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

Ieronimakis N, Pantoja M, Hays AL, Dosey TL, Qi J, Fischer KA, Hoofnagle AN, Sadilek M, Chamberlain JS, Ruohola-Baker H, Reyes M - Skelet Muscle (2013)

Bottom Line: Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1).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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, School of Medicine, University of Washington, Seattle, WA 98195, USA. hannele@u.washington.edu.

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 injection of S1P increased the number of Myf5nlacz/+ 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.

No MeSH data available.


Related in: MedlinePlus

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 mdx4cv:Myf5nlacZ/+ 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.
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Figure 5: 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 mdx4cv:Myf5nlacZ/+ 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.

Mentions: Right and left TAs of three 3-MO male mdx4cv:Myf5nlacZ/+ were injured once more with 10 nM CTX (Figure 5). S1P (Enzo Life Sciences, Farmingdale, NY, USA; Calbiochem) preparation was undertaken according to manufacturer’s instructions. Briefly, S1P was dissolved in methanol (0.5 mg/ml) and aliquoted, then the solvent was evaporated with a stream of nitrogen to deposit a thin film on the inside of the tube. Prior to use, aliquots were resuspended in PBS with 4 mg/ml BSA (fatty acid free) to a concentration of 500 μM. Directly following CTX injection, 20 μl 500 μM S1P was injected in left TAs, daily until day 3 post injury, at which time animals were euthanized and muscles were harvested for freezing. Right TAs were injected with an equal volume of PBS with 4 mg/ml BSA as vehicle controls. In a separate experiment (Figure 6), TAs of four 2.5-MO female mdx4cv were injected with S1P or vehicle under the same conditions stated above, in the absence of injury. AJ/SCID mice (n = 4, 9-MO, B6. Cg-DysfprmdPrkdcscid/J) were also injected for 3 days with S1P or vehicle in TAs post CTX injury, following the same concentration and injection regimen used in mdx4cv. For measurement of S1P muscle content (Figure 7A) following intramuscular injections, 11-MO mdx4cv (n = 3) were injected 20 μl 500 μM S1P in left TAs and 20 μl vehicle in right TAs. Muscles were harvested and frozen in liquid nitrogen 15 minutes post injection, and then processed using the aforementioned methods for analyzing S1P in muscle by LC-MS/MS. For injection of biotinylated-S1P, TAs from 11-MO mdx4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA). TAs were harvested and frozen in OCT compound 15 minutes following injection.


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

Ieronimakis N, Pantoja M, Hays AL, Dosey TL, Qi J, Fischer KA, Hoofnagle AN, Sadilek M, Chamberlain JS, Ruohola-Baker H, Reyes M - Skelet Muscle (2013)

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 mdx4cv:Myf5nlacZ/+ 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750760&req=5

Figure 5: 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 mdx4cv:Myf5nlacZ/+ 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.
Mentions: Right and left TAs of three 3-MO male mdx4cv:Myf5nlacZ/+ were injured once more with 10 nM CTX (Figure 5). S1P (Enzo Life Sciences, Farmingdale, NY, USA; Calbiochem) preparation was undertaken according to manufacturer’s instructions. Briefly, S1P was dissolved in methanol (0.5 mg/ml) and aliquoted, then the solvent was evaporated with a stream of nitrogen to deposit a thin film on the inside of the tube. Prior to use, aliquots were resuspended in PBS with 4 mg/ml BSA (fatty acid free) to a concentration of 500 μM. Directly following CTX injection, 20 μl 500 μM S1P was injected in left TAs, daily until day 3 post injury, at which time animals were euthanized and muscles were harvested for freezing. Right TAs were injected with an equal volume of PBS with 4 mg/ml BSA as vehicle controls. In a separate experiment (Figure 6), TAs of four 2.5-MO female mdx4cv were injected with S1P or vehicle under the same conditions stated above, in the absence of injury. AJ/SCID mice (n = 4, 9-MO, B6. Cg-DysfprmdPrkdcscid/J) were also injected for 3 days with S1P or vehicle in TAs post CTX injury, following the same concentration and injection regimen used in mdx4cv. For measurement of S1P muscle content (Figure 7A) following intramuscular injections, 11-MO mdx4cv (n = 3) were injected 20 μl 500 μM S1P in left TAs and 20 μl vehicle in right TAs. Muscles were harvested and frozen in liquid nitrogen 15 minutes post injection, and then processed using the aforementioned methods for analyzing S1P in muscle by LC-MS/MS. For injection of biotinylated-S1P, TAs from 11-MO mdx4cv (n = 2) were injected intramuscularly with 20 μl 500 μM S1P-biotin or vehicle (Echelon Biosciences, Salt Lake City, UT, USA). TAs were harvested and frozen in OCT compound 15 minutes following injection.

Bottom Line: Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1).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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, School of Medicine, University of Washington, Seattle, WA 98195, USA. hannele@u.washington.edu.

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 injection of S1P increased the number of Myf5nlacz/+ 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.

No MeSH data available.


Related in: MedlinePlus