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Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice Background: Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice. Methods: We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles. Results: Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer- term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular nlacz/+ injection of S1P increased the number of Myf5 positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function. Conclusions: These data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD. Background degeneration, leading to muscle wasting over time. Duchenne muscular dystrophy (DMD) is a muscle wast- Since no effective treatment presently exists and the im- ing disease for which there is no cure. This severe X- mune response to dystrophin has hampered gene ther- linked recessive disease affects 1 in 3,500 male births apy approaches, new advances for the treatment of [1]. In dystrophic muscles, rounds of contractions result DMD are imperative [2,3]. in degeneration/regeneration cycles. In turn, dystrophic Previously, sphingosine-1-phosphate (S1P) has been im- muscle cannot regenerate sufficiently to overcome plicated in muscle repair, satellite cell proliferation, myo- blast differentiation in vitro and in non-diseased mouse models in vivo [2,4-6]. These essential roles for S1P in * Correspondence: hannele@u.washington.edu; morayma@u.washington.edu skeletal muscle regeneration suggested that elevation of Department of Biochemistry, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA S1P may have therapeutically beneficial effects in models Department of Pathology, School of Medicine, University of Washington, of disease [7]. More recently, S1P has been shown benefi- Seattle, WA 98195, USA cial for activating satellite cells in dystrophic muscles [8]. Full list of author information is available at the end of the article © 2013 Ieronimakis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 2 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Furthermore, an unbiased genetic modifier screen in Injections were 6 hours apart. This injection regimen and Drosophila revealed that by increasing S1P levels via re- dose was repeated for all subsequent experiments involv- duction of the lipid phosphate phosphatase 3 (LPP3) ing THI, but for longer-treatment durations as outlined. 4cv homolog, wunen, or the S1P lyase, sply, prevents to a large Six 5-MO mdx males were used for the experiments in degree dystrophic muscle wasting in flies [9]. In mice, Figure 1B, and Additional file 1: Figure S1 and S2. For elevation of S1P by the genetic reduction of S1P lyase Figures 2 and 3, and Additional file 1: Figures S3 to S7, six 4cv can be phenocopied pharmacologically via treatment 11-MO females and seven 16-MO males mdx were with the small molecule 2-acetyl-4(5)-tetrahydroxybutyl used for these experiments. In these mice, the left tibialis imidazole (THI) [10,11]. Furthermore, in Drosophila, anterior (TA) and quadriceps femoris (quads) were injured THI treatment also significantly suppresses the dys- with 10 nM CTX (Calbiochem, Darmstadt, Germany) trophic muscle phenotype [9]. from Naja nigricollis. Once more, THI-treated mice were Utilizing the mdx mouse model, we initiated studies injected IP with 250 μl 0.15 mg/ml THI in PBS, twice daily on the effect of increasing S1P levels in dystrophic mice, (injections 6 hours apart) immediately after injury and for and found that short-term treatment with THI improves the first 3 days following injury. The vehicle controls were 4cv muscle integrity and function following acute injury with injected IP with PBS. On day 4 post injury, 5-MO mdx cardiotoxin (CTX). THI treatment also leads to signi- animals were euthanized for S1P and creatine kinase (CK) 4cv ficant improvements of the pathology of dystrophic analysis. On day 17 post CTX, 11-MO and 16-MO mdx muscles, as indicated by the reduced accumulation of fi- mice were also injected IP with 1% Evans Blue dye (EBD) brosis and fat deposition in acutely injured muscles. In to label persistently damaged (dye permeable) muscle fi- turn, intramuscular injection of S1P resulted in an in- bers [12], and euthanized on day 18 post injury for his- creased number of myogenic cells and newly regenerat- topathology analysis. Muscles for S1P and expression 4cv ing fibers in vivo. S1P receptor 1 (S1PR1) is expressed by analysis (from 5-MO mdx ) were frozen directly in liquid many muscle cell types, particularly muscle fibers, and nitrogen, while muscles taken for histopathology were fro- phosphorylated S1PR1 is localized in the plasma mem- zen under liquid nitrogen cooled isopentane in optimal brane and intracellularly (perinuclear localization) of cutting temperature (OCT) compound. All myofibers muscle fibers. Intramuscular S1P administration results were measured for the minimum diameters on the cross- in increased levels of total and phosphorylated S1PR1 sections of mouse quadriceps muscle using ImageJ software and ribosomal protein S6 (rpS6). This suggests that in- (Bethesda, MD, USA). Between 750 and 850 myofibers creases in fiber size are mediated by anabolic pathways were counted for three mice treated with PBS or THI, that promote greater skeletal muscle mass and function, with or without CTX injury. For functional analysis potentially through S1PR1 signaling. Furthermore, ex vivo outlined in Figure 4B, 4.75- to 5-MO male mdx on a mdx/J administration of S1P improved specific force in uninjured C57BL/10 background (C57BL/10ScSn-Dmd )were dystrophic muscle. Similarly, longer-term THI treatment used for the 14-day treatment of THI or vehicle. Following of uninjured young mdx mice resulted in increased exten- the same dose and treatment regimen, mdx were treated sor digitorum longus (EDL) muscle force in the absence of with THI (n = 10) or vehicle (n = 9) for 14 days following CTX injury. Altogether, S1P acts at multiple levels in mus- CTX injury to left TAs and quadriceps. The same mdx cles, particularly in myogenic cells and muscle fibers, and strain was compared to wt C57BL/10 animals in Figure 4C collectively the actions of S1P in muscle are beneficial for and for exogenous S1P treatment depicted in Figure 4D. regeneration in the setting of muscular dystrophy. Animals used to evaluate the degree of CTX injury in EDL (Additional file 1: Figure S8) were 4-MO female mdx mdx/J Methods (n = 4, C57BL/10ScSn-Dmd background), injected in Animal procedure left TAs with CTX and with approximately 3 μlIndia ink, Experiments involving animals were undertaken in ac- added to the tip of the needle to mark injection penetra- cordance with approved guidelines and ethical approval tion. Following CTX injections, mice were immediately from the Institutional Animal Care and Use Committee, injected IP with 1% EBD. Both left (injured) and contralat- University of Washington, Seattle, WA, USA. eral uninjured TA and EDL muscles were harvested and frozen in OCT compound 12 hours post injury. THI injections in injured mice Peripheral blood cells from 1.5-month-old (MO) wild THI treatment in drinking water of young, uninjured mdx type (wt) C57BL/k6 and mdx mice on a C57BL/k6 back- mice mdx-4Cv 4cv ground (B6Ros.Cg-Dmd /J, herein referred to as Beginning at 4 weeks of age, male mdx were treated 4cv mdx ) were analyzed (Figure 1A). Blood was collected with THI (n = 4) or vehicle (n = 3) for 4 weeks, and ana- before and 12 hours following the last of two 250 μl in- lyzed by EDL myography at 8 weeks of age. For this traperitoneal (IP) injections of 0.15 mg/ml THI in PBS. treatment we followed the dose and conditions described Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 3 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 1 IP injection of THI reduces peripheral blood leukocytes and increases S1P levels in most tissues. (A) Leukocytes were analyzed 4cv from the peripheral blood of 1.5-MO mdx mice (n = 3) before and 12 hours following treatment with THI (2 × 250 μl 0.15 mg/ml IP injections, 6 hours apart). IP administration of THI significantly reduced circulating leukocytes to values below or near age-matched wt (n = 4). The average value of each population is listed in the table below the bar graph. Values between pre and post THI, and wt were also significant by ANOVA 4cv (P <0.05) for all leukocytes except monocytes. (B) mdx mice (n = 6, 5-MO) were treated with THI or vehicle for 3 days (2 × 250 μl 0.15 mg/ml IP injections per day) following CTX injury to assess changes in S1P muscle content. Muscles and spleens were harvested on day 4 post injury for S1P analysis by LC-MS/MS. Results indicate S1P levels in spleen and injured quadriceps (quads) were significantly elevated with THI treatment. Interestingly, uninjured quadriceps did not show a significant increase of S1P, whereas uninjured TA muscles did. *P <0.05 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; IP, intraperitoneal; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole; wt, wild type. by Schwab et al. [11]. Briefly, 50 mg/l THI was adminis- 20 μl per sample using the Hemavet 950 FS system tered ad libitum. The vehicle consisted of water at pH (Drew Scientific, Dallas, TX, USA). 2.8 containing 10 g/l glucose. Analysis of gene expression by quantitative reverse Peripheral blood cell analysis transcription-PCR (RT-PCR) Blood was collected via retro-orbital blood collection Total RNA (RNeasy Kit, Qiagen, Venlo, Netherlands) 4cv using heparinized capillaries and transferred to blood was prepared from mdx TA muscles homogenized collection tubes containing a final concentration of 1.6 under liquid nitrogen by mortar and pestle. Methods for mg/ml EDTA (SARSTEDT, Nümbrecht, Germany) for RNA isolation and cDNA generation were in accordance analysis. Analysis of whole blood was undertaken with with manufacturer’s protocols using reverse transcriptase Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 4 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 2 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 5 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 2 Dystrophic pathology following muscle injury is improved with THI treatment. (A) Experimental schematic of THI (0.075 μg/day) 4cv and PBS (vehicle)-treated mdx mice injected IP twice daily for the first 72 hours following CTX injury. Muscles from aged mdx mice (n = 7, THI-treated: 3 × 11-MO females, 4 × 16-MO males; n = 6 vehicle-treated: 3 × 11-MO females, 3 × 16-MO males) were harvested for histopathology analysis 18 days post CTX injury. (B) Histological quantification of picrosirius red staining indicates lower fibrotic accumulation following injury in both TA and quadriceps (quads) muscles from mice treated with THI. For CTX-injected muscles, damaged regions of muscle (for example fields with the greatest accumulation of sirius red staining) were quantified for both THI and vehicle-treated mice. The level of fibrosis was not significantly different between treated and control (vehicle) uninjured quadriceps; however, uninjured TA muscles from 11-MO THI-treated mice had lower fibrosis compared to control TA muscles. For each muscle, three separate sections (200 μm apart in longitudinal distance) were analyzed. (C) Representative photographs of injured quadriceps stained with picrosirius red and fast green depict collagen deposition (red staining), while muscle morphology and organization is depicted with hematoxylin and eosin staining. Scale bars = 50 μm. (D) Oil Red O staining depicts fat deposits (arrows) over the entire CSA of THI-treated and vehicle-injured quadriceps from 16-MO males. Scale bars = 500 μm. (E) The ratio of fat deposition in injured TAs over uninjured contralateral TAs quantified from Oil Red O staining was significantly 4cv reduced in THI-treated versus control animals in 11-MO (*) but not 16-MO mdx mice. In contrast, the ratio of injured over uninjured fat deposits in quadriceps was significantly reduced in 16-MO (#) but not in 11-MO mdx mice. *P <0.05, **P <0.01 by student’s t-test. Error bars represent SEM. CSA, cross-sectional area; CTX, cardiotoxin; IP, intraperitoneal; MO, month-old; SEM, standard error of the mean; TA, tibialis anterior; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. (Applied Biosystems, Carlsbad, CA, USA) as previ- 450 ms train duration [17]. Force frequency was carried ously described [13]. RNA (0.5 μg) was reverse tran- out using the same pulse duration at 10, 20, 40, 60, 80, scribed using the Omniscript RT Kit (Qiagen). For 100 and 120 Hz, as outlined in the x-axis of Figure 3B. reverse transcription-PCR (RT-PCR), 10 ng cDNA was Specific force was calculated as previously described combined with SYBR Green (Thermo Scientific, Waltham, [18] by normalizing to the muscle cross-sectional area MA, USA) following published conditions and primer (CSA). CSA is the quotient of dry muscle mass (mg) sequences for S1P-related genes by Grabski et al.[14] over Lo (mm), which is defined as the product of Lf and by Au et al.[15]for 18S. with the fiber length ratio (0.44 for EDL) and mamma- lian muscle density (1.06 mg/mm ). Functional analysis: myography Animals treated with THI or PBS (vehicle) via IP injec- Measurement of S1P in mouse tissue tion as aforementioned for 14 days were analyzed be- S1P was quantified in tissue after homogenization and tween 1 and 4 days following the final day of injection. extraction using liquid chromatography-tandem mass Prior to euthanasia animals were anesthetized with 0.5 spectrometry (LC-MS/MS). Tissue was pulverized in mg/g weight avertin diluted in PBS. EDLs were then ex- liquid nitrogen using a mortar and pestle. Collected tis- cised and equilibrated in Ringer’s solution (120 mM sue was weighed and an internal standard (C17 base NaCl, 4.7 mM KCl, 3.15 mM MgCl , 1.3 mM NaH PO , D-erythro-sphingosine-1-phosphate in methanol (Avanti 2 2 4 25 mM NaHCO , 11 mM glucose, 1.25 mM CaCl , Polar Lipids, Alabaster, AL, USA)) was added at 1 pmol/ 3 2 pH 7.2) with 95% O /5% CO for a minimum of 15 mi- mg tissue. Tissue was then vortexed/extracted in 16 vol- 2 2 nutes prior to stimulation [16]. For assessment of direct umes (mg/μl) of acetonitrile:water (80:20, v/v) for 10 mi- S1P administration, EDL muscles from uninjured and nutes at room temperature. Supernatants were collected mdx/J untreated 3.5-MO male mdx (C57BL/10ScSn-Dmd ) after centrifugation (10 minutes at 14,000 rpm) and con- were incubated with oxygenated Ringer’s solution centrated to dryness using a SpeedVac Concentrator containing 10 μM S1P or vehicle (PBS with 4 mg/ml (Thermo Scientific). Pellets were resuspended in metha- fatty acid free BSA) for 15 minutes prior to stimulation nol to a calculated concentration of 0.05 μM C17 base [16]. All functional experiments were carried out with D-erythro-sphingosine-1-phosphate. Then 10 μlwas buffer solutions at 25°C under constant oxygenation. analyzed by LC-MS/MS using C17 base D-erythro- Myography was conducted using a 820S myograph sphingosine-1-phosphate plus C18 base D-erythro- (DMT, Ann Arbor, MI, USA) and data was recorded sphingosine-1-phosphate (both at 0.05 μM) as a standard. using a PowerLab 4/30 acquisition system with LabChart Separation of analytes was undertaken by liquid chro- Pro software v7.3.1 (both from ADInstruments, Dunedin, matography using a Chromolith RP-C18e 100 × 2 mm New Zealand). Stimulations were conducted with S88X column (EMD, Gibbstown, NJ, USA) and analysis by dual systems (Grass Technologies, Middleton, WI, USA). tandem mass spectrometry with a Quattro Micro mass Muscles were stimulated to establish optimal fiber spectrometer (Waters, Milford, MA, USA) in positive length (Lf) and voltage at which maximum tetanic force ion mode. The HPLC gradient using two pumps was wasmeasuredat120 Hz using4.15mspulseswithin linear from 50% MeOH to 99% MeOH using solvent A Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 6 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 3 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 7 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 3 Elevating S1P levels with THI increases muscle fiber size. (A) Staining for laminin (green) and DAPI (blue) depict a dramatic increase in muscle fiber size in both injured and uninjured quadriceps (quads) with THI treatment. Depicted are quadriceps muscles from 11-MO 4cv mdx mice. Scale bars = 50 μm. (B,C,D) Quantification of minimum muscle fiber diameter reveals a significant increase in myofiber size in THI- 4cv treated animals. Increased myofiber diameter was observed in both (B) injured and (C) uninjured quadriceps from THI-treated 11-MO mdx 4cv mice, whereas only (D) uninjured quadriceps in THI-treated 16-MO mdx mice showed increased myofiber size compared to vehicle controls. As indicated by the distributions, mean and median values of muscle fiber minimum diameters, there is an overall increase in muscle fiber size with THI treatment. Quantifications were undertaken in random fields in both injured and uninjured muscles in order to obtain an overall representation of fiber size increase for each muscle.*P <0.05, ***P <0.0005 by student’s t-test. Error bars represent SEM. DAPI, 4',6-diamidino-2- phenylindole; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. (water, 0.1% formic acid) and solvent B (MeOH, 0.1% with a dwell time of 0.07 seconds. Data collection was formic acid) over 1 minute at a flow rate of 0.35 ml/ by MassLynx software (Waters) and processed with min. To wash the column, the gradient was repeated QuanLynx software (Waters). twice before equilibrating for 3 minutes before running the next sample. The transitions analyzed were 380.25 Measurement of S1P in mouse plasma >264.50 and 380.25 >82.00 for endogenous S1P, and S1P was quantified in plasma using butanol extraction 366.25 >250.50 and 366.25 >82.00 for internal standard and liquid LC-MS/MS [19]. Internal standard (5 μl3 μM mdx/J Figure 4 S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmd ) muscle. (A) Experimental schematic of longer-term, 14-day treatment of THI or PBS (vehicle) following CTX injury. THI was administered following the aforementioned dose and injection regimen. Following treatment, EDL muscles were harvested and specific isometric force was analyzed by in vitro myography from both injured and uninjured limbs. (B) Force frequency analysis reveals that EDL muscles isolated from injured limbs of THI-treated animals (n = 10) have significantly greater specific force compared to injured vehicle controls (n = 9). (C) Analysis of untreated and uninjured wt (C57BL/10ScSn) and mdx/J mdx (C57BL/10ScSn-Dmd ) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured mdx/J and untreated mdx (C57BL/10ScSn-Dmd ) EDL muscles with a high concentration of S1P (10 μM) leads to a significant increase in maximal specific force. *P <0.05, **P <0.005 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; EDL, extensor digitorum longus; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole; wt, wild type. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 8 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 C17 base D-erythro-sphingosine-1-phosphate in ethanol manufacturer’s instructions. Briefly, S1P was dissolved (Avanti Polar Lipids)) was added to 10 μl EDTA- in methanol (0.5 mg/ml) and aliquoted, then the solvent anticoagulated plasma and mixed thoroughly on an or- was evaporated with a stream of nitrogen to deposit a bital shaker (Thermomixer, Eppendorf, Hauppauge, NY, thin film on the inside of the tube. Prior to use, aliquots USA) for 10 minutes at 1,400 rpm at 20°C. The sample were resuspended in PBS with 4 mg/ml BSA (fatty acid was then acidified using 50 μl 30 mM citric acid/40 mM free) to a concentration of 500 μM. Directly following Na HPO , pH 4.0, and extracted for 10 minutes at 1,400 CTX injection, 20 μl500 μMS1P wasinjectedinleft 2 4 rpm at 20°C with 125 μl water-saturated butanol (Fisher TAs, daily until day 3 post injury, at which time animals Scientific, Waltham, MA, USA). The butanol layer was were euthanized and muscles were harvested for freez- removed and lyophilized in a centrifugal evaporator at ing. Right TAs were injected with an equal volume of 20°C. The residue was stored at −20°C until analyzed. PBS with 4 mg/ml BSA as vehicle controls. In a separate The residue was resuspended in 125 μl HPLC buffer A experiment (Figure 6), TAs of four 2.5-MO female 4cv (50% methanol, 1% formic acid, 5 mM ammonium mdx were injected with S1P or vehicle under the formate in water (JT Baker) and sonicated in a bath same conditions stated above, in the absence of injury. prmd scid sonicator for 1 minute at 20°C. Analytes in a portion of AJ/SCID mice (n = 4, 9-MO, B6. Cg-Dysf Prkdc /J) the sample (10 μl) were then separated using liquid were also injected for 3 days with S1P or vehicle in TAs chromatography (Shimadzu, Nakagyo-ku, Kyoto, Japan) post CTX injury, following the same concentration and 4cv with a Luna 3 μm C18(2) 100Ǻ 50 × 2 mm column injection regimen used in mdx . For measurement of (Phenomenex, Torrance, CA, USA) and analyzed by tan- S1P muscle content (Figure 7A) following intramuscu- 4cv dem mass spectrometry on a 4000 QTRAP mass spec- lar injections, 11-MO mdx (n = 3) were injected 20 μl trometer (AB SCIEX, Framingham, MA, USA) in 500 μM S1P in left TAs and 20 μl vehicle in right TAs. positive ion mode. The HPLC gradient was linear from Muscles were harvested and frozen in liquid nitrogen buffer A to buffer B (10% isopropyl alcohol, 1% formic 15 minutes post injection, and then processed using the acid, 5 mM ammonium formate in methanol) over 1 mi- aforementioned methods for analyzing S1P in muscle nute at a flow rate of 0.4 ml/min. To wash the column, by LC-MS/MS. For injection of biotinylated-S1P, TAs 4cv the gradient was repeated twice before equilibrating for from 11-MO mdx (n =2)wereinjectedintramuscu- the next sample. The transitions analyzed were 380.3/ larly with 20 μl500 μM S1P-biotin or vehicle (Echelon 264.3 and 380.3/81.9 for endogenous S1P, and 366.2/ Biosciences, Salt Lake City, UT, USA). TAs were 93.0, 366.2/82.0 and 366.2/250.3 for internal standard harvested and frozen in OCT compound 15 minutes fol- with a dwell time of 15 milliseconds. Calibrators were in lowing injection. mouse plasma (C18 base D-erythro-sphingosine-1-phos- phate, Avanti Polar Lipids). Between-day coefficient of Mouse histology and immunohistochemistry variation was 7.7%. Pertinent instrument specific param- All mouse muscles were frozen directly in OCT com- eters were empirically derived and included curtain gas: pound with liquid nitrogen cooled in isopentane and 15, ion source voltage: 5000 V, emitter temperature: 550°C, sectioned 8 μm thick. Tissue for X-gal staining was fixed desolvation gas 1: 20, desolvation gas 2: 70, collision gas: 6, for 10 minutes with 2% formaldehyde/0.2% glutaralde- entrance potential: 10, and collision cell exit potential: 10. hyde and incubated overnight at 37°C with staining Chromatographic data were analyzed using Analyst 1.4.2 buffer (PBS with 1 mg/ml X-gal, 5 mM potassium ferri- (AB SCIEX) by summing transitions for each analyte. cyanide, 5 mM potassium ferrocyanide, 2 mM CaCl (all from Fisher Scientific)). Picrosirius red with fast green, Creatine kinase (CK) assay hematoxylin and eosin, and Oil Red O staining were 4cv mdx mouse plasma samples were diluted 1:50 and conducted following established protocols [21]. Fibrosis total CK activity was measured by an enzymatic rate was quantified as percentage of area stained red within method at the clinical laboratory of the Department of each 20 × field analyzed using ImageJ v1.40 or Adobe Laboratory Medicine, University of Washington, using Photoshop CS2 (San Jose, CA, USA). For evaluating fi- the Beckman Coulter instrument (Brea, CA, USA) as brosis, the mean value from three separate sections (200 previously described [20]. Relative levels were then nor- μm apart in longitudinal distance) were analyzed from malized to body weight. each muscle and used to calculate the overall mean for each muscle group outlined in the x-axis of Figure 1D. S1P injections Lipid accumulation was quantified with the ImageJ cell 4cv nlacZ/+ Right and left TAs of three 3-MO male mdx :Myf5 counter plugin by counting fatty infiltrates in montages were injured once more with 10 nM CTX (Figure 5). (stitched from 10 × photos) covering the entire CSA of S1P (Enzo Life Sciences, Farmingdale, NY, USA; each muscle. Muscles injected with S1P-biotin or vehicle Calbiochem) preparation was undertaken according to were cut 8 μm thick, fixed for 5 minutes with 4% Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 9 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 5 Direct administration of S1P promotes muscle regeneration following acute injury. (A) Experimental schematic of S1P and PBS 4cv nlacZ/+ (vehicle) injected daily for the first 72 hours into TAs of 3-MO mdx :Myf5 mice (n = 3, left TAs injected S1P, right TAs injected PBS) following CTX injury. (B) Top row: X-gal staining reveals an increased number of β-galactosidase+ nuclei at the sites of injury in S1P-treated TA muscles compared to vehicle controls. Bottom row: staining for eMyHC with DAB reveals a significant increase in the number of newly regenerated muscle fibers in S1P-treated TA muscles. Scale bars = 50 μm. (C) Left graph: quantification of β-galactosidase+ nuclei indicates the number of Myf5+ cells is significantly increased at the site of injury in S1P-treated compared to untreated muscles. Middle graph: a significant increase in β-galactosidase+ nuclei was also observed over the entire CSA of each S1P-treated TA muscle. Right graph: quantification of the number of eMyHC fibers within areas of regeneration was significantly greater with S1P treatment. *P <0.05 by student’s t-test. Error bars represent SEM. CSA, cross-sectional area; CTX, cardiotoxin; DAB, 3,3'-diaminobenzidine; eMyHC, embryonic myosin heavy chain; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior. formaldehyde, and then stained with streptavidin conju- were fixed overnight in 4% formaldehyde (from parafor- gated to Alexa Fluor 594 (Life Technologies, Carlsbad, maldehyde powder) at 4°C. Following fixation, antigen CA, USA) at 1:1000 in PBS and 1% BSA for 1 hour. retrieval was performed with 10 mM citrate buffer (with0.05% Tween20atpH6.0)warmedin a water bath at 90°C for 20 minutes. Slides were then perme- Immunohistological staining 4cv ated with ice cold methanol for 5 minutes at room Staining was undertaken using freshly frozen mdx temperature. Streptavidin/biotin blocking (Vector Labora- muscles. Pax7 staining was performed as outlined by tories, Burlingame, CA, USA) was performed according to Clever et al. [22] with slight modification. Sections Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 10 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 6 Administration of S1P leads to increased levels of S1PR1 and P-rpS6 in vivo. (A) Experimental schematic of S1P and PBS (vehicle) 4cv injected daily for the first 72 hours into TAs of uninjured mdx mice (n = 4, 2.5-MO, left TAs injected S1P, right TAs injected PBS). (B) Western 4cv blot analysis of injected TAs (n = 3, 2.5-MO mdx ) indicates that administration of S1P significantly increases S1PR1 levels. (C) Western blot 4cv analysis of injected TAs (n = 4, 2.5-MO mdx ) for total, and P-Akt, P-mTOR and P-rpS6, reveals that total and P-rpS6 were significantly higher with S1P treatment. Increased levels of total and P-rpS6 suggest that S1P administration promotes protein synthesis in mdx muscles. *P <0.05 by student’s t-test. Error bars represent SEM. MO, month-old; P-Akt, phosphorylated Akt; P-mTOR, phosphorylated mammalian target of rapamycin; P-rpS6, phosphorylated ribosomal protein S6; rpS6, ribosomal protein S6; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; TA, tibialis anterior. manufacturer’s instructions. Staining was undertaken using monoclonal anti-CD3e (clone 145-2C11, eBioscience, San the Mouse on Mouse (MOM) Kit (Vector Laboratories) Diego, CA, USA) at 1:100 dilution, followed by anti-rat with immunoglobulin G (IgG) blocking for 5 hours at 4°C IgG conjugated to Alexa Fluor 594 at 1:1000 dilution. prior to addition of mouse monoclonal anti-Pax7 (clone For laminin staining, tissue was also fixed with 2% for- PAX7, R&D Systems, Minneapolis, MN, USA) diluted at maldehyde for 5 minutes then treated with polyclonal 1:20 and incubated overnight at 4°C. Biotinylated anti- rabbit anti-laminin (Sigma-Aldrich, St Louis, MO, USA) mouse secondary was supplied with and used as pre- for 1 hour at 1:400 dilution in PBS and 1% BSA. Follow- scribed by MOM Kit instructions. Streptavidin conjugated ing washes, Alexa Fluor 488 conjugated goat anti-rabbit to Alexa Fluor 488 (Life Technologies) was added at IgG (Life Technologies) was administered at 1:800 dilu- 1:1000. As a negative control for Pax7 staining, a mouse tion for 1 hour. Controls omitting the primary antibody IgG isotype was applied to separate ribbons and treated in were included with all staining. For embryonic myosin parallel. For BS1 staining, muscles were initially fixed with heavy chain (eMyHC), tissue was first fixed with 2% for- 4% formaldehyde for 5 minutes at room temperature then maldehyde for 5 minutes, treated with streptavidin/ stained with BS1 directly conjugated to fluorescein iso- avidin blocking and blocked with IgG block from MOM thiocyanate (FITC), diluted at 1:400 in PBS with 1% BSA Kit for 5 hours at 4°C. Following blockade, concentrated and applied for 1 hour at room temperature. Following mouse anti-eMyHC (clone F1.652, received concen- BS1 staining, wheat germ agglutinin (WGA) directly con- trated at 357 μg/ml IgG, Developmental Studies Hybrid- jugated to rhodamine was administered at 1:400 dilution oma Bank (DSHB), University of Iowa, IA, USA) was as a counterstain for identifying myofibers. CD3e staining administered at 1:400 dilution overnight at 4°C. The was undertaken in the same manner as BS1, using rat remainder of the staining was undertaken following Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 11 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 7 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 12 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 7 Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. (A) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5) of S1P in left TAs and vehicle in right TAs 4cv of uninjured mdx (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. (B) To visualize the location of S1P following injection, biotinylated-S1P was injected in left 4cv TAs versus vehicle in right TAs of uninjured mdx mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining 4cv with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. (C) Staining of mdx TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). (D) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5) reveals S1PR1 is 4cv present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. **P <0.005 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; eMyHC, embryonic myosin heavy chain; IgG, immunoglobulin G; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; S1PR3, S1P receptor 3; SEM, standard error of the mean; TA, tibialis anterior. MOM Kit staining instruction. 3,3'-diaminobenzidine complete protease inhibitor cocktail (Roche, Basel, (DAB) was used for visualizing and quantifying eMyHC Switzerland), and complete phosphatase inhibitor cocktails fibers. For fluorescence, eMyHC was visualized using 1 and 2 (Sigma-Aldrich). Protein extracts were separated streptavidin conjugated to Alexa Fluor 594 used at using Ready Gel Tris–HCl (BioRad, Hercules, CA, USA), 1:1000 dilution for 1 hour. For S1P receptor staining, 4 to 20% linear gradient and transferred to polyvinylidene slides were fixed with 4% formaldehyde for 5 minutes fluoride (PVDF) membranes with a wet transfer system and stained with rabbit polyclonal IgG antibodies (BioRad). Membranes were blocked for 1 hour with against S1PR1, S1PR3 (Cayman Chemical, Ann Arbor, Tris-buffered saline with 0.1% (v/v) Tween 20 containing MI, USA) and phosphorylated S1PR1 (raised against 5% (w/v) BSA. For S1PR1 analysis, rabbit polyclonal Thr236, Assay Biotechnology, Sunnyvale, CA, USA), all anti-S1PR1 was used at a 1:500 dilution (Santa Cruz Bio- applied at a dilution of 1:200 for 2 hours. Following re- technology, Santa Cruz, CA, USA). Rabbit polyclonal anti- ceptor staining, goat anti-rabbit IgG conjugated to Alexa bodies were used to blot against phosphorylated (Thr308) Fluor 488 was added at 1:1000 for 1 hour. In parallel, we Akt, total Akt, phosphorylated (Ser2448) mammalian tar- stained additional slides with rabbit polyclonal IgG isotype get of rapamycin (mTOR), total mTOR, phosphorylated at the same final concentrations to exclude non-specific (Ser240/Ser244) rpS6, total rpS6 (1:1000, Cell Signaling 4cv staining of these antibodies in mdx muscles. Technology, Danvers, MA, USA) and β-actin (1:10000, Staining quantifications were all undertaken using Sigma-Aldrich). The signals were detected using an en- ImageJ cell counter plugin. Calculations, statistics and hanced chemiluminescence kit (Millipore, Billerica, MA, graphs were generated with Microsoft Excel (Redmond, USA) and CL-XPosure films (Thermo Scientific) were an- WA, USA). Bright field photographs were captured using alyzed using ImageJ. either a Fisher Scientific Micromaster digital inverted or upright microscopes with Micron software. Fluorescent Statistics photographs were captured with a monochromatic camera Student’s t-test was used to determine statistical signifi- using an Axiovert 200 microscope (Zeiss, Oberkochen, cance for the majority of experiments. P values gener- Germany). Individual fluorescent channels were colored ated by analysis of variance (ANOVA) are specified in and merged using Adobe Photoshop. Brightness contrast the text. levels were adjusted to increase visibility and reduce back- ground in most photographs. Results Alterations of S1P regulation and content following IP Western blot analysis injection of THI in mdx mice Tissue for western blot analysis was snap frozen in liquid To determine the effect of elevating S1P levels in dys- nitrogen and subsequently homogenized. Freshly iso- trophic animals, we studied the effects of THI in the lated TA muscles were harvested and snap frozen in li- mdx mouse model for DMD [23,24]. Recently, Loh et al. quid nitrogen prior to homogenization with disposable (2012) showed that compared to wt, mdx muscles are in tissue grinders. Tissue was homogenized under liquid a state of S1P deprivation as they exhibit increased levels nitrogen then resuspended in lysis buffer containing of the enzymes that degrade S1P (S1P lyase and S1P 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, phosphatase 1) [8]. THI is a hydrophilic small molecule 5 mM NaF, 0.25% (w/v) sodium deoxycholate, 2 mM that increases S1P levels by inhibiting the lyase that irre- NaVO , 1% Triton X-100 (v/v), supplemented with versibly degrades S1P [11,25,26]. In turn, low doses of 3 Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 13 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 THI may be sufficient to cause mild lymphocytopenia but significantly elevated in the injured muscles from THI- the presumable increase of S1P levels in muscle have not treated animals. These results suggest that acute injury 4cv been reported [8,11]. To corroborate the effects of THI in in mdx muscles induces upregulation of enzymes that 4cv mdx mice, we analyzed changes in lymphocytes before regulate S1P metabolism. In turn, elevated expression of and after treatment, and measured S1P content in muscle both S1P kinases with THI treatment may be beneficial (Figure 1). THI has low oral bioavailability; Bagdanoff et for muscle regeneration in mdx mice. However, with al. showed 10 to 12% bioavailability of THI when adminis- THI treatment S1P phosphatase 1 and lyase expression tered orally [10]. Thus we evaluated IP injections of THI were also greatly increased. Therefore we examined S1P as a parenteral delivery route for elevating systemic levels content, to determine if THI treatment results in in- of THI. Peripheral blood was collected and analyzed be- creased intramuscular S1P levels and in turn promotes fore and 12 hours after two IP injections of THI (each in- muscle regeneration following CTX injury. jection was 250 μl 0.15 mg/ml THI, administered 6 hours In order to determine if THI treatment results in in- apart). Following THI treatment, we observed a significant creased intramuscular S1P levels, a second group of 4cv 4cv drop of all leukocytes except monocytes in mdx (n = 3, mdx animals was treated with THI or PBS (n = 6, 1.5-MO) (Figure 1A). Of note, prior to treatment with 5-MO males), following the same dosing schedule (2 × IP THI, the total number of white blood cells and amount of injections per day for the first 3 days post CTX injury) and individual leukocyte populations except monocytes, was sacrificed at day 4 to analyze the efficacy of THI in 4cv significantly elevated in 1.5-MO mdx mice (n = 3) ver- increasing S1P levels (Figure 1B). In concordance with sus age-matched wt mice (n = 4). Interestingly, the num- published work, treatment with THI increased S1P levels 4cv ber of platelets was also elevated twofold in mdx versus in spleen but not plasma (Figure 1B, Additional file 1: wt, but declined to near wt following THI administration Figure S3A) [10,11]. S1P levels were also significantly in- (Additional file 1: Figure S1). This systemic effect in creased in CTX-injured quadriceps from THI-treated ani- lymphocyte count indicates that THI functions efficiently mals (Figure 1B). This indicates that despite increased when delivered systemically via IP injection. In addition, expression of S1P phosphatase 1 and lyase following in- for short-term treatments, IP administration is desirable jury, the counteracting increased expression of both S1P to ensure that all mice received the same dose. Thus for kinases results in elevated levels of intramuscular S1P. In the majority of experiments described herein, we opted to addition, we also observed increased S1P levels in the un- administer THI via IP administration. injured TA muscles from mice treated with THI compared Loh et al. also demonstrated that following acute in- to vehicles. To examine if such extravascular increases of jury, the expression of S1P lyase increases in wt muscle S1P correlated with a beneficial effect in dystrophic mice, [8]. Thus we analyzed the expression of enzymes that we analyzed the level of plasma CK, which are elevated in regulate S1P production and degradation following CTX humans and mice with muscular dystrophy activity in the 4cv injury in the mdx background with and without THI same group of THI-treated mdx mice [31]. Results indi- treatment. Right TA and quadriceps muscles were unin- cate a trending, but not statistically significant decline in jured, while left counterparts were injured using CTX, a CK activity levels in plasma collected on day 4 post injury well characterized model of acute injury where initial from THI versus vehicle-treated mice (Additional file 1: muscle destruction is followed by a rapid myogenic re- Figure S3B). 4cv sponse [27-30]. mdx mice (n = 6, 3.5-MO males) were injected IP immediately following CTX and thereafter Reduction of dystrophic muscle pathology in acutely five additional times during a 3-day period (for example injured mdx muscles via administration of THI IP 2 × IP injections per day) with either the previously used Although young mdx mice exhibit robust muscle repair, dose of THI or vehicle. For this analysis, muscles were regeneration becomes impaired with aging, resulting in harvested at day 4 post injury; the peak of myogenic muscle atrophy and dystrophy [3]. Therefore, in a third gene expression following CTX-induced damage [28]. In experiment, the effects of THI on histopathology were the absence of THI, expression of the S1P lyase was sig- assessed in injured and uninjured muscles from two 4cv nificantly elevated following injury (Additional file 1: groups of aged mdx mice (n = 6, 11-MO females; n = 7, Figure S2A). Surprisingly, expression of S1P phosphatase 16-MO males), to determine the effects of increasing 1 and lyase were greater in the injured muscles with THI levels of S1P in dystrophic animals at a stage of severe treatment, suggesting a possible compensation in the muscle wasting. Importantly, it has been reported that S1P degradation pathways in response to the inhibition mdx females older than 6 months of age exhibit greater fi- of the S1P lyase. Analogous to these results, expression brosis than males [32]. Once more, right TA and quadri- levels of S1P kinase 1 were also increased with injury and ceps muscles were uninjured, while left counterparts were at higher levels with THI (Additional file 1: Figure S2B). injured with CTX (Figure 2A). Regeneration following In contrast, the expression of S1P kinase 2 was only CTX injury is well orchestrated in normal muscle but Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 14 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 impaired in older mdx mice [29]. Therefore in these reduced fat deposition resulting from injury in 11-MO studies we analyzed the muscles from 11- and 16-MO female TAs and 16-MO male quadriceps. These results mdx mice 18 days following CTX injury, a time point demonstrate that THI treatment reduces injury-induced expected for non-diseased muscles to fully regenerate fat deposition and fibrosis in mdx muscles. 4cv [28]. In the 16-MO mice, muscles were weighed imme- Further analysis of THI-treated mdx mice revealed an diately after collection and normalized to body weight increase in muscle fiber size in quadriceps (Figure 3A). (grams muscle weight over grams mouse weight). As Although mdx mice undergo muscle hypertrophy as com- expected, injured muscles were lighter than uninjured pared to wild type, we observed a significant increase in muscles in vehicle mice, an approximate weight loss the minimum fiber diameter with THI treatment in dia- greater than 20% (Additional file 1: Figure S4A). However, phragms, and in both uninjured and injured quadriceps of in the THI-treated mice the weight of injured quadriceps 11-MO mice (Figure 3B,C and Additional file 1: Figure S7) was similar to uninjured quadriceps (muscle weight [37]. Uninjured quadriceps of THI-treated 16-MO males ratio injured/uninjured approximates one), suggesting also showed a significant increase in fiber size (Figure 3D). that THI treatment promotes muscle repair and pro- In summary, 3 days of THI treatment is sufficient to in- tects from muscle loss following acute injury. crease muscle fiber size in older mdx mice. Fibrosis and fat deposition are both hallmarks of muscle To assess if increases in muscle fiber size observed with wasting and dystrophic muscle pathology [32,33]. In THI treatment are accompanied by an increase in the addition, when regeneration is impaired, fibrosis and fat number of satellite cells, we quantified the number of accumulate in place of muscle following acute injury Pax7+ cells. Within skeletal muscle, Pax7 is specifically [34,35]. Histological quantification revealed that THI expressed by satellite cells, which have been reported to 4cv treatment reduced accumulation of both fibrosis and fat decline in older mdx muscles [38-40]. As expected, few deposition following acute injury in quadriceps and TA satellite cells (Pax7+ nuclei) were visible in cross-sections muscles (Figure 2B,C). Results for lower fibrosis were con- of 11-MO mdx muscles. However, there was a significant firmed by third party hydroxyproline analysis of injured increase in the mean number of Pax7+ nuclei, collectively TAs from 16-MO animals (Additional file 1: Figure S4B). in limb muscles (TAs and quadriceps) from THI-treated Interestingly, fibrosis was also significantly lower in unin- 11-MO animals (Additional file 1: Figure S8). jured TAs of 11-MO females, which correlates with the S1P is a potent angiogenic factor [41-43]. Thus we capacity of THI to elevate S1P levels in uninjured TAs studied the effects of THI treatment on the skeletal (Figure 1B, Additional file 1: Figure S5). Although only left muscle microvasculature. We quantified the number of TAs and quadriceps were injected with CTX, fibrosis ac- vessels using BS1, a lectin that highlights endothelial cumulation in uninjured muscles was likely elevated as cells [44]. In contrast to the increase in Pax7+ cells, we mice disuse injured limbs and bear most of the use/weight did not observe an increase in BS1+ vessels in injured on the uninjured contralateral limb. Therefore, the differ- 11-MO TA muscles. Quantitative RT-PCR analysis of ences observed in uninjured TAs are likely due to reduc- endothelial related genes eNOS and CD31 in 5-MO 4cv tions in the amount of fibrotic deposition that would mdx TA muscles at day 4 post injury, show no signifi- otherwise accumulate without THI treatment, since it is cant difference in the levels of expression of these endo- unlikely THI can reverse already accrued fibrosis. Along thelial associated genes in THI treatment compared to with lower fibrosis observed in injured muscles, the overall vehicle (Additional file 1: Figure S9). This suggests that morphology appeared more organized with THI treatment THI benefits on muscle repair do not depend on in- compared to vehicle-treated animals (Figure 2C). In creasing microvasculature density. addition, the number of EBD-positive fibers, an indicator of muscle fiber damage, was lower in injured 11-MO mus- THI treatment elevates isometric force in acutely injured cles and significantly reduced in uninjured 11-MO quadri- mdx EDL muscles ceps (Additional file 1: Table S1) [12,36]. In these muscles To assess if increasing S1P levels promotes dystrophic the number of centrally nucleated fibers was comparable muscle function, in a fourth experiment we conducted between THI and vehicle-treated animals (Additional myography analysis following longer treatment with file 1: Figure S6). THI. For this experiment, another group of mdx mice mdx/J To test whether THI-treated mice show decreased fat (male 4.75- to 5-MO C57BL/10ScSn-Dmd ) was in- deposition in injured muscles, we quantified the fat de- jured and treated with daily IP injections using the same posits within entire cross-sections of THI and vehicle- THI dose and injection interval, for 14 consecutive treated muscles (Figure 2D). The ratio of fat deposits days; the maximum duration for IP administration between injured and uninjured contralateral muscles allowed by our approved animal protocol. Animals were was then compared to THI and vehicle-treated mice treated with THI (n = 10) or vehicle (n = 9) for 14 days (Figure 2E). This analysis indicates that THI significantly following injury, and analyzed between day 15 and 19 Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 15 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (Figure 4A). EDL muscles from injured and uninjured Direct administration of S1P promotes muscle contralateral limbs were analyzed for isometric specific regeneration in mdx mice following CTX injury force; a physiological measurement of muscle force that S1P is essential for satellite cell turnover, myoblast dif- is reduced with muscular dystrophy in mice and ferentiation and muscle regeneration in non-diseased humans [18,45,46]. mice, and more recently shown to promote satellite cell To assess if the EDL is damaged as a consequence of activation in mdx muscle [4,5,8,47]. To determine if the CTX injection in the TA, we injured and analyzed a sep- increase in satellite cell number observed in the THI- arate group of mdx mice (n = 4) 12 hours post injury. treated muscles was a result of increased S1P muscle For this fifth experiment, CTX injections included India content, we examined the effects of direct S1P adminis- ink to label needle penetration [47]. To assess muscle tration following CTX-induced acute injury in dys- fiber damage, a consequence of CTX injury, animals trophic muscles. In order to identify satellite cells and 4cv nlacz/+ were injected IP with EBD immediately following CTX their progeny, we utilized mdx :Myf5 mice carry- injection. The presence of EBD indicates EDL muscles ing the nuclear lacZ reporter driven by the endogenous are damaged. However, EDL damage is not due to direct Myf5 gene, a marker of myogenic cells [49-51]. CTX was 4cv penetration by the needle since India ink was only applied to both TA muscles (n = 3, 3-MO mdx : nlacz/+ present in the CTX-injected TA muscles (Additional file 1: Myf5 males), then S1P was immediately injected Figure S10). intramuscularly into left TAs and a vehicle control into Force frequency analysis revealed a significantly higher right TAs. Injections were repeated daily for the first specific force by EDL muscles isolated from injured 72 hours following injury and TAs were harvested on limbs of THI-treated mice (Figure 4B). These values day 4 post injury, directly following the peak of injury- were similar to EDL muscles isolated from contralateral induced myogenic cell proliferation for analysis of uninjured limbs, indicating that THI prevented wasting Myf5+ nuclei (Figure 5A) [28]. S1P-treated muscles and preserved muscle function following acute injury showed a dramatic, fourfold increase in the number of (Figure 4B). However, the specific force observed after Myf5+ nuclei in areas with severe CTX damage com- THI treatment was still lower than wt control animals pared to vehicle controls (Figure 5B top row and 5C left (Figure 4C). Two weeks of THI treatment was not suf- graph). Furthermore, a significant increase in the number ficient to improve specific force in uninjured EDL mus- of Myf5+ nuclei was observed over the entire CSA of S1P- cles.However,as shown in Figure 1B,the THIdoseof treated TAs (Figure 5C middle graph, Additional file 1: 0.75 μg/day used for all our experiments does not sig- Figure S12). These data demonstrate that S1P treatment nificantly raise S1P levels in all uninjured mdx muscles. increases the number of myogenic cells in mdx muscles In addition, although peripheral lymphocytes declined following injury and suggests that S1P promotes satellite with THI (Figure 1A), we did not observe a decline of cell proliferation in vivo. CD3e+ T-cells present in the diaphragm following 2 We then determined whether the increase in myo- weeks of THI (Additional file 1: Figure S11) [48]. genic cells promotes dystrophic muscle repair by stain- Therefore, it is plausible that a higher dose of THI is ing for eMyHC, a marker of regenerating muscle fibers required to sufficiently elevate S1P levels needed to [27]. In concurrence with the rise of Myf5+myogenic improve specific force in uninjured mdx muscles. cells, a 3.6 fold increase in the number of eMyHC+ fibers However, since THI is insoluble in PBS at higher con- was observed in S1P-treated TAs (Figure 5B bottom centrations and has low oral bioavailability, we chose to row, 5C right graph). This increase in eMyHC+ fibers, directly study the effects of high levels of S1P on unin- corresponded with elevated numbers of centrally nucle- jured mdx muscles ex vivo. For this experiment, EDLs ated muscle fibers in the injured regions of S1P-treated from uninjured and untreated mdx mice were analyzed muscles (Additional file 1: Figure S13A). Furthermore, following incubation with 10 μM S1P [16]. Analysis of the size of regenerating myofibers in S1P-treated TAs the maximal specific force indicates that direct admin- was significantly greater, as indicated by the minimum istration of S1P significantly increases force output in diameter quantified for the largest eMyHC+ fibers uninjured mdx muscle (Figure 4D). Such results indi- (Additional file 1: Figure S13B). Collectively, these data cate that treatment with high concentrations of S1P show that local administration of S1P promotes dys- can promote functional improvement of dystrophic trophic muscle repair by improving satellite cell re- muscles. sponse and contribution to muscle fiber regeneration. Overall, reduction in fibrosis and fat deposition, and increase in myofiber size and satellite cell numbers, indi- S1P directly acts on mdx muscle fibers, and elevates cate that elevating S1P levels, pharmacologically or by levels of total and phosphorylated S1PR1 direct administration, has a profound benefit in dys- In mammals there are five S1P receptors that share trophic muscle repair and function. homology to G-protein coupled receptors [52]. It has Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 16 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 been recently reported that S1P receptor 2 (S1PR2) is spe- localized perinuclearly andlessso around the perim- cifically activated in myogenic cells and that downstream eter of eMyHC+ fibers (Figure 6E). These results indi- effectors of S1P action in satellite cells include compo- cate that S1PR1 signaling is active in regenerating nents of the JAK-STAT signaling pathway [8]. In contrast, muscle fibers and suggests that the beneficial actions our results and others, of exogenous S1P treatment that S1P exerts on mdx muscle fibers maybemediated resulting in increased EDL force, suggests that S1P also through S1PR1. acts directly on muscle fibers [16]. The amount of exogen- ous S1P added in the bath was super-physiological and S1P administration correlates with increased levels of thus we measured S1P muscle levels following intramus- S1PR1 and P-rpS6, an indicator of protein synthesis cular injection of S1P. In this experiment, left TAs from S1PR1 has been implicated in myoblast proliferation 4cv mdx mice (n = 3, 11-MO) were injected with the same and shown to steadily increase during the course of re- 4cv nlacz/+ dose of S1P as the mdx :Myf5 mice depicted in generation in non-diseased muscle [4,5]. Therefore to Figure 5A, while contralateral TAs received the same ve- gain more insight on the potential action that S1P ex- hicle. In contrast to the previous experiment depicted in erts via S1PR1 in dystrophic muscle, we injected S1P in 4cv Figure 5A, TA muscles were injected in the absence of in- uninjured TAs of mdx (n =3,2.5-MO),and quanti- jury and were harvested for S1P analysis 15 minutes post fied the level of S1PR1 and some downstream effectors injection (Figure 6A); the same time used for S1P incuba- (Figure 7A) [56]. In turn, S1P treatment resulted in 4cv tion prior to EDL force measurement shown in Figure 4D. significantly elevated levels of S1PR1 in mdx TAs Results indicate that within this timeframe, intramuscular (Figure 7B). In a separate experiment, we injected S1P 4cv injection of S1P does significantly increase S1P levels in in left TAs and vehicle in right TAs of mdx (n = 3, mdx muscle (Figure 6A). 10-MO), following the same dose and experimental de- To directly observe where S1P binds in the muscle, a sign (three injections, one per day, harvest on day 4), 4cv separate group of mdx (n = 2, 11-MO) were injected and analyzed TA muscles for phosphorylated S1PR1. with the same amount of biotinylated-S1P in left and ve- Results from this experiment show that phosphorylated hicle in right TAs. Once more, TAs were harvested 15 S1PR1 is also significantly elevated with S1P treatment minutes post injection for histological visualization of (Additional file 1: Figure S15). S1P. Staining with streptavidin conjugated to Alexa A result of S1P injection was larger eMyHC+ fibers Fluor 594 reveals that biotinylated-S1P is present in that were positive for phosphorylated S1PR1 (Figure 6E, many cells, but particularly localized to the perimeter of Additional file 1: Figure S13B). Therefore, we examined muscle fibers (Figure 5B). Among the three S1P recep- if elevated S1PR1 levels corresponded with known regu- tors (S1PR1, S1PR2, S1PR3) expressed in muscle, S1PR3 lators of cell size and protein synthesis; Akt, mTOR, S6 and S1PR1 are the most abundant in wt muscle [5]. Im- kinase and rpS6. S1P-induced hypertrophy has been portantly, expression of these three S1P receptors is re- described in cultured cardiomyocytes, which was ac- duced in mdx muscle cells, especially S1PR1, which companied by activation of Akt and S6 kinase [57]. In shows more than five fold reduction in relative mRNA addition, S1PR1 activation of S6 kinase via a Gi- 4cv levels (Additional file 1: Figure S14). Staining of mdx dependent pathway has been reported in vascular muscles (3.5-MO) for S1PR1 and S1PR3, reveals that smooth muscle cells [56]. Akt and mTOR signaling via S1PR1 is present at the perimeter of muscle fibers and S6 kinase, an activator of rpS6 implicated in protein myonuclei, whereas S1PR3 appears localized to the synthesis, has been described as sufficient to induce vasculature (Figure 6C). S1PR1 is a G protein-coupled skeletal muscle hypertrophy [58-60]. Therefore, we receptor (GPCR) that can be activated via phosphoryl- evaluated if direct injection of S1P induces activation of 4cv ation, resulting in translocation to the endosomal com- these pathways in uninjured TA muscles of mdx mice partment and/or the perinuclear compartment [53-55]. (n = 4, 2.5-MO). Western blot analysis of TA muscles Therefore, perinuclear localization of S1PR1 suggested injected for 3 days with S1P (Figure 7A) revealed that that in response to S1P treatment, receptor 1 signaling is the levels of phosphorylated Akt (P-Akt) and mTOR 4cv activated in mdx muscle fibers. To evaluate the pres- (P-mTOR), though increased, were not significantly ence of active S1PR1 signaling during muscle fiber re- higher in S1P-treated muscles (Figure 7C). However, the generation, we surveyed the same CTX-injured muscles levels of rpS6 and phosphorylated rpS6 (P-rpS6) were depicted in Figure 5A for the presence of phosphory- significantly increased with S1P treatment compared to lated S1PR1. Results indicate S1PR1 is localized around control muscles, suggesting an increase in protein syn- the perimeter of muscle fibers and intracellularly near or thesis. Although a more detailed study is required to within the myonuclei (perinuclear) of newly regenerated elucidate the role of S1P in skeletal muscle protein syn- eMyHC+ fibers (Figure 6D). In parallel, we observed thesis, our data suggest that S1P can activate muscle more concentrated staining for phosphorylated S1PR1 anabolic pathways in the mdx mouse. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 17 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Direct administration of S1P promotes muscle regeneration Figure S16). These results indicate that approaches in dysferlinopathy mice following acute injury aimed at elevating muscle S1P may be beneficial to The role of dysferlin is currently unknown, but its ab- promote muscle regeneration in additional muscle sence in humans and mice results in chronic muscle wasting diseases. wasting that primarily affects limb and girdle muscles [61-63]. Although dysferlinopathy is less severe than Longer-term treatment with THI shows a functional DMD [64], dysferlinopathy patients are often wheelchair benefit in uninjured mdx muscle bound between 30 and 40 years of age [65]. Much like To this point we have largely examined the role of S1P DMD, muscles in humans and mice lacking functional in promoting muscle regeneration in acutely injured dys- dysferlin exhibit chronic atrophy, resulting in the accu- trophic muscles. Since long-term intramuscular injec- mulation of fibrosis and fat [66]. Therefore we tested the tions of S1P are neither feasible nor practical (the effects of S1P administration after CTX injury in a injections also cause damage), we decided to revisit the model of dysferlinopathy (AJ/SCID) to evaluate if the use of THI for elevating S1P muscle content. Although benefits of S1P are exclusive to the mdx background or our initial experiments with THI showed little benefit in can be applied to other muscle wasting diseases [67]. uninjured mdx muscles, they were short-term and in We followed the same experimental design outlined in older animals with severe pathology (Figures 2, 3), or Figure 5A, injecting left TAs of AJ/SCID mice (n = 4, adult animals (Figure 4) at a point when hypertrophy 9-MO) with the same dose of S1P and vehicle in right and robust regeneration compensate for degeneration in TAs for 3 days following CTX injury. In contrast to the limb muscles [24,68,69]. Therefore, we examined longer- 4cv experiments in mdx , we harvested TAs on day 6 post term treatment of THI in younger mdx mice at 4 weeks injury in order to also evaluate the onset of fibrosis. In of age, a time point characterized by significant muscle accordance to the results observed in mdx, we observed degeneration prior to the compensatory period [70]. For 4cv improved muscle regeneration with the administration this experiment, uninjured mdx animals were treated of S1P in AJ muscles. Specifically, we observed lower for 1 month, beginning at 4 weeks of age, with THI or fibrosis and increased centrally nucleated fibers, as well vehicle in the drinking water (Figure 8A) [11]. At 8 weeks as improved muscle architecture in the damaged regions of age, we assessed the functional benefit of THI treat- of muscle with S1P administration (Additional file 1: ment by analyzing EDL specific force via myography. In Figure 8 Longer-term treatment with THI elevated muscle force in uninjured mdx EDL muscles. (A) Experimental schematic outlining the 4cv treatment regimen. Beginning at 4 weeks of age, mdx mice (1-MO males) were treated for 4 weeks ad libitum with 50 mg/l THI (n = 4) or vehicle (n = 3) in drinking water. (B) Myography analysis of EDL muscles reveals a significant increase in maximal specific force with THI treatment. *P <0.05 by student’s t-test. Error bars represent SEM. (C) Summary of findings: S1P can act to not only promote myogenic cell activation and muscle repair, but also enhance muscle fiber size and force, possibly through S1PR1 mediated signaling. EDL, extensor digitorum longus; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 18 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 turn, EDLs from THI-treated animals showed significantly reductions in HDAC activity result in an increase of greater specific force compared to vehicle-treated controls follistatin, an inhibitor of myostatin, which may explain (Figure 8B). This data demonstrates that elevating S1P the amelioration of DMD pathology [74]. Our data sup- levels is beneficial for the chronic muscle injury that port this possibility and suggest that the molecular occurs early in muscular dystrophy. mechanism for the suppression of muscle degeneration involves the anabolic pathways for muscle formation Discussion rpS6. These components have been shown to lie down- We have shown that systemic administration of the stream of myostatin and insulin-like growth factor [75]. pharmacological agent THI by IP injection to dystrophic mdx mice led to elevated levels of S1P in recovering in- Conclusion jured muscle tissue, as well as a reduction of fibrosis and Based on the work reported here, elevation of S1P may fat infiltration, both pathological indicators of muscle be a fruitful strategy for ameliorating the pathology wasting (Figure 2). Additionally, systemic THI led to a manifested in patients afflicted with DMD and possibly significant increase in muscle fiber size and specific force other muscle wasting diseases (for example dysferli- of CTX-injured muscles (Figures 3 and 4). In turn, nopathy). Therapies based on promoting S1P levels in ex vivo administration of high levels of S1P resulted in dystrophic muscle have the potential to improve path- specific force levels in uninjured mdx EDL muscles ology by promoting satellite cell and anabolic-mediated (Figure 4D). To pursue a better understanding of how regeneration. An obvious candidate for a small molecule elevated S1P reduces DMD pathology, we found that therapeutic is THI. Our work has shown that short- direct administration of S1P via intramuscular injection term treatment of THI has significant efficacy in doubles muscle S1P content compared to the S1P levels increasing regenerative capacity in the mdx mouse fol- reached with IP injections of THI. In addition, intramus- lowing acute muscle injury, while longer treatment can cular S1P injections led to an increase in myogenic cells improve muscle function in younger uninjured mdx (Myf5+) and induced phosphorylation of S1PR1, which muscle. Moreover, significant increases in muscle fiber was particularly abundant in newly regenerating fibers size have been suggested as a viable approach in (Figure 7, Additional file 1: Figure S15), as well as a sig- overcoming dystrophic muscle damage by promoting nificant increase in rpS6 and P-rpS6 levels (Figure 6). strength and function [76]. Additionally, there are other These results suggest that S1P not only works to activate THI derivatives with increased oral bioavailability that myogenic precursors but also elevates protein synthesis may be more effective at increasing and maintaining in muscle fibers, potentially through S1PR1 mediated high intramuscular S1P levels in long-term treatments, signaling (summarized in Figure 8C). In summary, THI/ which was necessary for functional improvement of un- S1P administration led to improved regeneration and injured EDL muscles [10]. Alternatively there are inhi- pathology, higher muscle specific force, an increase in bitors of lipid phosphate phosphatases and/or S1P the number of myogenic cells, and larger muscle fibers. phosphatases that may also increase intramuscular S1P Our results indicate that S1P mediates satellite cell- levels [10,77]. In addition, there are specific S1P recep- dependent and muscle fiber-dependent effects on skel- tor agonists (for example FTY720) that are currently etal muscle. If amelioration of muscle wasting occurs FDA approved or in clinical trials [78,79]. Based on our through receptor-mediated signaling then S1P, elevated present results and those of others, future studies fo- intracellularly via THI, must be exported to activate the cused on S1P-based therapeutics for the treatment of S1P receptors. THI has been reported to inhibit the S1P DMD and related myopathies are warranted. lyase, an enzyme whose active site is on the cytoplasmic side of the endoplasmic reticulum. Therefore elevations Additional file of S1P levels mediated via THI inhibition of the S1P lyase presumably occur within the cytoplasm [71]. S1P Additional file 1: Figure S1. Treatment with THI lowers mdx plasma may also act intracellularly before possible export to platelet levels. Figure S2. THI alters the expression of S1P regulatory promote muscle wasting suppression. This alternative is gene in mdx muscle. Figure S3. THI does not alter S1P plasma levels but lowers plasma CK activity. Figure S4. Muscle weight is preserved and supported by our work with Drosophila, which have no hydroxy proline is reduced in injured muscles from THI treated mdx mice. known S1P receptors [9], as well as by a recent report Figure S5. Fibrosis is lower in uninjured mdx TA muscles with THI that showed S1P interacts directly, intracellularly, with treatment. Figure S6. The number of centrally nucleated muscle fibers does not change with THI. Figure S7. Diaphragm muscle fibers size histone deacetylases (HDACs) [72]. As HDAC inhibitors increases with THI treatment. Figure S8. THI treated mdx mice have an have been previously shown to suppress dystrophic phe- elevated number of Pax7+ satellite cells. Figure S9. The microvasculature notypes in mdx mice, the actions of S1P on the sup- of mdx muscles did not increase with THI. Figure S10. CTX injected in the TA reaches and also damages the EDL. Figure S11. THI treatment pression of muscle wasting may occur in part through did not reduce T-cells in mdx diaphragms. Figure S12. Montages such mechanisms [73]. It has also been reported that Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 19 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 and Regenerative Medicine, University of Washington, Seattle, WA 98195, covering entire cross-sectional areas of each TA from S1P and vehicle USA. Department of Laboratory Medicine, School of Medicine, University of treated mdx4CV:Myf5nlacZ/+ animals, were created by combining Washington, Seattle, WA 98195, USA. Department of Chemistry, University of individual 10x photos. Figure S13. (A) Quantification of centrally Washington, Seattle, WA 98195, USA. Department of Neurology, Senator nucleated muscle fibers from the same injured TAs presented in Figure 5, Paul D Wellstone Muscular Dystrophy Cooperative Research Center, coincides with the number of newly regenerated fibers (eMyHC+ fibers) University of Washington, Seattle, WA 98195, USA. observed in S1P injected TA muscles. (B) Quantification of the minimum diameter of the largest eMyHC+ myofibers represented in Figure 5, Received: 27 November 2012 Accepted: 22 May 2013 indicates a significant increase in regenerated fiber size with S1P Published: 1 August 2013 treatment. Figure S14. The expression of S1P receptors is reduced in mdx muscle cells. Figure S15. Direct S1P administration results in elevated levels of phosphorylation S1PR1 in mdx muscles. Figure S16. References S1P promotes muscle regeneration in the A/J mouse model of 1. Deconinck N, Dan B: Pathophysiology of duchenne muscular dystrophy: dysferlinopathy. Table S1. Average number of Evans Blue+ muscle fibers current hypotheses. Pediatr Neurol 2007, 36:1–7. within each muscle group. 2. Mendell JR, Rodino-Klapac LR, Malik V: Molecular therapeutic strategies targeting Duchenne muscular dystrophy. J Child Neurol 2010, Abbreviations 25:1145–1148. ANOVA: Analysis of variance; BSA: Bovine serum albumin; CK: Creatine kinase; 3. 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PLoS One 2008, We thank Zack Usa, Alex Nelson and Jessica Becker for technical help. We 5:e10920. thank Dr Guenter Daum for advice and guidance on S1P based approaches. 14. Grabski AD, Shimizu T, Deou J, Mahoney WM Jr, Reidy MA, Daum G: We also thank Dr Guenter Daum and Dr LeBoeuf Renee for sharing Akt- and nlacZ/+ Sphingosine-1-phosphate receptor-2 regulates expression of smooth S1P-related antibodies. We thank Dr Zipora Yablonka-Reuveni for the Myf5 muscle alpha-actin after arterial injury. Arterioscler Thromb Vasc Biol 2009, reporter mice. Finally, we would like to thank Dr Nick Whitehead for his technical 29:1644–1650. guidance on myography. This work was supported by the University of 15. Au CG, Butler TL, Sherwood MC, Egan JR, North KN, Winlaw DS: Increased Washington Center for Commercialization (C4C), Departments of Pathology and connective tissue growth factor associated with cardiac fibrosis in the Laboratory Medicine, University of Washington; Provost Bridge grant to MR; mdx mouse model of dystrophic cardiomyopathy. Int J Exp Pathol 2011, University of Washington Nathan Shock Center of Excellence in the Basic Biology 92:57–65. of Aging and the Genetic Approaches to Aging Training Grant T32 AG00057 to 16. Danieli-Betto D, Germinario E, Esposito A, Megighian A, Midrio M, Ravara B, NI; and the American Recovery and Reinvestment Act of 2009 (ARRA) Challenge Damiani E, Libera LD, Sabbadini RA, Betto R: Sphingosine 1-phosphate Grant 5RC1AR058520, R01GM083867, R01GM097372 and 1P01GM081619 to HRB. protects mouse extensor digitorum longus skeletal muscle during Support was also received from the Washington Research Foundation, the fatigue. 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J Pharmacol Exp Ther 2011, • Thorough peer review 338:879–889. • No space constraints or color figure charges doi:10.1186/2044-5040-3-20 • Immediate publication on acceptance Cite this article as: Ieronimakis et al.: Increased sphingosine-1-phosphate • Inclusion in PubMed, CAS, Scopus and Google Scholar improves muscle regeneration in acutely injured mdx mice. Skeletal Muscle 2013 3:20. • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

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Copyright © 2013 by Ieronimakis et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-3-20
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23915702
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Abstract

Background: Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice. Methods: We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles. Results: Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer- term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular nlacz/+ injection of S1P increased the number of Myf5 positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function. Conclusions: These data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD. Background degeneration, leading to muscle wasting over time. Duchenne muscular dystrophy (DMD) is a muscle wast- Since no effective treatment presently exists and the im- ing disease for which there is no cure. This severe X- mune response to dystrophin has hampered gene ther- linked recessive disease affects 1 in 3,500 male births apy approaches, new advances for the treatment of [1]. In dystrophic muscles, rounds of contractions result DMD are imperative [2,3]. in degeneration/regeneration cycles. In turn, dystrophic Previously, sphingosine-1-phosphate (S1P) has been im- muscle cannot regenerate sufficiently to overcome plicated in muscle repair, satellite cell proliferation, myo- blast differentiation in vitro and in non-diseased mouse models in vivo [2,4-6]. These essential roles for S1P in * Correspondence: hannele@u.washington.edu; morayma@u.washington.edu skeletal muscle regeneration suggested that elevation of Department of Biochemistry, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA S1P may have therapeutically beneficial effects in models Department of Pathology, School of Medicine, University of Washington, of disease [7]. More recently, S1P has been shown benefi- Seattle, WA 98195, USA cial for activating satellite cells in dystrophic muscles [8]. Full list of author information is available at the end of the article © 2013 Ieronimakis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 2 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Furthermore, an unbiased genetic modifier screen in Injections were 6 hours apart. This injection regimen and Drosophila revealed that by increasing S1P levels via re- dose was repeated for all subsequent experiments involv- duction of the lipid phosphate phosphatase 3 (LPP3) ing THI, but for longer-treatment durations as outlined. 4cv homolog, wunen, or the S1P lyase, sply, prevents to a large Six 5-MO mdx males were used for the experiments in degree dystrophic muscle wasting in flies [9]. In mice, Figure 1B, and Additional file 1: Figure S1 and S2. For elevation of S1P by the genetic reduction of S1P lyase Figures 2 and 3, and Additional file 1: Figures S3 to S7, six 4cv can be phenocopied pharmacologically via treatment 11-MO females and seven 16-MO males mdx were with the small molecule 2-acetyl-4(5)-tetrahydroxybutyl used for these experiments. In these mice, the left tibialis imidazole (THI) [10,11]. Furthermore, in Drosophila, anterior (TA) and quadriceps femoris (quads) were injured THI treatment also significantly suppresses the dys- with 10 nM CTX (Calbiochem, Darmstadt, Germany) trophic muscle phenotype [9]. from Naja nigricollis. Once more, THI-treated mice were Utilizing the mdx mouse model, we initiated studies injected IP with 250 μl 0.15 mg/ml THI in PBS, twice daily on the effect of increasing S1P levels in dystrophic mice, (injections 6 hours apart) immediately after injury and for and found that short-term treatment with THI improves the first 3 days following injury. The vehicle controls were 4cv muscle integrity and function following acute injury with injected IP with PBS. On day 4 post injury, 5-MO mdx cardiotoxin (CTX). THI treatment also leads to signi- animals were euthanized for S1P and creatine kinase (CK) 4cv ficant improvements of the pathology of dystrophic analysis. On day 17 post CTX, 11-MO and 16-MO mdx muscles, as indicated by the reduced accumulation of fi- mice were also injected IP with 1% Evans Blue dye (EBD) brosis and fat deposition in acutely injured muscles. In to label persistently damaged (dye permeable) muscle fi- turn, intramuscular injection of S1P resulted in an in- bers [12], and euthanized on day 18 post injury for his- creased number of myogenic cells and newly regenerat- topathology analysis. Muscles for S1P and expression 4cv ing fibers in vivo. S1P receptor 1 (S1PR1) is expressed by analysis (from 5-MO mdx ) were frozen directly in liquid many muscle cell types, particularly muscle fibers, and nitrogen, while muscles taken for histopathology were fro- phosphorylated S1PR1 is localized in the plasma mem- zen under liquid nitrogen cooled isopentane in optimal brane and intracellularly (perinuclear localization) of cutting temperature (OCT) compound. All myofibers muscle fibers. Intramuscular S1P administration results were measured for the minimum diameters on the cross- in increased levels of total and phosphorylated S1PR1 sections of mouse quadriceps muscle using ImageJ software and ribosomal protein S6 (rpS6). This suggests that in- (Bethesda, MD, USA). Between 750 and 850 myofibers creases in fiber size are mediated by anabolic pathways were counted for three mice treated with PBS or THI, that promote greater skeletal muscle mass and function, with or without CTX injury. For functional analysis potentially through S1PR1 signaling. Furthermore, ex vivo outlined in Figure 4B, 4.75- to 5-MO male mdx on a mdx/J administration of S1P improved specific force in uninjured C57BL/10 background (C57BL/10ScSn-Dmd )were dystrophic muscle. Similarly, longer-term THI treatment used for the 14-day treatment of THI or vehicle. Following of uninjured young mdx mice resulted in increased exten- the same dose and treatment regimen, mdx were treated sor digitorum longus (EDL) muscle force in the absence of with THI (n = 10) or vehicle (n = 9) for 14 days following CTX injury. Altogether, S1P acts at multiple levels in mus- CTX injury to left TAs and quadriceps. The same mdx cles, particularly in myogenic cells and muscle fibers, and strain was compared to wt C57BL/10 animals in Figure 4C collectively the actions of S1P in muscle are beneficial for and for exogenous S1P treatment depicted in Figure 4D. regeneration in the setting of muscular dystrophy. Animals used to evaluate the degree of CTX injury in EDL (Additional file 1: Figure S8) were 4-MO female mdx mdx/J Methods (n = 4, C57BL/10ScSn-Dmd background), injected in Animal procedure left TAs with CTX and with approximately 3 μlIndia ink, Experiments involving animals were undertaken in ac- added to the tip of the needle to mark injection penetra- cordance with approved guidelines and ethical approval tion. Following CTX injections, mice were immediately from the Institutional Animal Care and Use Committee, injected IP with 1% EBD. Both left (injured) and contralat- University of Washington, Seattle, WA, USA. eral uninjured TA and EDL muscles were harvested and frozen in OCT compound 12 hours post injury. THI injections in injured mice Peripheral blood cells from 1.5-month-old (MO) wild THI treatment in drinking water of young, uninjured mdx type (wt) C57BL/k6 and mdx mice on a C57BL/k6 back- mice mdx-4Cv 4cv ground (B6Ros.Cg-Dmd /J, herein referred to as Beginning at 4 weeks of age, male mdx were treated 4cv mdx ) were analyzed (Figure 1A). Blood was collected with THI (n = 4) or vehicle (n = 3) for 4 weeks, and ana- before and 12 hours following the last of two 250 μl in- lyzed by EDL myography at 8 weeks of age. For this traperitoneal (IP) injections of 0.15 mg/ml THI in PBS. treatment we followed the dose and conditions described Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 3 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 1 IP injection of THI reduces peripheral blood leukocytes and increases S1P levels in most tissues. (A) Leukocytes were analyzed 4cv from the peripheral blood of 1.5-MO mdx mice (n = 3) before and 12 hours following treatment with THI (2 × 250 μl 0.15 mg/ml IP injections, 6 hours apart). IP administration of THI significantly reduced circulating leukocytes to values below or near age-matched wt (n = 4). The average value of each population is listed in the table below the bar graph. Values between pre and post THI, and wt were also significant by ANOVA 4cv (P <0.05) for all leukocytes except monocytes. (B) mdx mice (n = 6, 5-MO) were treated with THI or vehicle for 3 days (2 × 250 μl 0.15 mg/ml IP injections per day) following CTX injury to assess changes in S1P muscle content. Muscles and spleens were harvested on day 4 post injury for S1P analysis by LC-MS/MS. Results indicate S1P levels in spleen and injured quadriceps (quads) were significantly elevated with THI treatment. Interestingly, uninjured quadriceps did not show a significant increase of S1P, whereas uninjured TA muscles did. *P <0.05 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; IP, intraperitoneal; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole; wt, wild type. by Schwab et al. [11]. Briefly, 50 mg/l THI was adminis- 20 μl per sample using the Hemavet 950 FS system tered ad libitum. The vehicle consisted of water at pH (Drew Scientific, Dallas, TX, USA). 2.8 containing 10 g/l glucose. Analysis of gene expression by quantitative reverse Peripheral blood cell analysis transcription-PCR (RT-PCR) Blood was collected via retro-orbital blood collection Total RNA (RNeasy Kit, Qiagen, Venlo, Netherlands) 4cv using heparinized capillaries and transferred to blood was prepared from mdx TA muscles homogenized collection tubes containing a final concentration of 1.6 under liquid nitrogen by mortar and pestle. Methods for mg/ml EDTA (SARSTEDT, Nümbrecht, Germany) for RNA isolation and cDNA generation were in accordance analysis. Analysis of whole blood was undertaken with with manufacturer’s protocols using reverse transcriptase Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 4 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 2 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 5 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 2 Dystrophic pathology following muscle injury is improved with THI treatment. (A) Experimental schematic of THI (0.075 μg/day) 4cv and PBS (vehicle)-treated mdx mice injected IP twice daily for the first 72 hours following CTX injury. Muscles from aged mdx mice (n = 7, THI-treated: 3 × 11-MO females, 4 × 16-MO males; n = 6 vehicle-treated: 3 × 11-MO females, 3 × 16-MO males) were harvested for histopathology analysis 18 days post CTX injury. (B) Histological quantification of picrosirius red staining indicates lower fibrotic accumulation following injury in both TA and quadriceps (quads) muscles from mice treated with THI. For CTX-injected muscles, damaged regions of muscle (for example fields with the greatest accumulation of sirius red staining) were quantified for both THI and vehicle-treated mice. The level of fibrosis was not significantly different between treated and control (vehicle) uninjured quadriceps; however, uninjured TA muscles from 11-MO THI-treated mice had lower fibrosis compared to control TA muscles. For each muscle, three separate sections (200 μm apart in longitudinal distance) were analyzed. (C) Representative photographs of injured quadriceps stained with picrosirius red and fast green depict collagen deposition (red staining), while muscle morphology and organization is depicted with hematoxylin and eosin staining. Scale bars = 50 μm. (D) Oil Red O staining depicts fat deposits (arrows) over the entire CSA of THI-treated and vehicle-injured quadriceps from 16-MO males. Scale bars = 500 μm. (E) The ratio of fat deposition in injured TAs over uninjured contralateral TAs quantified from Oil Red O staining was significantly 4cv reduced in THI-treated versus control animals in 11-MO (*) but not 16-MO mdx mice. In contrast, the ratio of injured over uninjured fat deposits in quadriceps was significantly reduced in 16-MO (#) but not in 11-MO mdx mice. *P <0.05, **P <0.01 by student’s t-test. Error bars represent SEM. CSA, cross-sectional area; CTX, cardiotoxin; IP, intraperitoneal; MO, month-old; SEM, standard error of the mean; TA, tibialis anterior; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. (Applied Biosystems, Carlsbad, CA, USA) as previ- 450 ms train duration [17]. Force frequency was carried ously described [13]. RNA (0.5 μg) was reverse tran- out using the same pulse duration at 10, 20, 40, 60, 80, scribed using the Omniscript RT Kit (Qiagen). For 100 and 120 Hz, as outlined in the x-axis of Figure 3B. reverse transcription-PCR (RT-PCR), 10 ng cDNA was Specific force was calculated as previously described combined with SYBR Green (Thermo Scientific, Waltham, [18] by normalizing to the muscle cross-sectional area MA, USA) following published conditions and primer (CSA). CSA is the quotient of dry muscle mass (mg) sequences for S1P-related genes by Grabski et al.[14] over Lo (mm), which is defined as the product of Lf and by Au et al.[15]for 18S. with the fiber length ratio (0.44 for EDL) and mamma- lian muscle density (1.06 mg/mm ). Functional analysis: myography Animals treated with THI or PBS (vehicle) via IP injec- Measurement of S1P in mouse tissue tion as aforementioned for 14 days were analyzed be- S1P was quantified in tissue after homogenization and tween 1 and 4 days following the final day of injection. extraction using liquid chromatography-tandem mass Prior to euthanasia animals were anesthetized with 0.5 spectrometry (LC-MS/MS). Tissue was pulverized in mg/g weight avertin diluted in PBS. EDLs were then ex- liquid nitrogen using a mortar and pestle. Collected tis- cised and equilibrated in Ringer’s solution (120 mM sue was weighed and an internal standard (C17 base NaCl, 4.7 mM KCl, 3.15 mM MgCl , 1.3 mM NaH PO , D-erythro-sphingosine-1-phosphate in methanol (Avanti 2 2 4 25 mM NaHCO , 11 mM glucose, 1.25 mM CaCl , Polar Lipids, Alabaster, AL, USA)) was added at 1 pmol/ 3 2 pH 7.2) with 95% O /5% CO for a minimum of 15 mi- mg tissue. Tissue was then vortexed/extracted in 16 vol- 2 2 nutes prior to stimulation [16]. For assessment of direct umes (mg/μl) of acetonitrile:water (80:20, v/v) for 10 mi- S1P administration, EDL muscles from uninjured and nutes at room temperature. Supernatants were collected mdx/J untreated 3.5-MO male mdx (C57BL/10ScSn-Dmd ) after centrifugation (10 minutes at 14,000 rpm) and con- were incubated with oxygenated Ringer’s solution centrated to dryness using a SpeedVac Concentrator containing 10 μM S1P or vehicle (PBS with 4 mg/ml (Thermo Scientific). Pellets were resuspended in metha- fatty acid free BSA) for 15 minutes prior to stimulation nol to a calculated concentration of 0.05 μM C17 base [16]. All functional experiments were carried out with D-erythro-sphingosine-1-phosphate. Then 10 μlwas buffer solutions at 25°C under constant oxygenation. analyzed by LC-MS/MS using C17 base D-erythro- Myography was conducted using a 820S myograph sphingosine-1-phosphate plus C18 base D-erythro- (DMT, Ann Arbor, MI, USA) and data was recorded sphingosine-1-phosphate (both at 0.05 μM) as a standard. using a PowerLab 4/30 acquisition system with LabChart Separation of analytes was undertaken by liquid chro- Pro software v7.3.1 (both from ADInstruments, Dunedin, matography using a Chromolith RP-C18e 100 × 2 mm New Zealand). Stimulations were conducted with S88X column (EMD, Gibbstown, NJ, USA) and analysis by dual systems (Grass Technologies, Middleton, WI, USA). tandem mass spectrometry with a Quattro Micro mass Muscles were stimulated to establish optimal fiber spectrometer (Waters, Milford, MA, USA) in positive length (Lf) and voltage at which maximum tetanic force ion mode. The HPLC gradient using two pumps was wasmeasuredat120 Hz using4.15mspulseswithin linear from 50% MeOH to 99% MeOH using solvent A Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 6 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 3 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 7 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 3 Elevating S1P levels with THI increases muscle fiber size. (A) Staining for laminin (green) and DAPI (blue) depict a dramatic increase in muscle fiber size in both injured and uninjured quadriceps (quads) with THI treatment. Depicted are quadriceps muscles from 11-MO 4cv mdx mice. Scale bars = 50 μm. (B,C,D) Quantification of minimum muscle fiber diameter reveals a significant increase in myofiber size in THI- 4cv treated animals. Increased myofiber diameter was observed in both (B) injured and (C) uninjured quadriceps from THI-treated 11-MO mdx 4cv mice, whereas only (D) uninjured quadriceps in THI-treated 16-MO mdx mice showed increased myofiber size compared to vehicle controls. As indicated by the distributions, mean and median values of muscle fiber minimum diameters, there is an overall increase in muscle fiber size with THI treatment. Quantifications were undertaken in random fields in both injured and uninjured muscles in order to obtain an overall representation of fiber size increase for each muscle.*P <0.05, ***P <0.0005 by student’s t-test. Error bars represent SEM. DAPI, 4',6-diamidino-2- phenylindole; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. (water, 0.1% formic acid) and solvent B (MeOH, 0.1% with a dwell time of 0.07 seconds. Data collection was formic acid) over 1 minute at a flow rate of 0.35 ml/ by MassLynx software (Waters) and processed with min. To wash the column, the gradient was repeated QuanLynx software (Waters). twice before equilibrating for 3 minutes before running the next sample. The transitions analyzed were 380.25 Measurement of S1P in mouse plasma >264.50 and 380.25 >82.00 for endogenous S1P, and S1P was quantified in plasma using butanol extraction 366.25 >250.50 and 366.25 >82.00 for internal standard and liquid LC-MS/MS [19]. Internal standard (5 μl3 μM mdx/J Figure 4 S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmd ) muscle. (A) Experimental schematic of longer-term, 14-day treatment of THI or PBS (vehicle) following CTX injury. THI was administered following the aforementioned dose and injection regimen. Following treatment, EDL muscles were harvested and specific isometric force was analyzed by in vitro myography from both injured and uninjured limbs. (B) Force frequency analysis reveals that EDL muscles isolated from injured limbs of THI-treated animals (n = 10) have significantly greater specific force compared to injured vehicle controls (n = 9). (C) Analysis of untreated and uninjured wt (C57BL/10ScSn) and mdx/J mdx (C57BL/10ScSn-Dmd ) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured mdx/J and untreated mdx (C57BL/10ScSn-Dmd ) EDL muscles with a high concentration of S1P (10 μM) leads to a significant increase in maximal specific force. *P <0.05, **P <0.005 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; EDL, extensor digitorum longus; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole; wt, wild type. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 8 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 C17 base D-erythro-sphingosine-1-phosphate in ethanol manufacturer’s instructions. Briefly, S1P was dissolved (Avanti Polar Lipids)) was added to 10 μl EDTA- in methanol (0.5 mg/ml) and aliquoted, then the solvent anticoagulated plasma and mixed thoroughly on an or- was evaporated with a stream of nitrogen to deposit a bital shaker (Thermomixer, Eppendorf, Hauppauge, NY, thin film on the inside of the tube. Prior to use, aliquots USA) for 10 minutes at 1,400 rpm at 20°C. The sample were resuspended in PBS with 4 mg/ml BSA (fatty acid was then acidified using 50 μl 30 mM citric acid/40 mM free) to a concentration of 500 μM. Directly following Na HPO , pH 4.0, and extracted for 10 minutes at 1,400 CTX injection, 20 μl500 μMS1P wasinjectedinleft 2 4 rpm at 20°C with 125 μl water-saturated butanol (Fisher TAs, daily until day 3 post injury, at which time animals Scientific, Waltham, MA, USA). The butanol layer was were euthanized and muscles were harvested for freez- removed and lyophilized in a centrifugal evaporator at ing. Right TAs were injected with an equal volume of 20°C. The residue was stored at −20°C until analyzed. PBS with 4 mg/ml BSA as vehicle controls. In a separate The residue was resuspended in 125 μl HPLC buffer A experiment (Figure 6), TAs of four 2.5-MO female 4cv (50% methanol, 1% formic acid, 5 mM ammonium mdx were injected with S1P or vehicle under the formate in water (JT Baker) and sonicated in a bath same conditions stated above, in the absence of injury. prmd scid sonicator for 1 minute at 20°C. Analytes in a portion of AJ/SCID mice (n = 4, 9-MO, B6. Cg-Dysf Prkdc /J) the sample (10 μl) were then separated using liquid were also injected for 3 days with S1P or vehicle in TAs chromatography (Shimadzu, Nakagyo-ku, Kyoto, Japan) post CTX injury, following the same concentration and 4cv with a Luna 3 μm C18(2) 100Ǻ 50 × 2 mm column injection regimen used in mdx . For measurement of (Phenomenex, Torrance, CA, USA) and analyzed by tan- S1P muscle content (Figure 7A) following intramuscu- 4cv dem mass spectrometry on a 4000 QTRAP mass spec- lar injections, 11-MO mdx (n = 3) were injected 20 μl trometer (AB SCIEX, Framingham, MA, USA) in 500 μM S1P in left TAs and 20 μl vehicle in right TAs. positive ion mode. The HPLC gradient was linear from Muscles were harvested and frozen in liquid nitrogen buffer A to buffer B (10% isopropyl alcohol, 1% formic 15 minutes post injection, and then processed using the acid, 5 mM ammonium formate in methanol) over 1 mi- aforementioned methods for analyzing S1P in muscle nute at a flow rate of 0.4 ml/min. To wash the column, by LC-MS/MS. For injection of biotinylated-S1P, TAs 4cv the gradient was repeated twice before equilibrating for from 11-MO mdx (n =2)wereinjectedintramuscu- the next sample. The transitions analyzed were 380.3/ larly with 20 μl500 μM S1P-biotin or vehicle (Echelon 264.3 and 380.3/81.9 for endogenous S1P, and 366.2/ Biosciences, Salt Lake City, UT, USA). TAs were 93.0, 366.2/82.0 and 366.2/250.3 for internal standard harvested and frozen in OCT compound 15 minutes fol- with a dwell time of 15 milliseconds. Calibrators were in lowing injection. mouse plasma (C18 base D-erythro-sphingosine-1-phos- phate, Avanti Polar Lipids). Between-day coefficient of Mouse histology and immunohistochemistry variation was 7.7%. Pertinent instrument specific param- All mouse muscles were frozen directly in OCT com- eters were empirically derived and included curtain gas: pound with liquid nitrogen cooled in isopentane and 15, ion source voltage: 5000 V, emitter temperature: 550°C, sectioned 8 μm thick. Tissue for X-gal staining was fixed desolvation gas 1: 20, desolvation gas 2: 70, collision gas: 6, for 10 minutes with 2% formaldehyde/0.2% glutaralde- entrance potential: 10, and collision cell exit potential: 10. hyde and incubated overnight at 37°C with staining Chromatographic data were analyzed using Analyst 1.4.2 buffer (PBS with 1 mg/ml X-gal, 5 mM potassium ferri- (AB SCIEX) by summing transitions for each analyte. cyanide, 5 mM potassium ferrocyanide, 2 mM CaCl (all from Fisher Scientific)). Picrosirius red with fast green, Creatine kinase (CK) assay hematoxylin and eosin, and Oil Red O staining were 4cv mdx mouse plasma samples were diluted 1:50 and conducted following established protocols [21]. Fibrosis total CK activity was measured by an enzymatic rate was quantified as percentage of area stained red within method at the clinical laboratory of the Department of each 20 × field analyzed using ImageJ v1.40 or Adobe Laboratory Medicine, University of Washington, using Photoshop CS2 (San Jose, CA, USA). For evaluating fi- the Beckman Coulter instrument (Brea, CA, USA) as brosis, the mean value from three separate sections (200 previously described [20]. Relative levels were then nor- μm apart in longitudinal distance) were analyzed from malized to body weight. each muscle and used to calculate the overall mean for each muscle group outlined in the x-axis of Figure 1D. S1P injections Lipid accumulation was quantified with the ImageJ cell 4cv nlacZ/+ Right and left TAs of three 3-MO male mdx :Myf5 counter plugin by counting fatty infiltrates in montages were injured once more with 10 nM CTX (Figure 5). (stitched from 10 × photos) covering the entire CSA of S1P (Enzo Life Sciences, Farmingdale, NY, USA; each muscle. Muscles injected with S1P-biotin or vehicle Calbiochem) preparation was undertaken according to were cut 8 μm thick, fixed for 5 minutes with 4% Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 9 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 5 Direct administration of S1P promotes muscle regeneration following acute injury. (A) Experimental schematic of S1P and PBS 4cv nlacZ/+ (vehicle) injected daily for the first 72 hours into TAs of 3-MO mdx :Myf5 mice (n = 3, left TAs injected S1P, right TAs injected PBS) following CTX injury. (B) Top row: X-gal staining reveals an increased number of β-galactosidase+ nuclei at the sites of injury in S1P-treated TA muscles compared to vehicle controls. Bottom row: staining for eMyHC with DAB reveals a significant increase in the number of newly regenerated muscle fibers in S1P-treated TA muscles. Scale bars = 50 μm. (C) Left graph: quantification of β-galactosidase+ nuclei indicates the number of Myf5+ cells is significantly increased at the site of injury in S1P-treated compared to untreated muscles. Middle graph: a significant increase in β-galactosidase+ nuclei was also observed over the entire CSA of each S1P-treated TA muscle. Right graph: quantification of the number of eMyHC fibers within areas of regeneration was significantly greater with S1P treatment. *P <0.05 by student’s t-test. Error bars represent SEM. CSA, cross-sectional area; CTX, cardiotoxin; DAB, 3,3'-diaminobenzidine; eMyHC, embryonic myosin heavy chain; MO, month-old; S1P, sphingosine-1-phoshate; SEM, standard error of the mean; TA, tibialis anterior. formaldehyde, and then stained with streptavidin conju- were fixed overnight in 4% formaldehyde (from parafor- gated to Alexa Fluor 594 (Life Technologies, Carlsbad, maldehyde powder) at 4°C. Following fixation, antigen CA, USA) at 1:1000 in PBS and 1% BSA for 1 hour. retrieval was performed with 10 mM citrate buffer (with0.05% Tween20atpH6.0)warmedin a water bath at 90°C for 20 minutes. Slides were then perme- Immunohistological staining 4cv ated with ice cold methanol for 5 minutes at room Staining was undertaken using freshly frozen mdx temperature. Streptavidin/biotin blocking (Vector Labora- muscles. Pax7 staining was performed as outlined by tories, Burlingame, CA, USA) was performed according to Clever et al. [22] with slight modification. Sections Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 10 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 6 Administration of S1P leads to increased levels of S1PR1 and P-rpS6 in vivo. (A) Experimental schematic of S1P and PBS (vehicle) 4cv injected daily for the first 72 hours into TAs of uninjured mdx mice (n = 4, 2.5-MO, left TAs injected S1P, right TAs injected PBS). (B) Western 4cv blot analysis of injected TAs (n = 3, 2.5-MO mdx ) indicates that administration of S1P significantly increases S1PR1 levels. (C) Western blot 4cv analysis of injected TAs (n = 4, 2.5-MO mdx ) for total, and P-Akt, P-mTOR and P-rpS6, reveals that total and P-rpS6 were significantly higher with S1P treatment. Increased levels of total and P-rpS6 suggest that S1P administration promotes protein synthesis in mdx muscles. *P <0.05 by student’s t-test. Error bars represent SEM. MO, month-old; P-Akt, phosphorylated Akt; P-mTOR, phosphorylated mammalian target of rapamycin; P-rpS6, phosphorylated ribosomal protein S6; rpS6, ribosomal protein S6; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; TA, tibialis anterior. manufacturer’s instructions. Staining was undertaken using monoclonal anti-CD3e (clone 145-2C11, eBioscience, San the Mouse on Mouse (MOM) Kit (Vector Laboratories) Diego, CA, USA) at 1:100 dilution, followed by anti-rat with immunoglobulin G (IgG) blocking for 5 hours at 4°C IgG conjugated to Alexa Fluor 594 at 1:1000 dilution. prior to addition of mouse monoclonal anti-Pax7 (clone For laminin staining, tissue was also fixed with 2% for- PAX7, R&D Systems, Minneapolis, MN, USA) diluted at maldehyde for 5 minutes then treated with polyclonal 1:20 and incubated overnight at 4°C. Biotinylated anti- rabbit anti-laminin (Sigma-Aldrich, St Louis, MO, USA) mouse secondary was supplied with and used as pre- for 1 hour at 1:400 dilution in PBS and 1% BSA. Follow- scribed by MOM Kit instructions. Streptavidin conjugated ing washes, Alexa Fluor 488 conjugated goat anti-rabbit to Alexa Fluor 488 (Life Technologies) was added at IgG (Life Technologies) was administered at 1:800 dilu- 1:1000. As a negative control for Pax7 staining, a mouse tion for 1 hour. Controls omitting the primary antibody IgG isotype was applied to separate ribbons and treated in were included with all staining. For embryonic myosin parallel. For BS1 staining, muscles were initially fixed with heavy chain (eMyHC), tissue was first fixed with 2% for- 4% formaldehyde for 5 minutes at room temperature then maldehyde for 5 minutes, treated with streptavidin/ stained with BS1 directly conjugated to fluorescein iso- avidin blocking and blocked with IgG block from MOM thiocyanate (FITC), diluted at 1:400 in PBS with 1% BSA Kit for 5 hours at 4°C. Following blockade, concentrated and applied for 1 hour at room temperature. Following mouse anti-eMyHC (clone F1.652, received concen- BS1 staining, wheat germ agglutinin (WGA) directly con- trated at 357 μg/ml IgG, Developmental Studies Hybrid- jugated to rhodamine was administered at 1:400 dilution oma Bank (DSHB), University of Iowa, IA, USA) was as a counterstain for identifying myofibers. CD3e staining administered at 1:400 dilution overnight at 4°C. The was undertaken in the same manner as BS1, using rat remainder of the staining was undertaken following Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 11 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Figure 7 (See legend on next page.) Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 12 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (See figure on previous page.) Figure 7 Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. (A) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5) of S1P in left TAs and vehicle in right TAs 4cv of uninjured mdx (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. (B) To visualize the location of S1P following injection, biotinylated-S1P was injected in left 4cv TAs versus vehicle in right TAs of uninjured mdx mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining 4cv with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. (C) Staining of mdx TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). (D) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5) reveals S1PR1 is 4cv present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. **P <0.005 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; eMyHC, embryonic myosin heavy chain; IgG, immunoglobulin G; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; S1PR3, S1P receptor 3; SEM, standard error of the mean; TA, tibialis anterior. MOM Kit staining instruction. 3,3'-diaminobenzidine complete protease inhibitor cocktail (Roche, Basel, (DAB) was used for visualizing and quantifying eMyHC Switzerland), and complete phosphatase inhibitor cocktails fibers. For fluorescence, eMyHC was visualized using 1 and 2 (Sigma-Aldrich). Protein extracts were separated streptavidin conjugated to Alexa Fluor 594 used at using Ready Gel Tris–HCl (BioRad, Hercules, CA, USA), 1:1000 dilution for 1 hour. For S1P receptor staining, 4 to 20% linear gradient and transferred to polyvinylidene slides were fixed with 4% formaldehyde for 5 minutes fluoride (PVDF) membranes with a wet transfer system and stained with rabbit polyclonal IgG antibodies (BioRad). Membranes were blocked for 1 hour with against S1PR1, S1PR3 (Cayman Chemical, Ann Arbor, Tris-buffered saline with 0.1% (v/v) Tween 20 containing MI, USA) and phosphorylated S1PR1 (raised against 5% (w/v) BSA. For S1PR1 analysis, rabbit polyclonal Thr236, Assay Biotechnology, Sunnyvale, CA, USA), all anti-S1PR1 was used at a 1:500 dilution (Santa Cruz Bio- applied at a dilution of 1:200 for 2 hours. Following re- technology, Santa Cruz, CA, USA). Rabbit polyclonal anti- ceptor staining, goat anti-rabbit IgG conjugated to Alexa bodies were used to blot against phosphorylated (Thr308) Fluor 488 was added at 1:1000 for 1 hour. In parallel, we Akt, total Akt, phosphorylated (Ser2448) mammalian tar- stained additional slides with rabbit polyclonal IgG isotype get of rapamycin (mTOR), total mTOR, phosphorylated at the same final concentrations to exclude non-specific (Ser240/Ser244) rpS6, total rpS6 (1:1000, Cell Signaling 4cv staining of these antibodies in mdx muscles. Technology, Danvers, MA, USA) and β-actin (1:10000, Staining quantifications were all undertaken using Sigma-Aldrich). The signals were detected using an en- ImageJ cell counter plugin. Calculations, statistics and hanced chemiluminescence kit (Millipore, Billerica, MA, graphs were generated with Microsoft Excel (Redmond, USA) and CL-XPosure films (Thermo Scientific) were an- WA, USA). Bright field photographs were captured using alyzed using ImageJ. either a Fisher Scientific Micromaster digital inverted or upright microscopes with Micron software. Fluorescent Statistics photographs were captured with a monochromatic camera Student’s t-test was used to determine statistical signifi- using an Axiovert 200 microscope (Zeiss, Oberkochen, cance for the majority of experiments. P values gener- Germany). Individual fluorescent channels were colored ated by analysis of variance (ANOVA) are specified in and merged using Adobe Photoshop. Brightness contrast the text. levels were adjusted to increase visibility and reduce back- ground in most photographs. Results Alterations of S1P regulation and content following IP Western blot analysis injection of THI in mdx mice Tissue for western blot analysis was snap frozen in liquid To determine the effect of elevating S1P levels in dys- nitrogen and subsequently homogenized. Freshly iso- trophic animals, we studied the effects of THI in the lated TA muscles were harvested and snap frozen in li- mdx mouse model for DMD [23,24]. Recently, Loh et al. quid nitrogen prior to homogenization with disposable (2012) showed that compared to wt, mdx muscles are in tissue grinders. Tissue was homogenized under liquid a state of S1P deprivation as they exhibit increased levels nitrogen then resuspended in lysis buffer containing of the enzymes that degrade S1P (S1P lyase and S1P 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, phosphatase 1) [8]. THI is a hydrophilic small molecule 5 mM NaF, 0.25% (w/v) sodium deoxycholate, 2 mM that increases S1P levels by inhibiting the lyase that irre- NaVO , 1% Triton X-100 (v/v), supplemented with versibly degrades S1P [11,25,26]. In turn, low doses of 3 Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 13 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 THI may be sufficient to cause mild lymphocytopenia but significantly elevated in the injured muscles from THI- the presumable increase of S1P levels in muscle have not treated animals. These results suggest that acute injury 4cv been reported [8,11]. To corroborate the effects of THI in in mdx muscles induces upregulation of enzymes that 4cv mdx mice, we analyzed changes in lymphocytes before regulate S1P metabolism. In turn, elevated expression of and after treatment, and measured S1P content in muscle both S1P kinases with THI treatment may be beneficial (Figure 1). THI has low oral bioavailability; Bagdanoff et for muscle regeneration in mdx mice. However, with al. showed 10 to 12% bioavailability of THI when adminis- THI treatment S1P phosphatase 1 and lyase expression tered orally [10]. Thus we evaluated IP injections of THI were also greatly increased. Therefore we examined S1P as a parenteral delivery route for elevating systemic levels content, to determine if THI treatment results in in- of THI. Peripheral blood was collected and analyzed be- creased intramuscular S1P levels and in turn promotes fore and 12 hours after two IP injections of THI (each in- muscle regeneration following CTX injury. jection was 250 μl 0.15 mg/ml THI, administered 6 hours In order to determine if THI treatment results in in- apart). Following THI treatment, we observed a significant creased intramuscular S1P levels, a second group of 4cv 4cv drop of all leukocytes except monocytes in mdx (n = 3, mdx animals was treated with THI or PBS (n = 6, 1.5-MO) (Figure 1A). Of note, prior to treatment with 5-MO males), following the same dosing schedule (2 × IP THI, the total number of white blood cells and amount of injections per day for the first 3 days post CTX injury) and individual leukocyte populations except monocytes, was sacrificed at day 4 to analyze the efficacy of THI in 4cv significantly elevated in 1.5-MO mdx mice (n = 3) ver- increasing S1P levels (Figure 1B). In concordance with sus age-matched wt mice (n = 4). Interestingly, the num- published work, treatment with THI increased S1P levels 4cv ber of platelets was also elevated twofold in mdx versus in spleen but not plasma (Figure 1B, Additional file 1: wt, but declined to near wt following THI administration Figure S3A) [10,11]. S1P levels were also significantly in- (Additional file 1: Figure S1). This systemic effect in creased in CTX-injured quadriceps from THI-treated ani- lymphocyte count indicates that THI functions efficiently mals (Figure 1B). This indicates that despite increased when delivered systemically via IP injection. In addition, expression of S1P phosphatase 1 and lyase following in- for short-term treatments, IP administration is desirable jury, the counteracting increased expression of both S1P to ensure that all mice received the same dose. Thus for kinases results in elevated levels of intramuscular S1P. In the majority of experiments described herein, we opted to addition, we also observed increased S1P levels in the un- administer THI via IP administration. injured TA muscles from mice treated with THI compared Loh et al. also demonstrated that following acute in- to vehicles. To examine if such extravascular increases of jury, the expression of S1P lyase increases in wt muscle S1P correlated with a beneficial effect in dystrophic mice, [8]. Thus we analyzed the expression of enzymes that we analyzed the level of plasma CK, which are elevated in regulate S1P production and degradation following CTX humans and mice with muscular dystrophy activity in the 4cv injury in the mdx background with and without THI same group of THI-treated mdx mice [31]. Results indi- treatment. Right TA and quadriceps muscles were unin- cate a trending, but not statistically significant decline in jured, while left counterparts were injured using CTX, a CK activity levels in plasma collected on day 4 post injury well characterized model of acute injury where initial from THI versus vehicle-treated mice (Additional file 1: muscle destruction is followed by a rapid myogenic re- Figure S3B). 4cv sponse [27-30]. mdx mice (n = 6, 3.5-MO males) were injected IP immediately following CTX and thereafter Reduction of dystrophic muscle pathology in acutely five additional times during a 3-day period (for example injured mdx muscles via administration of THI IP 2 × IP injections per day) with either the previously used Although young mdx mice exhibit robust muscle repair, dose of THI or vehicle. For this analysis, muscles were regeneration becomes impaired with aging, resulting in harvested at day 4 post injury; the peak of myogenic muscle atrophy and dystrophy [3]. Therefore, in a third gene expression following CTX-induced damage [28]. In experiment, the effects of THI on histopathology were the absence of THI, expression of the S1P lyase was sig- assessed in injured and uninjured muscles from two 4cv nificantly elevated following injury (Additional file 1: groups of aged mdx mice (n = 6, 11-MO females; n = 7, Figure S2A). Surprisingly, expression of S1P phosphatase 16-MO males), to determine the effects of increasing 1 and lyase were greater in the injured muscles with THI levels of S1P in dystrophic animals at a stage of severe treatment, suggesting a possible compensation in the muscle wasting. Importantly, it has been reported that S1P degradation pathways in response to the inhibition mdx females older than 6 months of age exhibit greater fi- of the S1P lyase. Analogous to these results, expression brosis than males [32]. Once more, right TA and quadri- levels of S1P kinase 1 were also increased with injury and ceps muscles were uninjured, while left counterparts were at higher levels with THI (Additional file 1: Figure S2B). injured with CTX (Figure 2A). Regeneration following In contrast, the expression of S1P kinase 2 was only CTX injury is well orchestrated in normal muscle but Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 14 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 impaired in older mdx mice [29]. Therefore in these reduced fat deposition resulting from injury in 11-MO studies we analyzed the muscles from 11- and 16-MO female TAs and 16-MO male quadriceps. These results mdx mice 18 days following CTX injury, a time point demonstrate that THI treatment reduces injury-induced expected for non-diseased muscles to fully regenerate fat deposition and fibrosis in mdx muscles. 4cv [28]. In the 16-MO mice, muscles were weighed imme- Further analysis of THI-treated mdx mice revealed an diately after collection and normalized to body weight increase in muscle fiber size in quadriceps (Figure 3A). (grams muscle weight over grams mouse weight). As Although mdx mice undergo muscle hypertrophy as com- expected, injured muscles were lighter than uninjured pared to wild type, we observed a significant increase in muscles in vehicle mice, an approximate weight loss the minimum fiber diameter with THI treatment in dia- greater than 20% (Additional file 1: Figure S4A). However, phragms, and in both uninjured and injured quadriceps of in the THI-treated mice the weight of injured quadriceps 11-MO mice (Figure 3B,C and Additional file 1: Figure S7) was similar to uninjured quadriceps (muscle weight [37]. Uninjured quadriceps of THI-treated 16-MO males ratio injured/uninjured approximates one), suggesting also showed a significant increase in fiber size (Figure 3D). that THI treatment promotes muscle repair and pro- In summary, 3 days of THI treatment is sufficient to in- tects from muscle loss following acute injury. crease muscle fiber size in older mdx mice. Fibrosis and fat deposition are both hallmarks of muscle To assess if increases in muscle fiber size observed with wasting and dystrophic muscle pathology [32,33]. In THI treatment are accompanied by an increase in the addition, when regeneration is impaired, fibrosis and fat number of satellite cells, we quantified the number of accumulate in place of muscle following acute injury Pax7+ cells. Within skeletal muscle, Pax7 is specifically [34,35]. Histological quantification revealed that THI expressed by satellite cells, which have been reported to 4cv treatment reduced accumulation of both fibrosis and fat decline in older mdx muscles [38-40]. As expected, few deposition following acute injury in quadriceps and TA satellite cells (Pax7+ nuclei) were visible in cross-sections muscles (Figure 2B,C). Results for lower fibrosis were con- of 11-MO mdx muscles. However, there was a significant firmed by third party hydroxyproline analysis of injured increase in the mean number of Pax7+ nuclei, collectively TAs from 16-MO animals (Additional file 1: Figure S4B). in limb muscles (TAs and quadriceps) from THI-treated Interestingly, fibrosis was also significantly lower in unin- 11-MO animals (Additional file 1: Figure S8). jured TAs of 11-MO females, which correlates with the S1P is a potent angiogenic factor [41-43]. Thus we capacity of THI to elevate S1P levels in uninjured TAs studied the effects of THI treatment on the skeletal (Figure 1B, Additional file 1: Figure S5). Although only left muscle microvasculature. We quantified the number of TAs and quadriceps were injected with CTX, fibrosis ac- vessels using BS1, a lectin that highlights endothelial cumulation in uninjured muscles was likely elevated as cells [44]. In contrast to the increase in Pax7+ cells, we mice disuse injured limbs and bear most of the use/weight did not observe an increase in BS1+ vessels in injured on the uninjured contralateral limb. Therefore, the differ- 11-MO TA muscles. Quantitative RT-PCR analysis of ences observed in uninjured TAs are likely due to reduc- endothelial related genes eNOS and CD31 in 5-MO 4cv tions in the amount of fibrotic deposition that would mdx TA muscles at day 4 post injury, show no signifi- otherwise accumulate without THI treatment, since it is cant difference in the levels of expression of these endo- unlikely THI can reverse already accrued fibrosis. Along thelial associated genes in THI treatment compared to with lower fibrosis observed in injured muscles, the overall vehicle (Additional file 1: Figure S9). This suggests that morphology appeared more organized with THI treatment THI benefits on muscle repair do not depend on in- compared to vehicle-treated animals (Figure 2C). In creasing microvasculature density. addition, the number of EBD-positive fibers, an indicator of muscle fiber damage, was lower in injured 11-MO mus- THI treatment elevates isometric force in acutely injured cles and significantly reduced in uninjured 11-MO quadri- mdx EDL muscles ceps (Additional file 1: Table S1) [12,36]. In these muscles To assess if increasing S1P levels promotes dystrophic the number of centrally nucleated fibers was comparable muscle function, in a fourth experiment we conducted between THI and vehicle-treated animals (Additional myography analysis following longer treatment with file 1: Figure S6). THI. For this experiment, another group of mdx mice mdx/J To test whether THI-treated mice show decreased fat (male 4.75- to 5-MO C57BL/10ScSn-Dmd ) was in- deposition in injured muscles, we quantified the fat de- jured and treated with daily IP injections using the same posits within entire cross-sections of THI and vehicle- THI dose and injection interval, for 14 consecutive treated muscles (Figure 2D). The ratio of fat deposits days; the maximum duration for IP administration between injured and uninjured contralateral muscles allowed by our approved animal protocol. Animals were was then compared to THI and vehicle-treated mice treated with THI (n = 10) or vehicle (n = 9) for 14 days (Figure 2E). This analysis indicates that THI significantly following injury, and analyzed between day 15 and 19 Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 15 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 (Figure 4A). EDL muscles from injured and uninjured Direct administration of S1P promotes muscle contralateral limbs were analyzed for isometric specific regeneration in mdx mice following CTX injury force; a physiological measurement of muscle force that S1P is essential for satellite cell turnover, myoblast dif- is reduced with muscular dystrophy in mice and ferentiation and muscle regeneration in non-diseased humans [18,45,46]. mice, and more recently shown to promote satellite cell To assess if the EDL is damaged as a consequence of activation in mdx muscle [4,5,8,47]. To determine if the CTX injection in the TA, we injured and analyzed a sep- increase in satellite cell number observed in the THI- arate group of mdx mice (n = 4) 12 hours post injury. treated muscles was a result of increased S1P muscle For this fifth experiment, CTX injections included India content, we examined the effects of direct S1P adminis- ink to label needle penetration [47]. To assess muscle tration following CTX-induced acute injury in dys- fiber damage, a consequence of CTX injury, animals trophic muscles. In order to identify satellite cells and 4cv nlacz/+ were injected IP with EBD immediately following CTX their progeny, we utilized mdx :Myf5 mice carry- injection. The presence of EBD indicates EDL muscles ing the nuclear lacZ reporter driven by the endogenous are damaged. However, EDL damage is not due to direct Myf5 gene, a marker of myogenic cells [49-51]. CTX was 4cv penetration by the needle since India ink was only applied to both TA muscles (n = 3, 3-MO mdx : nlacz/+ present in the CTX-injected TA muscles (Additional file 1: Myf5 males), then S1P was immediately injected Figure S10). intramuscularly into left TAs and a vehicle control into Force frequency analysis revealed a significantly higher right TAs. Injections were repeated daily for the first specific force by EDL muscles isolated from injured 72 hours following injury and TAs were harvested on limbs of THI-treated mice (Figure 4B). These values day 4 post injury, directly following the peak of injury- were similar to EDL muscles isolated from contralateral induced myogenic cell proliferation for analysis of uninjured limbs, indicating that THI prevented wasting Myf5+ nuclei (Figure 5A) [28]. S1P-treated muscles and preserved muscle function following acute injury showed a dramatic, fourfold increase in the number of (Figure 4B). However, the specific force observed after Myf5+ nuclei in areas with severe CTX damage com- THI treatment was still lower than wt control animals pared to vehicle controls (Figure 5B top row and 5C left (Figure 4C). Two weeks of THI treatment was not suf- graph). Furthermore, a significant increase in the number ficient to improve specific force in uninjured EDL mus- of Myf5+ nuclei was observed over the entire CSA of S1P- cles.However,as shown in Figure 1B,the THIdoseof treated TAs (Figure 5C middle graph, Additional file 1: 0.75 μg/day used for all our experiments does not sig- Figure S12). These data demonstrate that S1P treatment nificantly raise S1P levels in all uninjured mdx muscles. increases the number of myogenic cells in mdx muscles In addition, although peripheral lymphocytes declined following injury and suggests that S1P promotes satellite with THI (Figure 1A), we did not observe a decline of cell proliferation in vivo. CD3e+ T-cells present in the diaphragm following 2 We then determined whether the increase in myo- weeks of THI (Additional file 1: Figure S11) [48]. genic cells promotes dystrophic muscle repair by stain- Therefore, it is plausible that a higher dose of THI is ing for eMyHC, a marker of regenerating muscle fibers required to sufficiently elevate S1P levels needed to [27]. In concurrence with the rise of Myf5+myogenic improve specific force in uninjured mdx muscles. cells, a 3.6 fold increase in the number of eMyHC+ fibers However, since THI is insoluble in PBS at higher con- was observed in S1P-treated TAs (Figure 5B bottom centrations and has low oral bioavailability, we chose to row, 5C right graph). This increase in eMyHC+ fibers, directly study the effects of high levels of S1P on unin- corresponded with elevated numbers of centrally nucle- jured mdx muscles ex vivo. For this experiment, EDLs ated muscle fibers in the injured regions of S1P-treated from uninjured and untreated mdx mice were analyzed muscles (Additional file 1: Figure S13A). Furthermore, following incubation with 10 μM S1P [16]. Analysis of the size of regenerating myofibers in S1P-treated TAs the maximal specific force indicates that direct admin- was significantly greater, as indicated by the minimum istration of S1P significantly increases force output in diameter quantified for the largest eMyHC+ fibers uninjured mdx muscle (Figure 4D). Such results indi- (Additional file 1: Figure S13B). Collectively, these data cate that treatment with high concentrations of S1P show that local administration of S1P promotes dys- can promote functional improvement of dystrophic trophic muscle repair by improving satellite cell re- muscles. sponse and contribution to muscle fiber regeneration. Overall, reduction in fibrosis and fat deposition, and increase in myofiber size and satellite cell numbers, indi- S1P directly acts on mdx muscle fibers, and elevates cate that elevating S1P levels, pharmacologically or by levels of total and phosphorylated S1PR1 direct administration, has a profound benefit in dys- In mammals there are five S1P receptors that share trophic muscle repair and function. homology to G-protein coupled receptors [52]. It has Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 16 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 been recently reported that S1P receptor 2 (S1PR2) is spe- localized perinuclearly andlessso around the perim- cifically activated in myogenic cells and that downstream eter of eMyHC+ fibers (Figure 6E). These results indi- effectors of S1P action in satellite cells include compo- cate that S1PR1 signaling is active in regenerating nents of the JAK-STAT signaling pathway [8]. In contrast, muscle fibers and suggests that the beneficial actions our results and others, of exogenous S1P treatment that S1P exerts on mdx muscle fibers maybemediated resulting in increased EDL force, suggests that S1P also through S1PR1. acts directly on muscle fibers [16]. The amount of exogen- ous S1P added in the bath was super-physiological and S1P administration correlates with increased levels of thus we measured S1P muscle levels following intramus- S1PR1 and P-rpS6, an indicator of protein synthesis cular injection of S1P. In this experiment, left TAs from S1PR1 has been implicated in myoblast proliferation 4cv mdx mice (n = 3, 11-MO) were injected with the same and shown to steadily increase during the course of re- 4cv nlacz/+ dose of S1P as the mdx :Myf5 mice depicted in generation in non-diseased muscle [4,5]. Therefore to Figure 5A, while contralateral TAs received the same ve- gain more insight on the potential action that S1P ex- hicle. In contrast to the previous experiment depicted in erts via S1PR1 in dystrophic muscle, we injected S1P in 4cv Figure 5A, TA muscles were injected in the absence of in- uninjured TAs of mdx (n =3,2.5-MO),and quanti- jury and were harvested for S1P analysis 15 minutes post fied the level of S1PR1 and some downstream effectors injection (Figure 6A); the same time used for S1P incuba- (Figure 7A) [56]. In turn, S1P treatment resulted in 4cv tion prior to EDL force measurement shown in Figure 4D. significantly elevated levels of S1PR1 in mdx TAs Results indicate that within this timeframe, intramuscular (Figure 7B). In a separate experiment, we injected S1P 4cv injection of S1P does significantly increase S1P levels in in left TAs and vehicle in right TAs of mdx (n = 3, mdx muscle (Figure 6A). 10-MO), following the same dose and experimental de- To directly observe where S1P binds in the muscle, a sign (three injections, one per day, harvest on day 4), 4cv separate group of mdx (n = 2, 11-MO) were injected and analyzed TA muscles for phosphorylated S1PR1. with the same amount of biotinylated-S1P in left and ve- Results from this experiment show that phosphorylated hicle in right TAs. Once more, TAs were harvested 15 S1PR1 is also significantly elevated with S1P treatment minutes post injection for histological visualization of (Additional file 1: Figure S15). S1P. Staining with streptavidin conjugated to Alexa A result of S1P injection was larger eMyHC+ fibers Fluor 594 reveals that biotinylated-S1P is present in that were positive for phosphorylated S1PR1 (Figure 6E, many cells, but particularly localized to the perimeter of Additional file 1: Figure S13B). Therefore, we examined muscle fibers (Figure 5B). Among the three S1P recep- if elevated S1PR1 levels corresponded with known regu- tors (S1PR1, S1PR2, S1PR3) expressed in muscle, S1PR3 lators of cell size and protein synthesis; Akt, mTOR, S6 and S1PR1 are the most abundant in wt muscle [5]. Im- kinase and rpS6. S1P-induced hypertrophy has been portantly, expression of these three S1P receptors is re- described in cultured cardiomyocytes, which was ac- duced in mdx muscle cells, especially S1PR1, which companied by activation of Akt and S6 kinase [57]. In shows more than five fold reduction in relative mRNA addition, S1PR1 activation of S6 kinase via a Gi- 4cv levels (Additional file 1: Figure S14). Staining of mdx dependent pathway has been reported in vascular muscles (3.5-MO) for S1PR1 and S1PR3, reveals that smooth muscle cells [56]. Akt and mTOR signaling via S1PR1 is present at the perimeter of muscle fibers and S6 kinase, an activator of rpS6 implicated in protein myonuclei, whereas S1PR3 appears localized to the synthesis, has been described as sufficient to induce vasculature (Figure 6C). S1PR1 is a G protein-coupled skeletal muscle hypertrophy [58-60]. Therefore, we receptor (GPCR) that can be activated via phosphoryl- evaluated if direct injection of S1P induces activation of 4cv ation, resulting in translocation to the endosomal com- these pathways in uninjured TA muscles of mdx mice partment and/or the perinuclear compartment [53-55]. (n = 4, 2.5-MO). Western blot analysis of TA muscles Therefore, perinuclear localization of S1PR1 suggested injected for 3 days with S1P (Figure 7A) revealed that that in response to S1P treatment, receptor 1 signaling is the levels of phosphorylated Akt (P-Akt) and mTOR 4cv activated in mdx muscle fibers. To evaluate the pres- (P-mTOR), though increased, were not significantly ence of active S1PR1 signaling during muscle fiber re- higher in S1P-treated muscles (Figure 7C). However, the generation, we surveyed the same CTX-injured muscles levels of rpS6 and phosphorylated rpS6 (P-rpS6) were depicted in Figure 5A for the presence of phosphory- significantly increased with S1P treatment compared to lated S1PR1. Results indicate S1PR1 is localized around control muscles, suggesting an increase in protein syn- the perimeter of muscle fibers and intracellularly near or thesis. Although a more detailed study is required to within the myonuclei (perinuclear) of newly regenerated elucidate the role of S1P in skeletal muscle protein syn- eMyHC+ fibers (Figure 6D). In parallel, we observed thesis, our data suggest that S1P can activate muscle more concentrated staining for phosphorylated S1PR1 anabolic pathways in the mdx mouse. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 17 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 Direct administration of S1P promotes muscle regeneration Figure S16). These results indicate that approaches in dysferlinopathy mice following acute injury aimed at elevating muscle S1P may be beneficial to The role of dysferlin is currently unknown, but its ab- promote muscle regeneration in additional muscle sence in humans and mice results in chronic muscle wasting diseases. wasting that primarily affects limb and girdle muscles [61-63]. Although dysferlinopathy is less severe than Longer-term treatment with THI shows a functional DMD [64], dysferlinopathy patients are often wheelchair benefit in uninjured mdx muscle bound between 30 and 40 years of age [65]. Much like To this point we have largely examined the role of S1P DMD, muscles in humans and mice lacking functional in promoting muscle regeneration in acutely injured dys- dysferlin exhibit chronic atrophy, resulting in the accu- trophic muscles. Since long-term intramuscular injec- mulation of fibrosis and fat [66]. Therefore we tested the tions of S1P are neither feasible nor practical (the effects of S1P administration after CTX injury in a injections also cause damage), we decided to revisit the model of dysferlinopathy (AJ/SCID) to evaluate if the use of THI for elevating S1P muscle content. Although benefits of S1P are exclusive to the mdx background or our initial experiments with THI showed little benefit in can be applied to other muscle wasting diseases [67]. uninjured mdx muscles, they were short-term and in We followed the same experimental design outlined in older animals with severe pathology (Figures 2, 3), or Figure 5A, injecting left TAs of AJ/SCID mice (n = 4, adult animals (Figure 4) at a point when hypertrophy 9-MO) with the same dose of S1P and vehicle in right and robust regeneration compensate for degeneration in TAs for 3 days following CTX injury. In contrast to the limb muscles [24,68,69]. Therefore, we examined longer- 4cv experiments in mdx , we harvested TAs on day 6 post term treatment of THI in younger mdx mice at 4 weeks injury in order to also evaluate the onset of fibrosis. In of age, a time point characterized by significant muscle accordance to the results observed in mdx, we observed degeneration prior to the compensatory period [70]. For 4cv improved muscle regeneration with the administration this experiment, uninjured mdx animals were treated of S1P in AJ muscles. Specifically, we observed lower for 1 month, beginning at 4 weeks of age, with THI or fibrosis and increased centrally nucleated fibers, as well vehicle in the drinking water (Figure 8A) [11]. At 8 weeks as improved muscle architecture in the damaged regions of age, we assessed the functional benefit of THI treat- of muscle with S1P administration (Additional file 1: ment by analyzing EDL specific force via myography. In Figure 8 Longer-term treatment with THI elevated muscle force in uninjured mdx EDL muscles. (A) Experimental schematic outlining the 4cv treatment regimen. Beginning at 4 weeks of age, mdx mice (1-MO males) were treated for 4 weeks ad libitum with 50 mg/l THI (n = 4) or vehicle (n = 3) in drinking water. (B) Myography analysis of EDL muscles reveals a significant increase in maximal specific force with THI treatment. *P <0.05 by student’s t-test. Error bars represent SEM. (C) Summary of findings: S1P can act to not only promote myogenic cell activation and muscle repair, but also enhance muscle fiber size and force, possibly through S1PR1 mediated signaling. EDL, extensor digitorum longus; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; SEM, standard error of the mean; THI, 2-acetyl-4(5)-tetrahydroxybutyl imidazole. Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 18 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 turn, EDLs from THI-treated animals showed significantly reductions in HDAC activity result in an increase of greater specific force compared to vehicle-treated controls follistatin, an inhibitor of myostatin, which may explain (Figure 8B). This data demonstrates that elevating S1P the amelioration of DMD pathology [74]. Our data sup- levels is beneficial for the chronic muscle injury that port this possibility and suggest that the molecular occurs early in muscular dystrophy. mechanism for the suppression of muscle degeneration involves the anabolic pathways for muscle formation Discussion rpS6. These components have been shown to lie down- We have shown that systemic administration of the stream of myostatin and insulin-like growth factor [75]. pharmacological agent THI by IP injection to dystrophic mdx mice led to elevated levels of S1P in recovering in- Conclusion jured muscle tissue, as well as a reduction of fibrosis and Based on the work reported here, elevation of S1P may fat infiltration, both pathological indicators of muscle be a fruitful strategy for ameliorating the pathology wasting (Figure 2). Additionally, systemic THI led to a manifested in patients afflicted with DMD and possibly significant increase in muscle fiber size and specific force other muscle wasting diseases (for example dysferli- of CTX-injured muscles (Figures 3 and 4). In turn, nopathy). Therapies based on promoting S1P levels in ex vivo administration of high levels of S1P resulted in dystrophic muscle have the potential to improve path- specific force levels in uninjured mdx EDL muscles ology by promoting satellite cell and anabolic-mediated (Figure 4D). To pursue a better understanding of how regeneration. An obvious candidate for a small molecule elevated S1P reduces DMD pathology, we found that therapeutic is THI. Our work has shown that short- direct administration of S1P via intramuscular injection term treatment of THI has significant efficacy in doubles muscle S1P content compared to the S1P levels increasing regenerative capacity in the mdx mouse fol- reached with IP injections of THI. In addition, intramus- lowing acute muscle injury, while longer treatment can cular S1P injections led to an increase in myogenic cells improve muscle function in younger uninjured mdx (Myf5+) and induced phosphorylation of S1PR1, which muscle. Moreover, significant increases in muscle fiber was particularly abundant in newly regenerating fibers size have been suggested as a viable approach in (Figure 7, Additional file 1: Figure S15), as well as a sig- overcoming dystrophic muscle damage by promoting nificant increase in rpS6 and P-rpS6 levels (Figure 6). strength and function [76]. Additionally, there are other These results suggest that S1P not only works to activate THI derivatives with increased oral bioavailability that myogenic precursors but also elevates protein synthesis may be more effective at increasing and maintaining in muscle fibers, potentially through S1PR1 mediated high intramuscular S1P levels in long-term treatments, signaling (summarized in Figure 8C). In summary, THI/ which was necessary for functional improvement of un- S1P administration led to improved regeneration and injured EDL muscles [10]. Alternatively there are inhi- pathology, higher muscle specific force, an increase in bitors of lipid phosphate phosphatases and/or S1P the number of myogenic cells, and larger muscle fibers. phosphatases that may also increase intramuscular S1P Our results indicate that S1P mediates satellite cell- levels [10,77]. In addition, there are specific S1P recep- dependent and muscle fiber-dependent effects on skel- tor agonists (for example FTY720) that are currently etal muscle. If amelioration of muscle wasting occurs FDA approved or in clinical trials [78,79]. Based on our through receptor-mediated signaling then S1P, elevated present results and those of others, future studies fo- intracellularly via THI, must be exported to activate the cused on S1P-based therapeutics for the treatment of S1P receptors. THI has been reported to inhibit the S1P DMD and related myopathies are warranted. lyase, an enzyme whose active site is on the cytoplasmic side of the endoplasmic reticulum. Therefore elevations Additional file of S1P levels mediated via THI inhibition of the S1P lyase presumably occur within the cytoplasm [71]. S1P Additional file 1: Figure S1. Treatment with THI lowers mdx plasma may also act intracellularly before possible export to platelet levels. Figure S2. THI alters the expression of S1P regulatory promote muscle wasting suppression. This alternative is gene in mdx muscle. Figure S3. THI does not alter S1P plasma levels but lowers plasma CK activity. Figure S4. Muscle weight is preserved and supported by our work with Drosophila, which have no hydroxy proline is reduced in injured muscles from THI treated mdx mice. known S1P receptors [9], as well as by a recent report Figure S5. Fibrosis is lower in uninjured mdx TA muscles with THI that showed S1P interacts directly, intracellularly, with treatment. Figure S6. The number of centrally nucleated muscle fibers does not change with THI. Figure S7. Diaphragm muscle fibers size histone deacetylases (HDACs) [72]. As HDAC inhibitors increases with THI treatment. Figure S8. THI treated mdx mice have an have been previously shown to suppress dystrophic phe- elevated number of Pax7+ satellite cells. Figure S9. The microvasculature notypes in mdx mice, the actions of S1P on the sup- of mdx muscles did not increase with THI. Figure S10. CTX injected in the TA reaches and also damages the EDL. Figure S11. THI treatment pression of muscle wasting may occur in part through did not reduce T-cells in mdx diaphragms. Figure S12. Montages such mechanisms [73]. It has also been reported that Ieronimakis et al. Skeletal Muscle 2013, 3:20 Page 19 of 21 http://www.skeletalmusclejournal.com/content/3/1/20 and Regenerative Medicine, University of Washington, Seattle, WA 98195, covering entire cross-sectional areas of each TA from S1P and vehicle USA. Department of Laboratory Medicine, School of Medicine, University of treated mdx4CV:Myf5nlacZ/+ animals, were created by combining Washington, Seattle, WA 98195, USA. Department of Chemistry, University of individual 10x photos. Figure S13. (A) Quantification of centrally Washington, Seattle, WA 98195, USA. Department of Neurology, Senator nucleated muscle fibers from the same injured TAs presented in Figure 5, Paul D Wellstone Muscular Dystrophy Cooperative Research Center, coincides with the number of newly regenerated fibers (eMyHC+ fibers) University of Washington, Seattle, WA 98195, USA. observed in S1P injected TA muscles. (B) Quantification of the minimum diameter of the largest eMyHC+ myofibers represented in Figure 5, Received: 27 November 2012 Accepted: 22 May 2013 indicates a significant increase in regenerated fiber size with S1P Published: 1 August 2013 treatment. Figure S14. The expression of S1P receptors is reduced in mdx muscle cells. Figure S15. Direct S1P administration results in elevated levels of phosphorylation S1PR1 in mdx muscles. Figure S16. References S1P promotes muscle regeneration in the A/J mouse model of 1. Deconinck N, Dan B: Pathophysiology of duchenne muscular dystrophy: dysferlinopathy. Table S1. Average number of Evans Blue+ muscle fibers current hypotheses. Pediatr Neurol 2007, 36:1–7. within each muscle group. 2. Mendell JR, Rodino-Klapac LR, Malik V: Molecular therapeutic strategies targeting Duchenne muscular dystrophy. J Child Neurol 2010, Abbreviations 25:1145–1148. ANOVA: Analysis of variance; BSA: Bovine serum albumin; CK: Creatine kinase; 3. 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PLoS One 2008, We thank Zack Usa, Alex Nelson and Jessica Becker for technical help. We 5:e10920. thank Dr Guenter Daum for advice and guidance on S1P based approaches. 14. Grabski AD, Shimizu T, Deou J, Mahoney WM Jr, Reidy MA, Daum G: We also thank Dr Guenter Daum and Dr LeBoeuf Renee for sharing Akt- and nlacZ/+ Sphingosine-1-phosphate receptor-2 regulates expression of smooth S1P-related antibodies. We thank Dr Zipora Yablonka-Reuveni for the Myf5 muscle alpha-actin after arterial injury. Arterioscler Thromb Vasc Biol 2009, reporter mice. Finally, we would like to thank Dr Nick Whitehead for his technical 29:1644–1650. guidance on myography. This work was supported by the University of 15. Au CG, Butler TL, Sherwood MC, Egan JR, North KN, Winlaw DS: Increased Washington Center for Commercialization (C4C), Departments of Pathology and connective tissue growth factor associated with cardiac fibrosis in the Laboratory Medicine, University of Washington; Provost Bridge grant to MR; mdx mouse model of dystrophic cardiomyopathy. Int J Exp Pathol 2011, University of Washington Nathan Shock Center of Excellence in the Basic Biology 92:57–65. of Aging and the Genetic Approaches to Aging Training Grant T32 AG00057 to 16. Danieli-Betto D, Germinario E, Esposito A, Megighian A, Midrio M, Ravara B, NI; and the American Recovery and Reinvestment Act of 2009 (ARRA) Challenge Damiani E, Libera LD, Sabbadini RA, Betto R: Sphingosine 1-phosphate Grant 5RC1AR058520, R01GM083867, R01GM097372 and 1P01GM081619 to HRB. protects mouse extensor digitorum longus skeletal muscle during Support was also received from the Washington Research Foundation, the fatigue. 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J Pharmacol Exp Ther 2011, • Thorough peer review 338:879–889. • No space constraints or color figure charges doi:10.1186/2044-5040-3-20 • Immediate publication on acceptance Cite this article as: Ieronimakis et al.: Increased sphingosine-1-phosphate • Inclusion in PubMed, CAS, Scopus and Google Scholar improves muscle regeneration in acutely injured mdx mice. Skeletal Muscle 2013 3:20. • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Aug 1, 2013

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