contrast, GSK-3beta activity in plantaris muscle was not affected during RL compared with baseline, which was in line with the constant p-GSK-3beta-to-total GSK-3beta ratio during RL. Despite a lower GSK-3beta abundance, baseline values of GSK-3beta activity were approximately twofold higher in soleus muscle compared with plantaris muscle (data not shown). To ensure equal input, we subjected immunoprecipitation lysates used for GSK-3beta activity assay to Western blot analysis (data not shown).

mRNA abundance of proliferation and differentiation markers after HS and RL. Soleus and plantaris muscles recover differently from atrophy (38). To evaluate MB proliferation and differentiation, mRNA levels of H3.2 and MyHC-p were assessed during HS and RL (Fig. 3) with RPA and Q-PCR, respectively (MyHC-p was not detectable in plantaris by RPA). MyHC-p is a sensitive differentiation marker (49), whereas the expression of H3.2 mRNA is restricted to the S phase of the cell cycle (58) and absent once MB differentiate (59). H3.2 and MyHC-p were not statistically affected after HS in soleus, whereas after 3 days of RL a strong increase in H3.2 expression (636 ± 32% of baseline) was observed. This increase was sustained up to 7 days of RL. MyHC-p mRNA abundance in soleus muscle increased after RL, reaching maximal expression at day 7 (426 ± 49% of baseline). Although a slight but significant increase in H3.2 and MyHC-p expression was also observed in plantaris muscle at days 3 and 5 of RL, respectively, these changes were three- to fourfold smaller compared with the increases observed in the soleus muscle during RL. These data suggest active participation of regeneration in muscle regrowth in soleus, but not plantaris, muscle.


Figure 3
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  		Fig. 3. mRNA status of proliferation and differentiation markers after HS and RL. Total RNA was isolated from soleus and plantaris muscle to assess mRNA abundance of histone 3.2 (H3.2) by ribonuclease protection assay (RPA; A) or myosin heavy chain-perinatal (MyHC-peri) by Q-PCR (B). For RPA, signal intensity was determined by PhosphorImager analysis, normalized to GAPDH, and expressed relative (%) to BL. mRNA abundance measured by Q-PCR was normalized to beta-actin (QuantumRNA technology, Ambion). Statistically significant differences within each muscle compared with BL (*P < 0.05) or between soleus and plantaris muscle ({dagger}P < 0.05) were determined by 1-way ANOVA or independent Student's t-tests, respectively.

 
Myogenic mRNAs after HS and RL. Recently it was shown that inhibition of GSK-3beta in cultured muscle cells results in increased expression of various myogenic mRNAs (55). To evaluate whether changes in GSK-3beta phosphorylation and activity are also inversely related to myogenic mRNA expression during regrowth of skeletal muscle, the abundance of the mRNA transcripts encoding the MRFs MyoD, Myf-5, myogenin, and MRF4 were determined after HS and RL in soleus and plantaris muscle (Fig. 4). Table 1 shows the baseline mRNA transcripts for the MRFs. After HS, MyoD, Myf-5, myogenin and MRF4 were not significantly affected in soleus or plantaris muscle. However, after 3 days of RL, MyoD (375 ± 61% of baseline; Fig. 4A), myogenin (1,250 ± 43% of baseline; Fig. 4C), and MRF4 (281 ± 40% of baseline; Fig. 4D) mRNA levels were strongly upregulated in soleus muscle. In contrast, at day 3 of RL in the plantaris muscle MyoD was not induced, and increases in myogenin (455 ± 229% of baseline) and MRF4 (159 ± 50% of baseline) were less pronounced compared with the soleus muscle. In fact, the induction of MyoD and myogenin expression after 3 days of RL was four- and threefold stronger in soleus compared with plantaris muscle. Myf-5 mRNA levels (Fig. 4B) were not significantly affected during HS or RL in either muscle.


Figure 4
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  		Fig. 4. mRNA expression of muscle regulatory factors (MRFs) after HS and RL. Total RNA was isolated from soleus and plantaris muscle and subjected to RPA to assess mRNA abundance of MyoD (A), Myf-5 (B), myogenin (C), and MRF4 (D). Signal intensity of the various mRNA bands was determined with PhosphorImager analysis, normalized to GAPDH, and expressed as relative (%) to BL. Statistically significant differences within each muscle compared with BL (*P < 0.05) or between soleus and plantaris muscle ({dagger}P < 0.05) were determined by 1-way ANOVA or independent Student's t-tests, respectively.

 

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  		Table 1. Baseline muscle-specific mRNA transcripts: soleus vs. plantaris

 
GSK-3beta phosphorylation and MRF expression in cultured myoblasts and myotubes. IGF-I is an important mediator in skeletal muscle (re)growth and signaling by Akt, and its downstream mediators are essential for (re)growth of skeletal muscle (25, 26). To evaluate whether the differential regulation of GSK-3beta phosphorylation and activity in reloaded soleus and plantaris muscle, despite similar activation of Akt, could be related to the differentiating MB, Akt and downstream signaling was induced in differentiating MB and fully differentiated MT by treatment with IGF-I. Both MB and MT displayed a similar phosphorylation of Akt after IGF-I (data not shown). In contrast, despite similar basal levels of GSK-3beta phosphorylation, IGF-I treatment of differentiating MB resulted in a significantly higher GSK-3beta phosphorylation status compared with IGF-I-treated MT (Fig. 5A).

Since we and others reported previously that the phosphorylation of GSK-3beta in cultured muscle cells in response to IGF-I corresponds with GSK-3beta inactivation, we next addressed whether the differential phosphorylation/inactivation of GSK-3beta by IGF-I in MB and MT corresponded with a differential response of MRF expression to IGF-I. Treatment of differentiating MB with IGF-I resulted in a robust increase of MyoD and myogenin compared with control (Fig. 5B). In contrast, MyoD and myogenin expression in fully differentiated MT did not change in response to IGF-I. Myf-5 expression did not respond to IGF-I in MB or in MT. MRF4 was not detectable in C2C12 cells. These data suggest that the inactivation of GSK-3beta after IGF-I/Akt signaling is associated with MB during regrowth of atrophied skeletal muscle, facilitating the induction of MyoD and myogenin expression and subsequent differentiation of these cells as part of skeletal muscle regeneration.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that the IGF-I signaling pathway is an important regulatory conduit of skeletal muscle growth (40). In this study molecular events during muscle regrowth, including the activity of the Akt-GSK-3beta signaling module of the IGF-I pathway, were compared between two muscles recovering from different extents of atrophy. Despite almost similar patterns of IGF-IEa expression and Akt phosphorylation during regrowth of soleus and plantaris muscle after atrophy, this study revealed differences between these muscles in GSK-3beta phosphorylation status and activity that were related to the extent of atrophy and the presence or absence of a regenerative response. Specifically, inactivation of GSK-3beta was observed during regrowth of soleus muscle only, which involved muscle regeneration based on markers of MB proliferation and differentiation and a strong induction of MRF expression. Experiments in cultured muscle cells suggested that the induction of MRF expression is facilitated by inactivation of GSK-3beta and selectively occurs in the MB population. Inactivity is an important determinant of muscle atrophy in the elderly and in chronic wasting diseases (24). HS is a well-established model for skeletal muscle atrophy (39). In accordance with other studies, a significant decrease in both soleus and plantaris muscle weight was observed after 14 days of HS (22, 37), which was most pronounced in the soleus muscle. Reloading of skeletal muscle after a period of disuse will initiate a muscle regrowth program that may involve elements of muscle hypertrophy and regeneration. In both soleus and plantaris muscle CSA is decreased after disuse-induced atrophy, but interestingly the myonuclear number is reduced only in soleus muscle and in plantaris muscle it is not affected (38). Consequently, full recovery of soleus muscle from HS-induced muscle atrophy requires myonuclear accretion by incorporation of satellite cell-derived MB into existing muscle fibers, in addition to elements of hypertrophy, including increased mRNA translation, that also occur in plantaris muscle regrowth. This notion is supported by studies using {gamma}-irradiation of the hindlimb muscles to inactivate the satellite cells present in soleus and plantaris muscle, which demonstrated that in contrast to plantaris muscle {gamma}-irradiated soleus muscle recovered only half of its lost mass (38). However, in a model of compensatory hypertrophy resulting in a 40% increase in plantaris muscle weight proliferative markers were upregulated (3), indicative of satellite cell activation and proliferation. This suggests that myonuclear accretion during muscle (re)growth can also occur in plantaris muscle and is not fiber type dependent but rather determined by the magnitude of the (re)growth response to meet increased functional demand imposed on a particular muscle. Previous studies have shown that muscle RL induces fiber injury and inflammation (32, 52). However, because these studies were performed in soleus muscle only, the contribution of potential differences in muscle injury during RL to the differential regrowth response observed in soleus and plantaris muscle cannot be excluded. After release from HS followed by RL both muscles recovered almost completely, in accordance with the literature (38). IGF-I is an important growth factor that can function in a autocrine/paracrine fashion to regulate muscle mass (30). In this study muscle IGF-IEa expression was not affected after HS in both soleus and plantaris. This has also been reported not to change in the gastrocnemius muscle in a similar model (27). Intracellularly, the protein kinase Akt is an essential mediator of IGF-I signaling toward growth and maintenance of muscle mass. Although Akt phosphorylation appeared to be attenuated after HS, this effect did not reach statistical significance in either muscle. This may be attributed to the low baseline levels of Akt phosphorylation in both muscles, because previous studies showed that skeletal muscle unloading or inactivity is accompanied by decreased Akt phosphorylation (29, 53). Subsequently, consequent to decreased Akt activity, reduced mRNA translation (2, 10) and increased protein degradation resulting from decreased suppression of atrogin expression (46) likely contributed to the loss of muscle mass. It has been shown that Akt plays a role in preservation of muscle mass not only in mice; a recent study in human subjects revealed a reduction in Akt activity in amyotrophic lateral sclerosis, a degenerating muscle disease (35).

Akt negatively regulates protein kinase GSK-3beta (16). In accordance with a previous study, HS did not affect GSK-3beta phosphorylation in soleus muscle (12) or in plantaris muscle. In line with unchanged GSK-3beta phosphorylation, GSK-3beta kinase activity was not affected by HS in soleus and plantaris muscle. Baseline levels of protein abundance of GSK-3beta were ~50% less in soleus muscle compared with plantaris muscle, although activity levels in soleus were significantly higher. Similar observations have been made for the protein phosphatase calcineurin, which demonstrates highest activity in soleus muscle despite far greater calcineurin expression levels in fast-twitch muscles compared with soleus (20, 51). Reloading of atrophied muscle resulted in a rapid and strong increase in IGF-IEa expression and Akt phosphorylation in both muscles. Although some differences in induction and kinetics between the soleus and plantaris muscles were observed, the IGF-IEa expression coincided with the Akt phosphorylation, which both returned to baseline values at later time points of RL in both muscles. To our knowledge, we report for the first time that IGF-IEa expression is enhanced during muscle regrowth after atrophy. Various studies have demonstrated the growth-promoting effect of IGF-I overexpression in muscle (4, 15, 40). In addition, muscle IGF-IEa expression is increased during compensatory overload after synergist ablation in soleus or plantaris muscle (3, 8), suggesting a local role of muscle-produced IGF-IEa in muscle growth. In line with this, Akt phosphorylation is increased in the overloaded muscle in these models, coinciding with muscle growth (10). In our study we measured Akt1 phosphorylation, because this isoform is required for growth (14), whereas Akt2 has mostly been associated with the regulation of glucose metabolism (13, 47), although some reports suggest a role for Akt2 in muscle differentiation (54, 56). Various studies have linked increased Akt phosphorylation to stimulated mRNA translation and protein synthesis in hypertrophic muscle via its downstream targets p70s6k and 4E-BP (10, 12). Phosphorylation and inactivation of GSK-3beta by Akt also contributes to muscle hypertrophy (44, 57) by facilitating translation through eIF2B (31). Additionally, our group (55) recently showed that IGF-I/Akt signaling stimulates myogenic differentiation and MT formation, which was recapitulated by the inactivation of GSK-3beta, suggesting a central role for GSK-3beta in the regulation of muscle growth, as it may be involved in muscle hypertrophy and regeneration.

Consistent with the large increase observed in Akt phosphorylation, GSK-3beta phosphorylation was increased after RL in soleus muscle. Surprisingly, despite similar increases in Akt phosphorylation, GSK-3beta phosphorylation was not affected in plantaris muscle after RL. In soleus muscle the IGF-IEa induction was significantly stronger compared with plantaris muscle. Possibly, the IGF-IEa levels in the plantaris muscle were adequate to induce Akt activation but not sufficient to establish GSK-3beta phosphorylation and inactivation, although this would be somewhat surprising considering that GSK-3beta is a direct substrate of Akt. Another explanation for the dissociation of increased IGF-IEa and Akt phosphorylation levels with GSK-3beta phosphorylation and activity in plantaris muscle could be that there appear to be two distinct pools of GSK-3beta, which determine the availability of GSK-3beta for phosphorylation by Akt (23). In contrast to free GSK-3beta, GSK-3beta residing in the Wnt signaling complex cannot be phosphorylated by Akt (19). Interestingly, in plantaris muscle the interaction of GSK-3beta with this complex is associated with overload-induced hypertrophy (7).

In accordance with its phosphorylation status, GSK-3beta activity was decreased in soleus and unaffected in plantaris during RL. This demonstrates that in addition to the hypertrophy response observed in both reloaded muscles, characterized and probably regulated by increased IGF-IEa expression and Akt activity, soleus regrowth is accompanied by additional signal transduction that involves GSK-3beta. Since regrowth of inactivity-induced atrophied soleus, but not plantaris, muscle requires muscle regeneration based on the finding that myonuclear accretion is obligatory for full recovery of atrophied soleus muscle (38), the differential regulation of GSK-3beta may be associated with proliferating and differentiating MB during soleus regrowth. Indeed, H3.2 and MyHC-p mRNA transcripts were dramatically increased in soleus compared with plantaris during muscle regrowth, reflecting MB proliferation and differentiation, respectively (34), and indicative of myonuclear accretion and muscle regeneration that have been described for the soleus muscle under these conditions (38).

The family of MRFs, particularly MyoD and myogenin, govern myogenic differentiation and are essential for muscle regeneration (45). In line with other studies (18, 38), a large increase in mRNA transcripts encoding MyoD, myogenin, and MRF-4 was observed during RL in soleus muscle. In addition, our study demonstrates that these increases were absent or much less pronounced in plantaris muscle that displayed no evidence of muscle regeneration. Increased MRF expression probably stems from differentiating MB, because {gamma}-irradiation, which prevents MB proliferation and differentiation in regenerating soleus muscle, inhibits the induction of MyoD and myogenin expression (38). Experiments in cultured muscle cells further supported the notion that the induction of MRF expression occurred in differentiating MB and not in hypertrophying myofibers, because only cultured MB but not MT responded to IGF-I treatment with increased MyoD and myogenin expression, despite identical induction of Akt phosphorylation. In contrast to Akt, the extent of GSK-3beta phosphorylation after IGF-I differed markedly, suggesting that GSK-3beta is more receptive to inactivation by Akt in differentiating MB compared with fully differentiated MT, which recapitulates the differences between reloaded soleus and plantaris muscle. Moreover, the inverse relationship between GSK-3beta activity and MRF expression observed in regenerating soleus muscle is in line with recent findings by our group (55) showing that GSK-3beta is a negative regulator of myogenic differentiation, which may occur through suppression of MRF expression. Although the mechanisms by which GSK-3beta contributes to the regulation of muscle growth are not entirely known, recent reports implicate nuclear factor of activated T cells and translation initiation factor eIF2B as relevant substrates of GSK-3beta (42).

In conclusion, our study demonstrates that reloading of skeletal muscle after a period of disuse induces IGF-IEa mRNA expression and Akt signaling that is independent of the magnitude of muscle regrowth and likely initiates a muscle regrowth program involving elements of hypertrophy. Depending on the extent of atrophy, muscle regrowth also involves a regenerative response characterized by MB proliferation, differentiation, and increased MRF expression, which may be negatively regulated by GSK-3beta. These findings indicate that the development of therapeutic strategies aimed at restoration and preservation of muscle mass in health and disease should be based on the signal transduction of both muscle hypertrophy and muscle regeneration.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. J. Langen, Dept. of Respiratory Medicine, Maastricht University PO Box 5800, 6202 AZ Maastricht, The Netherlands (e-mail: r.langen{at}pul.unimaas.nl)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Adams GR. Autocrine and/or paracrine insulin-like growth factor-I activity in skeletal muscle. Clin Orthop 403: S188–S196, 2002.[CrossRef][Medline]

2. Adams GR, Caiozzo VJ, Haddad F, Baldwin KM. Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am J Physiol Cell Physiol 283: C1182–C1195, 2002.[Abstract/Free Full Text]

3. Adams GR, Haddad F, Baldwin KM. Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. J Appl Physiol 87: 1705–1712, 1999.[Abstract/Free Full Text]

4. Adams GR, McCue SA. Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84: 1716–1722, 1998.[Abstract/Free Full Text]

5. Allen DL, Monke SR, Talmadge RJ, Roy RR, Edgerton VR. Plasticity of myonuclear number in hypertrophied and atrophied mammalian skeletal muscle fibers. J Appl Physiol 78: 1969–1976, 1995.[Abstract/Free Full Text]

6. Allen DL, Roy RR, Edgerton VR. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22: 1350–1360, 1999.[CrossRef][Web of Science][Medline]

7. Armstrong DD, Esser KA. Wnt/beta-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy. Am J Physiol Cell Physiol 289: C853–C859, 2005.[Abstract/Free Full Text]

8. Awede B, Thissen J, Gailly P, Lebacq J. Regulation of IGF-I, IGFBP-4 and IGFBP-5 gene expression by loading in mouse skeletal muscle. FEBS Lett 461: 263–267, 1999.[CrossRef][Web of Science][Medline]

9. Baldwin KM, Haddad F. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle. J Appl Physiol 90: 345–357, 2001.[Abstract/Free Full Text]

10. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014–1019, 2001.[CrossRef][Web of Science][Medline]

11. Bondesen BA, Mills ST, Pavlath GK. The COX-2 pathway regulates growth of atrophied muscle via multiple mechanisms. Am J Physiol Cell Physiol 290: C1651–C1659, 2006.[Abstract/Free Full Text]

12. Childs TE, Spangenburg EE, Vyas DR, Booth FW. Temporal alterations in protein signaling cascades during recovery from muscle atrophy. Am J Physiol Cell Physiol 285: C391–C398, 2003.[Abstract/Free Full Text]

13. Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB 3rd, Kaestner KH, Bartolomei MS, Shulman GI, Birnbaum MJ. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science 292: 1728–1731, 2001.[Abstract/Free Full Text]

14. Cho H, Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ. Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276: 38349–38352, 2001.[Abstract/Free Full Text]

15. Coleman ME, DeMayo F, Yin KC, Lee HM, Geske R, Montgomery C, Schwartz RJ. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 270: 12109–12116, 1995.[Abstract/Free Full Text]

16. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789, 1995.[CrossRef][Medline]

17. Dangott B, Schultz E, Mozdziak PE. Dietary creatine monohydrate supplementation increases satellite cell mitotic activity during compensatory hypertrophy. Int J Sports Med 21: 13–16, 2000.[CrossRef][Web of Science][Medline]

18. Dapp C, Schmutz S, Hoppeler H, Fluck M. Transcriptional reprogramming and ultrastructure during atrophy and recovery of mouse soleus muscle. Physiol Genomics 20: 97–107, 2004.[Abstract/Free Full Text]

19. Ding VW, Chen RH, McCormick F. Differential regulation of glycogen synthase kinase 3beta by insulin and Wnt signaling. J Biol Chem 275: 32475–32481, 2000.[Abstract/Free Full Text]

20. Dunn SE, Burns JL, Michel RN. Calcineurin is required for skeletal muscle hypertrophy. J Biol Chem 274: 21908–21912, 1999.[Abstract/Free Full Text]

21. Florini JR, Ewton DZ, Roof SL. Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol 5: 718–724, 1991.[Abstract/Free Full Text]

22. Fluck M, Schmutz S, Wittwer M, Hoppeler H, Desplanches D. Transcriptional reprogramming during reloading of atrophied rat soleus muscle. Am J Physiol Regul Integr Comp Physiol 289: R4–R14, 2005.[Abstract/Free Full Text]

23. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359: 1–16, 2001.[CrossRef][Web of Science][Medline]

24. Franssen FM, Wouters EF, Schols AM. The contribution of starvation, deconditioning and ageing to the observed alterations in peripheral skeletal muscle in chronic organ diseases. Clin Nutr 21: 1–14, 2002.[CrossRef][Web of Science][Medline]

25. Glass DJ. Molecular mechanisms modulating muscle mass. Trends Mol Med 9: 344–350, 2003.[CrossRef][Web of Science][Medline]

26. Glass DJ. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol 5: 87–90, 2003.[CrossRef][Web of Science][Medline]

27. Haddad F, Adams GR, Bodell PW, Baldwin KM. Isometric resistance exercise fails to counteract skeletal muscle atrophy processes during the initial stages of unloading. J Appl Physiol 100: 433–441, 2006.[Abstract/Free Full Text]

28. Hall ZW, Ralston E. Nuclear domains in muscle cells. Cell 59: 771–772, 1989.[CrossRef][Web of Science][Medline]

29. Hilder TL, Tou JC, Grindeland RE, Wade CE, Graves LM. Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle. FEBS Lett 553: 63–67, 2003.[CrossRef][Web of Science][Medline]

30. Husmann I, Soulet L, Gautron J, Martelly I, Barritault D. Growth factors in skeletal muscle regeneration. Cytokine Growth Factor Rev 7: 249–258, 1996.[CrossRef][Medline]

31. Jefferson LS, Fabian JR, Kimball SR. Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle. Int J Biochem Cell Biol 31: 191–200, 1999.[CrossRef][Web of Science][Medline]

32. Krippendorf BB, Riley DA. Distinguishing unloading- versus reloading-induced changes in rat soleus muscle. Muscle Nerve 16: 99–108, 1993.[CrossRef][Web of Science][Medline]

33. Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-{kappa}B. FASEB J 15: 1169–1180, 2001.[Abstract/Free Full Text]

34. Langen RC, Van Der Velden JL, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM. Tumor necrosis factor-{alpha} inhibits myogenic differentiation through MyoD protein destabilization. FASEB J 18: 227–237, 2004.[Abstract/Free Full Text]

35. Leger B, Vergani L, Soraru G, Hespel P, Derave W, Gobelet C, D'Ascenzio C, Angelini C, Russell AP. Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1. FASEB J 20: 583–585, 2006.[Abstract/Free Full Text]

36. Machida S, Booth FW. Regrowth of skeletal muscle atrophied from inactivity. Med Sci Sports Exerc 36: 52–59, 2004.

37. Mitchell PO, Pavlath GK. Multiple roles of calcineurin in skeletal muscle growth. Clin Orthop Relat Res 403: S197–S202, 2002.[CrossRef][Medline]

38. Mitchell PO, Pavlath GK. A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy. Am J Physiol Cell Physiol 281: C1706–C1715, 2001.[Abstract/Free Full Text]

39. Morey-Holton E, Globus RK, Kaplansky A, Durnova G. The hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. Adv Space Biol Med 10: 7–40, 2005.[Medline]

40. Musaro A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton ER, Sweeney HL, Rosenthal N. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 27: 195–200, 2001.[CrossRef][Web of Science][Medline]

41. Olson EN. Signal transduction pathways that regulate skeletal muscle gene expression. Mol Endocrinol 7: 1369–1378, 1993.[Free Full Text]

42. Pap M, Cooper GM. Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta signaling pathway. Mol Cell Biol 22: 578–586, 2002.[Abstract/Free Full Text]

43. Pavlath GK, Rich K, Webster SG, Blau HM. Localization of muscle gene products in nuclear domains. Nature 337: 570–573, 1989.[CrossRef][Medline]

44. Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, Yancopoulos GD, Glass DJ. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 3: 1009–1013, 2001.[CrossRef][Web of Science][Medline]

45. Rudnicki MA. Marrow to muscle, fission versus fusion. Nat Med 9: 1461–1462, 2003.[CrossRef][Web of Science][Medline]

46. Sacheck JM, Ohtsuka A, McLary SC, Goldberg AL. IGF-I stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1. Am J Physiol Endocrinol Metab 287: E591–E601, 2004.[Abstract/Free Full Text]

47. Sakamoto K, Arnolds DE, Fujii N, Kramer HF, Hirshman MF, Goodyear LJ. Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle. Am J Physiol Endocrinol Metab 291: E1031–E1037, 2006.[Abstract/Free Full Text]

48. Schultz E, Jaryszak DL, Valliere CR. Response of satellite cells to focal skeletal muscle injury. Muscle Nerve 8: 217–222, 1985.[CrossRef][Web of Science][Medline]

49. Smith TH, Miller JB. Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro. Dev Biol 149: 16–26, 1992.[CrossRef][Web of Science][Medline]

50. Snow MH. Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. I. A fine structural study. Anat Rec 188: 181–199, 1977.[CrossRef][Medline]

51. Spangenburg EE, Williams JH, Roy RR, Talmadge RJ. Skeletal muscle calcineurin: influence of phenotype adaptation and atrophy. Am J Physiol Regul Integr Comp Physiol 280: R1256–R1260, 2001.[Abstract/Free Full Text]

52. St. Pierre BA, Tidball JG. Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension. J Appl Physiol 77: 290–297, 1994.[Abstract/Free Full Text]

53. Sugiura T, Abe N, Nagano M, Goto K, Sakuma K, Naito H, Yoshioka T, Powers SK. Changes in PKB/Akt and calcineurin signaling during recovery in atrophied soleus muscle induced by unloading. Am J Physiol Regul Integr Comp Physiol 288: R1273–R1278, 2005.[Abstract/Free Full Text]

54. Sumitani S, Goya K, Testa JR, Kouhara H, Kasayama S. Akt1 and Akt2 differently regulate muscle creatine kinase and myogenin gene transcription in insulin-induced differentiation of C2C12 myoblasts. Endocrinology 143: 820–828, 2002.[Abstract/Free Full Text]

55. Van der Velden JL, Langen RC, Kelders MC, Wouters EF, Janssen-Heininger YM, Schols AM. Inhibition of glycogen synthase kinase-3beta activity is sufficient to stimulate myogenic differentiation. Am J Physiol Cell Physiol 290: C453–C462, 2006.[Abstract/Free Full Text]

56. Vandromme M, Rochat A, Meier R, Carnac G, Besser D, Hemmings BA, Fernandez A, Lamb NJ. Protein kinase B beta/Akt2 plays a specific role in muscle differentiation. J Biol Chem 276: 8173–8179, 2001.[Abstract/Free Full Text]

57. Vyas DR, Spangenburg EE, Abraha TW, Childs TE, Booth FW. GSK-3beta negatively regulates skeletal myotube hypertrophy. Am J Physiol Cell Physiol 283: C545–C551, 2002.[Abstract/Free Full Text]

58. Wu RS, Tsai S, Bonner WM. Patterns of histone variant synthesis can distinguish G0 from G1 cells. Cell 31: 367–374, 1982.[CrossRef][Web of Science][Medline]

59. Wunsch AM, Lough J. Modulation of histone H3 variant synthesis during the myoblast-myotube transition of chicken myogenesis. Dev Biol 119: 94–99, 1987.[CrossRef][Web of Science][Medline]

60. Yaffe D, Saxel O. A myogenic cell line with altered serum requirements for differentiation. Differentiation 7: 159–166, 1977.[CrossRef][Web of Science][Medline]





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