Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: 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 mdx 4cv mice. Scale bars = 50 μm. ( B , C , D ) Quantification of minimum muscle fiber diameter reveals a significant increase in myofiber size in THI-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 4cv 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Staining

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: S1P promotes functional improvement of mdx ( C57BL/10ScSn-Dmd mdx/J ) 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 ( C57BL/10ScSn-Dmd mdx/J ) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. ( D ) Incubation of uninjured and untreated mdx ( C57BL/10ScSn-Dmd mdx/J ) 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Functional Assay, Injection, In Vitro, Isolation, Incubation, Concentration Assay

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Direct administration of S1P promotes muscle regeneration following acute injury. ( A ) Experimental schematic of S1P and PBS (vehicle) injected daily for the first 72 hours into TAs of 3-MO mdx 4cv :Myf5 nlacZ/+ 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Injection, Staining

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Administration of S1P leads to increased levels of S1PR1 and P-rpS6 in vivo . ( A ) Experimental schematic of S1P and PBS (vehicle) injected daily for the first 72 hours into TAs of uninjured mdx 4cv mice (n = 4, 2.5-MO, left TAs injected S1P, right TAs injected PBS). ( B ) Western blot analysis of injected TAs (n = 3, 2.5-MO mdx 4cv ) indicates that administration of S1P significantly increases S1PR1 levels. ( C ) Western blot analysis of injected TAs (n = 4, 2.5-MO mdx 4cv ) 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: In Vivo, Injection, Western Blot

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: 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 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (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 TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv 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 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Injection, Activation Assay, Liquid Chromatography with Mass Spectroscopy, Staining, Liquid Chromatography, Mass Spectrometry

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: IP injection of THI reduces peripheral blood leukocytes and increases S1P levels in most tissues. ( A ) Leukocytes were analyzed from the peripheral blood of 1.5-MO mdx 4cv 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 ( P <0.05) for all leukocytes except monocytes. ( B ) mdx 4cv 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Injection, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry

Journal: Skeletal Muscle

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

doi: 10.1186/2044-5040-3-20

Figure Lengend Snippet: Longer-term treatment with THI elevated muscle force in uninjured mdx EDL muscles. ( A ) Experimental schematic outlining the treatment regimen. Beginning at 4 weeks of age, mdx 4cv 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.

Article Snippet: For injection of biotinylated-S1P, TAs from 11-MO mdx 4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA).

Techniques: Activation Assay