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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 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 of uninjured mdx4cv (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 mdx4cv 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 mdx4cv 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 present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx4cv 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.
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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 of uninjured mdx4cv (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 mdx4cv 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 mdx4cv 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 present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx4cv 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.

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 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 of uninjured mdx4cv (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 mdx4cv 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 mdx4cv 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 present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx4cv 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.
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Related In: Results  -  Collection

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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 of uninjured mdx4cv (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 mdx4cv 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 mdx4cv 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 present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. (E) Staining for phosphorylated S1PR1 in the same mdx4cv 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.
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