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Am J Physiol Cell Physiol 294: C36-C46, 2008. First published October 17, 2007; doi:10.1152/ajpcell.00164.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Trophic action of sphingosine 1-phosphate in denervated rat soleus muscle

Marika Zanin,1 Elena Germinario,1,2 Luciano Dalla Libera,3 Dorianna Sandonà,4 Roger A. Sabbadini,5 Romeo Betto,2,3 and Daniela Danieli-Betto1,2

1Department of Human Anatomy and Physiology, University of Padua, Padua; 2Interuniversity Institute of Myology of Italy, 3Muscle Biology and Physiopathology Unit, Consiglio Nazionale delle Ricerche Institute of Neuroscience, Padua; 4Department of Biomedical Sciences University of Padua, Padua, Italy; and 5Department of Biology and Heart Institute, San Diego State University, San Diego, California

Submitted 17 April 2007 ; accepted in final form 10 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sphingosine 1-phosphate (S1P) mediates a number of cellular responses, including growth and proliferation. Skeletal muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors. The aim of this work was to localize S1P receptors in rat skeletal muscle and to investigate whether S1P exerts a trophic action on muscle fibers. RT-PCR and Western blot analyses demonstrated the expression of S1P1 and S1P3 receptors by soleus muscle. Immunofluorescence revealed that S1P1 and S1P3 receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes. The receptors also decorate the nuclear membrane. S1P1 receptors were also present at the neuromuscular junction. The possible trophic action of S1P was investigated by utilizing the denervation atrophy model. Rat soleus muscle was analyzed 7 and 14 days after motor nerve cut. During denervation, S1P was continuously delivered to the muscle through a mini osmotic pump. S1P and its precursor, sphingosine (Sph), significantly attenuated the progress of denervation-induced muscle atrophy. The trophic effect of Sph was prevented by N,N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that converts Sph into S1P. Neutralization of circulating S1P by a specific antibody further demonstrated that S1P was responsible for the trophic effects of S1P during denervation atrophy. Denervation produced the down regulation of S1P1 and S1P3 receptors, regardless of the presence of the receptor agonist. In conclusion, the results suggest that S1P acts as a trophic factor of skeletal muscle.

sphingosine 1-phosphate receptors; sphingomyelin derivatives; skeletal muscle atrophy

SPHINGOLIPIDS MEDIATE A NUMBER of cellular responses, including growth, proliferation, angiogenesis, and regulation of apoptosis (1, 52). The receptor-mediated activation of sphingomyelinase sequentially generates different biologically active compounds, namely, ceramide, sphingosine (Sph), and sphingosine 1-phosphate (S1P). S1P acts as a ligand of specific surface G protein-coupled receptors. In addition, intracellularly generated S1P can act in paracrine or autocrine fashion on its surface receptors (23, 44). Five S1P-specific receptors, named S1P1–SIP5, have been identified in mammals (9). Evidence shows that S1P1, S1P2, and S1P3 transcripts are ubiquitously expressed in several mammalian tissues, whereas S1P4 and SIP5 are mostly expressed by hematopoietic-derived cells (59).

Recent studies have revealed that sphingolipids are also active in skeletal muscle, where the full enzymatic machinery for sphingomyelin degradation is present (48). In fact, skeletal muscle possesses substantial endogenous levels of Sph and S1P (4, 17, 48, 49). Moreover, Sph and S1P are present at very high levels in the blood (12, 58). Skeletal muscle is a rich source of Sph kinase, the enzyme that converts Sph to S1P (42), providing for additional S1P generated at the muscle fiber surface. Northern blot analysis suggests the presence of S1P1, S1P2, and S1P3 receptors in skeletal muscle, with S1P1 expressed at the highest level (59). Consistent with the expression of S1P receptors in skeletal muscle, we have recently demonstrated that exogenous S1P exerts a protective action during muscle fatigue (13). Recent evidence demonstrates a significant role of S1P in the proliferation of the skeletal muscle cell line C2C12 (18, 34). Moreover, S1P has been proven to be critical for the activation of satellite cells (40). Altogether, these data suggest that S1P may have a role in the trophism of adult skeletal muscle, possibly through the activation of S1P receptors. Therefore, a first aim of present work was to demonstrate the expression and localize S1P receptor proteins in rat soleus skeletal muscle. Utilizing a muscle denervation model, we also investigated whether S1P has a role in muscle fiber trophism.

Muscle fiber size results from a balance between protein synthesis and protein breakdown. Signaling pathways that control protein synthesis play a major role in muscle hypertrophy, while accelerated proteolysis, mainly via the ubiquitin-proteasome pathway, is a major cause of muscle atrophy induced by denervation, disuse, and various pathological conditions (21). Exercise and diverse growth factors regulate muscle mass. Loss of activity, such as in denervation, produces a progressive and severe reduction of muscle mass and a transition of muscle fiber phenotype, as well as alterations in contractile properties (37, 38).

The present work demonstrates the expression and localization of S1P1 and S1P3 receptors in skeletal muscle and shows that S1P acts as a growth factor during denervation of skeletal muscle. Exogenous application of S1P counters the reduction of muscle mass caused by denervation, whereas neutralization of the extracellular lipid with a specific anti-S1P monoclonal antibody accelerated the atrophy caused by denervation. During denervation, significant changes in the expression level of S1P receptors also were observed.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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All procedures were performed in accordance with Italian laws. The study and the experimental protocols were approved by the Ethics Committee of the Medical Faculty of the University of Padua and by the Italian Health Ministry.

Surgical procedures. Forty-four male Wistar rats (2–3 mo old, 291 ± 10 g mean body weight) were used. All surgical procedures were performed in a single session. Rats were anesthetized by an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (20 mg/kg). The sciatic nerve was cut bilaterally at the level of trochanter. About 0.5–1 cm of the peripheral nerve stump was removed, and the proximal stump was sutured into a superficial muscle to avoid reinnervation and obtain a permanent denervation of the lower hindlimb. The treatment of innervated and denervated muscle with bioactive lipids was performed as previously described (36). Mini-osmotic pumps (Alzet 2002; 200-µl volume, 0.5 µl/h release rate, 14 days of discharge time; Cupertino, CA) were charged with physiological solution containing either 10 µM S1P (Avanti Polar Lipids, Alabaster, AL), 10 µM Sph (Sigma, St. Louis, MO), or 10 µM Sph and 20 µM N,N-dimethylsphingosine (DMS), an inhibitor of Sph kinase (Avanti Polar Lipids). The pump was implanted in the interscapular region and connected to left soleus muscle by means of a polyethylene catheter. By this procedure, the lipids were released in continuum on the muscle. Seven or 14 days after the injury, rats were euthanized by CO2 inhalation, and soleus muscle of both legs was removed, weighed, and frozen in isopentane cooled in liquid nitrogen. The release of the sole physiological solution from the pump did not affect muscle properties, indicating that all observed alterations were produced by the lipids (data not shown).

Animal treatment with the antibody specific for the lipid S1P was performed in eight Swiss male mice (2 mo old, 28 ± 2 g body weight). Denervation was produced as described above for rats. Anesthesia was carried out by the intraperitoneal injection of ketamine (14 mg/kg) and xylazine (14 mg/kg). The monoclonal antibody to S1P (Lpath Therapeutics, San Diego, CA) was administered intraperitoneally in four animals (10 mg/kg body wt) 2 days before and 1 and 4 days after the nerve was cut. Seven days after denervation, the right (innervated) and left (denervated) soleus muscles from treated and untreated animals were removed, weighed, and frozen in isopentane cooled in liquid nitrogen.

RT-PCR. Total RNA was isolated from soleus muscle using the RNeasy midi kit according to the manufacturer's instructions (Qiagen, Valencia, CA). RNA was reverse transcribed with a First-Strand cDNA synthesis RT-PCR kit (AMV) as described by the manufacturer (Roche, Basel, Switzerland). The following forward and reverse primers were used (31): S1P1, forward 5'-TCATCGTCCGGCATTACAACTA-3', reverse 5'-GAGTGAGCTTGTAGGTGGTG-3' (273 bp); S1P2, forward 5'-ATGGGCAGCTTGTACTCGGAG-3', reverse 5'-CAGCCAGCAGACGATAAAGAC-3' (720 bp); and S1P3, forward 5'-CTTGGTCATCTGCAGCTTCATC-3', reverse 5'-TGCTGATGCAGAAGGCAATGTA-3', (460 bp). The S16 ribosomal protein was utilized as an internal marker (720 bp), and the GeneRulers DNA ladder markers were used as a reference (Fermentas, Burlington, Ontario, Canada). After 2 min of predenaturation at 94°C, Taq DNA polymerase (Promega, Madison, WI) was added, and 30 amplification cycles were run, each consisting of denaturation for 30 s at 94°C, annealing for 1 min at 59°C, and elongation for 1 min at 72°C. The last elongation period was 5 min at 72°C. The products were separated on 1.0% (wt/vol) DNA agarose gels and stained with ethidium bromide. The fragments were excised from the gel, purified, quantified, and submitted for DNA sequencing at the University of Padua CRIBI Core Facility.

Antibodies. The rabbit polyclonal antibody specific for S1P1 receptor, previously characterized (41), was used for Western blotting at 1:3,000 dilution in 10% low-fat milk in Tris-buffered saline (TBS) and for immunofluorescence at 1:200 dilution in 10% low-fat milk in phosphate-buffered saline (PBS), incubated in both cases for 1 h. A rabbit polyclonal antibody specific for S1P3 receptor was obtained by using the peptide KLAGRLRDPPEGGTL derived from the NH2-terminal domain of the mouse receptor. The peptide corresponding to this sequence was synthesized by Sigma-Genosys (Woodlands, TX). A rabbit polyclonal anti-S1P3 antibody was generated using standard techniques and titered via ELISA. The serum from the final bleed was purified by protein A column chromatography (Bio-Rad, Hercules, CA). The anti-S1P3 antibody was utilized for Western blotting at 1:1,000 dilution in 10% low-fat milk in TBS and for immunofluorescence at 1:150 dilution in 10% low-fat milk in PBS, incubated for 1 h. The monoclonal antibody specific for {alpha}-actinin (EA-53; Sigma) was used for Western blotting at 1:4,000 dilution in 10% low-fat milk and 0.1% Tween 20 in TBS, incubated for 1 h. The monoclonal antibody specific for {alpha}-sarcoglycan (NCL-a-SARC; Novocastra, Newcastle Upon Tyne, UK) was used for immunofluorescence staining at 1:50 dilution [in 5% fetal bovine serum (Sigma) in PBS], incubated for 1 h. The monoclonal antibody specific for the {alpha}1S subunit of dihydropyridine (DHP) receptor (MAB 427; Chemicon, Temecula, CA) was used at 1:250 dilution (in PBS), incubated overnight at 4°C. The monoclonal antibody specific for MyoD (58A; Dako, Glostrup, Denmark) was used at 1:400 dilution (in 5% low-fat milk and 0.05% Tween 20 in TBS), incubated overnight at 4°C. The monoclonal antibody specific for myogenin (F5D; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was used at 1:100 dilution (in 10% low-fat milk and 0.1% Tween 20 in TBS), incubated overnight at 4°C.

Western blotting analysis. Western immunoblotting was performed on muscle fragments dissolved in SDS-PAGE buffer supplemented with Complete protease inhibitor cocktail (Roche). Muscle lysates were electrophoresed on 10% SDS-PAGE gels. Electroblotting was performed as previously described (19). Nitrocellulose filters were probed with the selected primary antibody at the conditions described above. The secondary antibody for Western blot revelation of S1P receptors was a peroxidase-conjugated anti-rabbit antibody (Chemicon) used at 1:4,000 dilution (in 2% low-fat milk and 0.1% Tween 20 in TBS), incubated for 1 h. The secondary antibody for Western blot revelation of MyoD, myogenin, and {alpha}-actinin was a peroxidase-conjugated anti-mouse antibody (Dako) used at 1:4,000 dilution (in the same buffer as the cognate primary antibody), incubated for 1 h. Visualization of reaction bands was performed by either diaminobenzidine staining or ECL chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). Signal intensities were evaluated by densitometry (GS-700 Imaging Densitometer; Bio-Rad). Anti-S1P monoclonal antibody (56) was a kind gift (Sphingomab) from Lpath Therapeutics.

Histological and immunofluorescence analysis. Muscles were frozen in liquid nitrogen in a slightly stretched position. Serial cross sections (8 µm thick) were cut in a cryostat microtome (Slee, London, UK) set at –24 ± 1°C. For the histochemical analysis, hematoxylin-eosin staining was performed on muscle sections to examine the general morphology and to determine the cross-sectional area (CSA) of individual fibers. Morphometric analysis was performed on digital photographs of muscle fibers analyzed using the NIH ImageJ imaging software. More than 300 fibers per muscle were measured from at least 5 randomly chosen areas. Immunofluorescence staining was performed as previously described (20). Briefly, muscle sections were incubated with selected antibodies under the conditions described above. Immunofluorescence double staining was performed by first incubating the cryostat section with the antibody specific for the S1P1 receptor, followed by the second antibody. To mark either sarcolemma or the T-tubule membranes, the section was incubated with the antibody specific for {alpha}-sarcoglycan (for 1 h) or the {alpha}1S subunit of DHP receptor (overnight at 4°C), respectively. To decorate the neuromuscular junction, after incubation with the antibody to the S1P1 receptor, the section was incubated with FITC-labeled {alpha}-bungarotoxin (1 µg/ml; Sigma). After rinsing, the appropriate secondary antibodies were incubated for 1 h at room temperature: FITC-conjugated anti-rabbit (1:80, diluted in 2% rat serum in PBS; Sigma), TRITC-conjugated anti-rabbit (1:100, diluted in 2% rat serum in PBS; Dako), or TRITC conjugated anti-mouse (1:100, diluted in 5% fetal bovine serum and 2% rat serum in PBS; Dako). Muscle sections were examined with a Leica RD100 fluorescence microscopy equipped with a digital camera.

Apoptotic nuclei were counted in muscle transverse sections by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay (In Situ Cell Death Detection kit; Roche) according to the manufacturer's instructions. Muscle sections were fixed with 4% formaldehyde for 20 min, rinsed with PBS, and permeabilized with 0.5% Triton X-100 for 5 min. After washing with PBS, the sections were saturated with 10% BSA for 30 min at room temperature and then incubated with a mixture of nucleotides and deoxynucleotidyl transferase enzyme in a humidified chamber at 37°C for 1 h. After washing, the nuclei were counterstained with Hoechst (Sigma).

Determination of Sph levels by reverse-phase HPLC. The determination of Sph levels in rat soleus muscle was performed by HPLC according to published procedures (13).

SDS-PAGE of whole muscle extracts. Analysis of myosin heavy chain (MyHC) isoforms of the whole muscle was performed using the SDS-PAGE method of Talmadge and Roy (53a), as previously described (14). Small muscle fragments were weighed, ground with a ceramic pestle in liquid nitrogen, and extracted at 2 mg/ml in SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2.3% SDS, 5% 2-mercaptoethanol, and 10% glycerol). Forty micrograms of each muscle sample were electrophoresed on 8% SDS-PAGE slab gels. MyHC protein bands were revealed by Coomassie brilliant blue staining. MyHC isoform percentage composition was determined by densitometry of gels using a Bio-Rad imaging densitometer (GS-670).

Statistical analysis. All values are means ± SE. All data originate from experimental values of one muscle and its contralateral. Comparisons of mean values were performed after analysis of variance (ANOVA) and Tukey's post hoc test. For the comparisons of muscle fiber CSA, more than 300 fibers from each muscle were measured. The mean CSA values from individual muscles were then pooled, and the resulting mean was compared with that of the contralateral muscle. After ANOVA, the significance of the results was determined using Student's t-test. Differences were considered significant at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression and localization of S1P receptors in soleus skeletal muscle. RT-PCR analysis demonstrated that soleus muscle expresses the transcripts of S1P1 and S1P3 receptors, with S1P1 at higher level (Fig. 1A), whereas that of S1P2 was not detectable. The expression of S1P1 and S1P3 receptor proteins was analyzed by Western blot in rat heart, as a positive control (41), and in soleus muscle homogenates. The results show that both S1P1 and S1P3 receptor proteins are expressed in soleus muscle, with S1P1 at the highest level (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Expression of sphingosine 1-phosphate (S1P) receptors in soleus muscle. A: typical RT-PCR analysis of S1P receptors in soleus skeletal muscle. Arrowheads indicate the expected size of S1P receptor RNA. Soleus muscle expresses the transcripts of S1P1 and S1P3 receptors, whereas that of S1P2 was not detected. The RT-PCR analysis was performed in 3 muscles. The DNA ladder marker, in 100-bp steps, is shown at left. The S16 ribosomal protein was used as the housekeeping reference. B: typical Western blot analysis of S1P1 and S1P3 receptor expression in rat tissue homogenates purified from heart and soleus muscle. {alpha}-Actinin was used as an internal reference. C: densitometric analysis of S1P1 and S1P3 receptor expression levels in rat heart (open bars) and soleus muscle (shaded bars). Data were obtained from 3 separate experiments (means ± SE) and normalized to the {alpha}-actinin level. *P < 0.05; **P < 0.01 compared with expression in the heart.

 
Immunofluorescence staining revealed that S1P1 and S1P3 receptors are expressed on soleus muscle sarcolemma (Fig. 2, A and D). Specific sarcolemma localization was confirmed by the colocalization of these S1P receptors with the transmembrane sarcolemmal protein {alpha}-sarcoglycan. As shown by the Western blot data, the expression of the S1P1 receptor appeared higher than that of S1P3. It is worth noting that both receptors decorated nuclei membranes (arrows in Fig. 2, A and D). In longitudinal sections of soleus muscle, both receptors colocalized with the DHP receptor, known to be specifically expressed at the T-tubule membrane level (Fig. 2, B and E). Transverse sections of soleus muscle stained by specific S1P1 antibody and FITC-labeled {alpha}-bungarotoxin revealed the expression of S1P1 receptor, also at the neuromuscular junction level (Fig. 2C).


Figure 2
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  		Fig. 2. Immunofluorescence localization of S1P1 and S1P3 receptors in rat soleus muscle. A: transverse cryostat sections from rat soleus muscle stained with the antibody specific for S1P1 receptor and with antibody specific for the sarcolemmal transmembrane protein {alpha}-sarcoglycan ({alpha}-SG). A merged image of the 2 reactions (m1) shows the clear colocalization of the receptor with {alpha}-SG and demonstrates that the receptor is localized at the plasma membrane. The antibody decorates the muscle fiber nuclear membrane (arrows), as also demonstrated by Hoechst nuclear staining (m2). B: longitudinal cryostat sections of soleus muscle stained with the antibody specific for S1P1 receptor (inset is a negative control) and with antibody specific for the dihydropyridine receptor (DHPR). The merged image of the 2 immunofluorescence reactions demonstrates the colocalization of the 2 receptors at the T-tubule membranes. C: transverse cryostat sections of soleus muscle stained with the antibody to S1P1 receptor and with FITC-labeled {alpha}-bungarotoxin ({alpha}-BTX), a selective neuromuscular junction probe. The colocalization (merge) of the 2 reactions demonstrates the presence of S1P1 receptor at the neuromuscular junction. D: transverse cryostat sections from rat soleus muscle stained with the antibody specific for S1P3 receptor (inset is a negative control) and with antibody specific for {alpha}-SG. The merged image of the 2 reactions (m1) shows that S1P3 receptor has a clear localization at the cell membrane. In addition, the receptor also is localized to the nuclear membrane (arrows), as shown by superimposing the merged image with Hoechst nuclear staining (m2). E: longitudinal cryostat sections of soleus muscle stained with the specific antibody for S1P3 receptor and with antibody specific for the DHPR. The merged image of the 2 immunofluorescence reactions demonstrates the colocalization of the 2 receptors at the T-tubule membranes.

 
Effects of exogenous S1P on denervated muscle. The presence of S1P-sensitive receptors at the muscle fiber surface reinforces the possibility of their physiological roles in skeletal muscle (13, 48). Because of the well-known role of S1P in promoting cell growth and hypertrophy (23, 47), we investigated possible effects produced by the extracellular addition of S1P on the progress of atrophy during denervation of rat soleus skeletal muscle.

Denervation of soleus produced a significant and progressive decreases in muscle weights, muscle-to-body weight ratios, and fiber CSA. After 7 and 14 days, soleus denervation caused, respectively, 45 and 72% reductions in muscle mass, 38 and 63% decreases in muscle-to-body weight ratios, and 46 and 75% decreases in muscle fiber CSA (Fig. 3). To test the trophic effects of S1P on soleus mass, S1P was supplemented by infusion from a mini-osmotic pump (filled with either 10 or 50 µM S1P) to the denervated muscle. The resulting infusion of S1P in rats produced significant positive effects on all growth parameters by attenuating muscle mass and CSA reduction both after 7 and 14 days of denervation (Fig. 3). Infusion with 10 µM S1P caused positive effects on denervation atrophy that were significant after 14 days of denervation (not shown), whereas infusion with high S1P (50 µM) was effective at both 7 and 14 days (Fig. 3).


Figure 3
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  		Fig. 3. Morphometric analysis of denervated soleus muscle treated with S1P receptor agonists. Muscle mass, muscle-to-body weight ratio, and muscle fiber cross sectional area (CSA) were measured in normally innervated soleus muscles and in muscles denervated for 7 and 14 days, either untreated (open bars) or (the contralateral muscle) treated with 50 µM S1P or 10 µM Sph (shaded bars). The lipids were delivered to the denervated muscle through mini-osmotic pumps, as described in METHODS. Values are means ± SE. Measurements of muscle and body mass were performed in 5 controls and in 5–6 denervated rats, and measurements of fiber CSA were performed in muscles from 5 different animals. At least 300 fibers were measured in each muscle. aP < 0.005; bP < 0.002 compared with control innervated muscles. *P < 0.05; **P < 0.01; ***P < 0.001 compared with untreated contralateral denervated muscles.

 
Since S1P has a short half-life in the extracellular fluid (2) and the circulating lipid is mostly bound to serum proteins, and, as a consequence, only partly bioavailable (39), we evaluated whether the S1P precursor Sph could be more efficacious in preventing denervation muscle atrophy. This hypothesis is based on the fact that Sph could be transformed into S1P by extracellular Sph kinase (13, 30, 32, 45) and that the kinase is expressed at the extracellular surfaces of skeletal muscle (35). Therefore, we expected that by delivering Sph to the muscle, a "local pulse" of S1P would be produced such that the concentration of S1P in the microenvironment would be transiently elevated.

As a consequence of Sph infusion with the mini-osmotic pump (10 µM), soleus muscles excised from denervated animals exhibited higher muscle weights and muscle-to-body weight ratios compared with contralateral, denervated muscles. S1P at 10 and 50 µM produced comparable positive effects in attenuating the atrophy of denervated muscle. The presence of either S1P or Sph attenuated by ~40% the fiber atrophy caused by 14 days of denervation compared with the atrophy produced in the contralateral denervated fibers (Figs. 3 and 4).


Figure 4
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  		Fig. 4. Protective action of S1P and Sph during denervation of soleus muscle. Hematoxylin-eosin staining of normally innervated (control) soleus muscle and of 14-day denervated muscles in either the absence (untreated) or presence of 50 µM S1P (+ S1P) or 10 µM Sph (+ Sph). The presence in the extracellular space of S1P and of Sph, the S1P precursor, alleviated the drop in muscle fiber CSA caused by denervation. The protective action of Sph during denervation was abolished by the contemporary presence of Sph and N,N-dimethylsphingosine (+ Sph & DMS), specific inhibitor of Sph kinase, the enzyme converting Sph into S1P. Inhibition of the kinase, however, reveals the cytotoxic action of Sph leading to the appearance of pathological signs, such as, for example, edema, endomysial proliferation, large heterogeneity of fiber size, fiber degeneration, central nuclei and fiber splitting. Bar, 100 µm.

 
In general, Sph is associated with cell growth arrest and apoptosis (1, 11, 43, 53). However, we (13) recently showed that in skeletal muscle, Sph, potentially cytotoxic, could be converted into S1P through a rheostat mechanism operated by Sph kinase. To confirm this, denervated muscles were treated with Sph together with DMS, a specific inhibitor of Sph kinase (57). In the presence of both Sph and DMS, the beneficial positive action of Sph was no longer evident, and pathological alterations on denervated fibers were observed instead (Fig. 4).

Since we recently demonstrated that endogenous Sph level could vary depending on muscle exercise (13), we also analyzed whether the lipid level varied during denervation. In contrast, endogenous Sph levels of soleus muscle at 7 (12.61 ± 2.28 nmol/g, n = 6) and 14 days (11.28 ± 2.51 nmol/g, n = 6) after denervation were similar to those of control muscles (12.72 ± 1.53 nmol/g, n = 12).

In addition, because of the proapoptotic action of Sph (1, 43, 53), and since apoptosis has been suggested to contribute to denervation atrophy (5, 6), we measured apoptotic nuclei in denervated and denervated-treated soleus muscles. Denervation produced a significant increase of apoptotic nuclei after 7 (1,248 ± 100 per µm3, P < 0.001, n = 6 muscles) and 14 days (2,193 ± 280 per µm3, P < 0.001, n = 7 muscles) with respect to the apoptotic nuclei present in the control innervated muscle (30 ± 15 per µm3, n = 5 muscles). However, neither supplementation with exogenous S1P nor that with Sph affected the number of apoptotic nuclei during denervation. In fact, the number of apoptotic nuclei after 7 and 14 days of denervation was, in the presence of S1P, 1,101 ± 50 (n = 3) and 1,991 ± 236 (n = 3), respectively, whereas in the presence of Sph, it was 980 ± 230 (n = 3) and 2,337 ± 358 (n = 3), respectively.

The above results indicate that S1P could exert a trophic action on the denervated muscle by attenuating the progress of muscle atrophy. Since considerable serum levels of S1P have been demonstrated (16, 58), we investigated the action of endogenous S1P on denervation atrophy by substantially reducing the circulating lipid by means of a specific monoclonal antibody that recognizes and neutralizes bioactive S1P in the extracellular space (56). The anti-S1P monoclonal antibody recognizes S1P from all species because S1P is one molecular form. However, the anti-S1P monoclonal antibody is a murine IgG1. Thus, to obviate the rat-against-mouse antibody reaction, denervation experiments using the murine anti-S1P antibody were carried out in mice. Animals were treated with the antibody specific for S1P at doses that neutralized the bioactive S1P (56). For the innervated muscles, systemic administration of the anti-S1P monoclonal antibody did not alter animal body weights (29.3 ± 1.6 g, n = 4, and 28.9 ± 0.9 g, n = 4, in untreated and treated mice, respectively), muscle mass (8.12 ± 0.57 and 7.45 ± 0.31 mg, n = 4), or muscle fiber CSA (Fig. 5B).


Figure 5
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  		Fig. 5. Low levels of circulating S1P aggravate soleus muscle denervation atrophy. A: hematoxylin-eosin staining of soleus muscle cryostat sections from innervated and 7-day denervated control mice and from 7-day denervated mice treated with the antibody specific for S1P. B: muscle fiber CSA of innervated and denervated soleus muscles from mice either untreated (open bars) or treated (shaded bars) with the antibody specific for S1P (anti-S1P). Values are means ± SE of pooled mean values from ~300 fibers measured in each muscle from 4 animals in each experimental group. aP < 0.005 compared with innervated muscles. bP < 0.05 compared with untreated denervated muscles.

 
As expected, 7 days after denervation, soleus muscle mass and fiber CSA decreased by ~24 and 40%, respectively, values similar to those produced by denervation of rat soleus (Fig. 5). However, neutralization of endogenous S1P with the anti-S1P antibody accelerated the reductions in CSA produced by 7 days of denervation with mean muscle fiber CSA reduced from 737 ± 69 to 538 ± 43 µm2 (P < 0.005) compared with denervated muscles from animals not treated with antibody (Fig. 5B).

S1P receptors expression level in denervated soleus muscle. The expression level of both S1P1 and S1P3 receptors significantly diminished during denervation of rat soleus muscle. Western blot analysis of whole muscle lysates demonstrated that protein level of both receptors decreased progressively during denervation, with S1P3 being almost undetectable 14 days after denervation (Fig. 6, A and D). Moreover, immunofluorescence analysis in rat denervated soleus fibers showed a reduced expression of both receptors at the plasma membrane (Fig. 6, B and C), whereas expression of the receptors at the nuclear membrane, particularly that of S1P3 receptor, appeared to be higher (Fig. 6C). The in vivo infusion with either S1P (both 10 and 50 µM) or Sph during denervation was ineffective on the overall expression levels of these receptors (not shown).


Figure 6
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  		Fig. 6. Expression level of S1P receptors during denervation of rat soleus muscle. A: Western blot analysis of S1P1 and S1P3 receptor levels in homogenates of innervated control soleus muscle and in the denervated muscle after 7 and 14 days. B: immunofluorescence analysis of S1P1 receptor in cryostat sections from innervated control (C) and the 7- and 14-day denervated rat soleus muscle. C: immunofluorescence analysis of S1P3 receptor in cryostat sections from innervated control (C) and the 7- and 14-day denervated soleus muscle. Bar, 50 µm. Images at bottom are higher magnifications of soleus muscle cryostat sections stained with the anti-S1P3 antibody, the antibody specific for {alpha}-SG, and the merge of the 2 reactions (m1) plus the Hoechst nuclear staining (m2). Arrowheads indicate the intense staining of the nuclear membrane produced by the anti-S1P3 antibody. D: densitometric analysis of S1P1 (open bars) and S1P3 (shaded bars) receptor expression during denervation. The expression levels of the 2 receptors were measured in homogenates of innervated soleus muscle (n = 6) and of muscles after 7 (n = 13) and 14 days (n = 13) of denervation. Values are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001 either compared with innervated muscle or between 7- and 14-day denervated muscles (as indicated).

 
Effect of S1P on MyoD and myogenin expression during denervation. Western blot analysis showed that the expression level of the myogenic transcription factors MyoD and myogenin progressively and significantly increased during denervation, as recently demonstrated by others (26). However, in vivo infusion of the denervated muscles with either S1P or Sph further increased the expression level of the two transcription factors. In the presence of S1P and Sph 7 days after denervation, the expression levels of both MyoD and myogenin were significantly greater than in the contralateral denervated muscles (Fig. 7). After 14 days, the presence of S1P and Sph during denervation caused large increases of MyoD expression but were completely ineffective on myogenin expression (Fig. 7).


Figure 7
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  		Fig. 7. Effects of S1P and Sph supplementation on the expression of MyoD and myogenin during denervation of rat soleus muscle. Densitometric analysis of MyoD and myogenin expression in innervated soleus muscle and in muscles denervated for 7 and 14 days, either untreated ({circ}) or treated (the contralateral) with 10 µM S1P (bullet), 50 µM S1P ({blacksquare}), or 10 µM Sph ({blacktriangleup}). The analysis was performed in 5 distinct control and treated muscles (means ± SE), and the expression of the myogenic factors was normalized to the {alpha}-actinin level. *P < 0.05; **P < 0.001 compared with contralateral denervated muscles.

 
Effect of S1P on myosin heavy chain isoforms expression during denervation. Inactivity of soleus muscle as a consequence of denervation has been shown to promote the progressive atrophy of muscle fibers (25). Atrophy is associated to substantial changes in the MyHC composition, particularly a decrease of the slow MyHC (type 1) compensated by the higher expression of fast isoforms (25). In agreement with these findings, Fig. 8 shows that after 14 days of denervation, soleus muscle exhibited significant increases of types 2X and 2B (almost undetectable in the innervated muscle) with detriment of types 1 and 2A MyHC. The infusion of denervated soleus with either S1P or Sph attenuated the slow-to-fast transformation due to inactivity. Interestingly, the de novo expression of type 2B MyHC was almost abolished in the presence of the two lipids (Fig. 8, A and B).


Figure 8
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  		Fig. 8. Effects of S1P and Sph supplementation on the expression of myosin heavy chain (MyHC) isoforms on the 14-day denervated rat soleus muscle. Densitometric analysis of MyHC expression in innervated soleus muscle and in muscles denervated for 14 days, either untreated or treated with S1P or Sph. MyHC isoforms 1, 2A, 2X, and 2B were separated by SDS-PAGE according to the method of Talmadge and Roy (53a). A: comparison of MyHC composition between 14-day denervated soleus muscle (14-Den; shaded bars) and its denervated contralateral infused with 50 µM S1P (solid bars, n = 4). Open bars represent the control innervated muscle (IN; n = 5) as reference. B: comparison of MyHC composition between 14-day denervated soleus muscle (shaded bars) and its denervated contralateral infused with 10 µM Sph (solid bars, n = 4). Open bars represent the control innervated muscle as reference. aP < 0.05; bP < 0.001 compared with control muscles. *P < 0.05 compared with untreated contralateral denervated muscles.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The present work was aimed at investigating the possible trophic action of the bioactive lipid S1P in skeletal muscle and, at the same time, the expression and localization of the S1P-specific receptors S1P1 and S1P3. The study demonstrates for the first time that S1P1 and S1P3 receptors are localized at the plasma membrane and the T-tubule membranes of skeletal muscle fibers. In addition, S1P1 receptor is also localized at the neuromuscular junction. The study shows that S1P reduces the development of muscle fibers atrophy produced by denervation and suggests that the lipid may play an important physiological role in protection against denervation atrophy.

The bioactive lipid S1P regulates diverse important cell function, such as growth, survival, vascular maturation, and angiogenesis (45, 52), by specifically stimulating five distinct G protein-coupled receptors, designated S1P1–S1P5, each activating distinct downstream pathways (9). S1P1 receptor is expressed at cardiomyocyte sarcolemma (41), and stimulation with S1P induces substantial hypertrophic changes, effects that are inhibited by an antibody specific for the S1P1 receptor (47). Our results demonstrate that S1P1 receptor is expressed at skeletal muscle sarcolemma and may well be a good candidate in mediating the trophic effects of S1P.

To investigate the possible trophic effect of S1P in skeletal muscle, we utilized the muscle denervation model, which causes rapid and marked fiber atrophy, especially in the soleus muscle (37, 46). Muscle atrophy is characterized by accelerated proteolysis, mainly via the ubiquitin-proteasome pathway (28), leading to progressive and severe reduction of muscle mass and alteration of contractile properties (21, 37). There is evidence for the involvement of several regulatory mechanisms in the breakdown of myofibrillar proteins and the resulting muscle atrophy. Activation of caspase-3 is probably an initial trigger of both muscle proteolysis and apoptosis in muscle atrophy (50). During denervation, the number of apoptotic nuclei transiently increases in the first 15 days (54). The exogenous addition of S1P and Sph, however, does not affect the number of apoptotic nuclei of the denervated muscle. This observation suggests that S1P does not protect the muscle from denervation-induced apoptosis, as it does in response to other stimuli (11, 32), but must exert its protective action by other means, likely involving trophic effects. As is the case for our results with S1P, IGF-1 also reduces the rate of denervation-induced myofiber atrophy (15, 22, 51), as also does ciliary neurotrophic factor (24). Hence, there is evidence from the literature that trophic influences can retard denervation atrophy by means not involving apoptosis.

Our results show that the exogenous supplementation of S1P or Sph to the denervated muscle exerted significant trophic effects. However, it is important to emphasize that to produce comparable effects, it was necessary to use fivefold higher concentrations of S1P than Sph. A possible explanation for this result is that extracellular S1P has a short half-life (2) and appears to be largely buffered by plasma proteins and thus only partly available (39). Thus it is probable that, for skeletal muscle, Sph may represent the main source for S1P, likely produced in the microenvironment of the muscle fiber surface through the action of Sph kinase. Consistent with this hypothesis is the finding that Sph kinase inhibitors could mitigate the Sph effect, suggesting that the protective action of the exogenous addition of Sph is the result of Sph conversion into S1P operated by the extracellular Sph kinase.

Since Sph is generated by the muscle during intense exercise and because of the presence at the cell surface of Sph kinase (12, 17, 35), it is possible that S1P exerts autocrine/paracrine trophic effects on skeletal muscle fibers and contributes to maintain muscle mass adequate to its activity. In the inactive denervated muscle, this autocrine effect is probably reduced, since the muscle does not produce the necessary extra doses of Sph. This concept was reinforced by the finding that neutralization of extracellular S1P with the anti-S1P antibody accelerated the denervation-induced reductions in CSA, suggesting that Sph conversion to S1P is important in retarding atrophy. These results reinforce the relevance of the "rheostat" mechanism operating in skeletal muscle cells by Sph kinase, especially during intense exercise when the endogenous Sph level increases (13, 17). It is worth noting that a similar rheostat mechanism was demonstrated in other cell types (32, 45) and probably also operates in cardiomyocytes, where a significant amount of the Sph produced is released in the extracellular medium and rapidly phosphorylated to S1P (8).

An early response of muscle denervation is the transient activation of satellite cells and changes of the expression of myogenic regulatory factors, generally considered as a compensatory response effort. Satellite cells are normally quiescent in adult muscles and undergo repetitive proliferation and differentiation cycles over the first 3–4 wk after denervation (29). However, it has been recently demonstrated that during denervation, only a minor population of satellite cells begin to proliferate (26). It is thus possible that overexpression of MyoD and myogenin is more related to atrophying myofibers than to satellite cells. In fact, it has been proposed that the role of denervation-induced overexpression of MyoD in myofibers could be to prevent denervation-induced atrophy (27). Our results are consistent with this hypothesis. Specifically, the stimulation of S1P receptors slows down denervation-induced atrophy and at the same time increases the expression of myogenic factors. It is interesting to note that both S1P1 and S1P3 receptors have a clear nuclear membrane localization that persists after denervation. We hypothesize that nuclear membrane S1P receptors could be involved in the increased expression of the myogenic factors and their protective action on denervation atrophy. However, further work is needed to confirm this.

We have shown that denervation is associated with a downregulation of both S1P1 and S1P3 receptors. It is possible that the lack of receptors could contribute to muscle atrophy by reducing the trophic influence of S1P and also could be responsible for the phenotype transitions observed during denervation. Inactivity and/or lack of stimulation probably represents a proper stimulus for the downregulation of S1P1 and S1P3 receptors. Inactivity of soleus muscle clearly modifies MyHC composition, causing the overall decrease of slow MyHC (type 1) compensated by the higher expression of fast isoforms (25). Although the initial trigger is still poorly understood, the main signaling pathways controlling muscle fiber-type specification involve the Ca2+/calmodulin-regulated calcineurin (33). Importantly, S1P3 receptor appears to be mainly involved in Ca2+ mobilization (45, 52), and it is possible that signaling through this receptor could supplement skeletal muscle with the necessary Ca2+ mobilization to activate calcineurin. Our results are consistent with this possibility. Despite the reduced expression of the receptors, supplementation with S1P and Sph significantly attenuates the slow-to-fast transformation of MyHC due to denervation.

Our results show that S1P1 and S1P3 receptors also are localized in the T-tubule membranes of skeletal muscle, the main site of excitation-contraction coupling. This finding suggests that in addition to the trophic action possibly exerted through the receptor located at sarcolemma, S1P can have additional physiological effects on muscle contractility. In fact, we recently demonstrated that the exogenous application of S1P attenuates the development of fatigue, an effect that we hypothesized to be receptor mediated (13). Also, exogenous S1P modifies Ca2+ influx through voltage-dependent L-type Ca2+ channels in isolated skeletal muscle fibers, at concentrations compatible with receptors activation (3). In addition, expression of S1P1 receptors also was demonstrated at the neuromuscular junction. Although there are no specific localization data for S1P1 receptors in nervous tissue and/or synapses, biochemical and functional data indicate an important role of S1P. For example, S1P enhances neurotransmitter release from the intact nerve terminal of frogs (7), and S1P1 receptor may play a role in neuronal migration and neuritogenesis (10, 55).

In conclusion, the substantial expression of S1P receptors by skeletal muscle observed in this study may explain many of the emerging pleiotropic effects of S1P on muscle function. In particular, we show presently that these receptors likely mediate S1P's trophic effect in countering muscle mass loss during denervation, suggesting that S1P operates as a growth factor for muscle fibers. Since muscle atrophy is also a secondary consequence of a multitude of pathological conditions, the identification of S1P as a potent endogenous growth factor attenuating muscle mass loss could have significant implications to conditions such as disuse atrophy, orthostatic intolerance, and various muscle dystrophies.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by grants from Programma di Ricerca Scientifica di Rilevante Interesse Nazionale Ministero dell'Istruzione dell'Università della Ricerca Project MIUR-PRIN 2003 (to D. Danieli-Betto), Association Française contre les Myopathies Grant 12055 (to D. Danieli-Betto), the Italian Space Agency (to R. Betto, D. Sandonà, and L. Dalla Libera), and Consiglio Nazionale delle Ricerca institutional funds (to R. Betto and L. Dalla Libera).


   ACKNOWLEDGMENTS
 
We thank Stefania Picunio for participating in some experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Danieli-Betto, Dept. of Human Anatomy and Physiology, Univ. of Padua, Via Marzolo 3, 35131 Padua, Italy (e-mail: daniela.danieli{at}unipd.it)

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.


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