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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

S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmdmdx/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-Dmdmdx/J) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured and untreated mdx (C57BL/10ScSn-Dmdmdx/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.
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Figure 4: S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmdmdx/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-Dmdmdx/J) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured and untreated mdx (C57BL/10ScSn-Dmdmdx/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.

Mentions: Peripheral blood cells from 1.5-month-old (MO) wild type (wt) C57BL/k6 and mdx mice on a C57BL/k6 background (B6Ros.Cg-Dmdmdx-4Cv/J, herein referred to as mdx4cv) were analyzed (Figure 1A). Blood was collected before and 12 hours following the last of two 250 μl intraperitoneal (IP) injections of 0.15 mg/ml THI in PBS. Injections were 6 hours apart. This injection regimen and dose was repeated for all subsequent experiments involving THI, but for longer-treatment durations as outlined. Six 5-MO mdx4cv males were used for the experiments in Figure 1B, and Additional file 1: Figure S1 and S2. For Figures 2 and 3, and Additional file 1: Figures S3 to S7, six 11-MO females and seven 16-MO males mdx4cv were used for these experiments. In these mice, the left tibialis anterior (TA) and quadriceps femoris (quads) were injured with 10 nM CTX (Calbiochem, Darmstadt, Germany) from Naja nigricollis. Once more, THI-treated mice were injected IP with 250 μl 0.15 mg/ml THI in PBS, twice daily (injections 6 hours apart) immediately after injury and for the first 3 days following injury. The vehicle controls were injected IP with PBS. On day 4 post injury, 5-MO mdx4cv animals were euthanized for S1P and creatine kinase (CK) analysis. On day 17 post CTX, 11-MO and 16-MO mdx4cv mice were also injected IP with 1% Evans Blue dye (EBD) to label persistently damaged (dye permeable) muscle fibers [12], and euthanized on day 18 post injury for histopathology analysis. Muscles for S1P and expression analysis (from 5-MO mdx4cv) were frozen directly in liquid nitrogen, while muscles taken for histopathology were frozen under liquid nitrogen cooled isopentane in optimal cutting temperature (OCT) compound. All myofibers were measured for the minimum diameters on the cross-sections of mouse quadriceps muscle using ImageJ software (Bethesda, MD, USA). Between 750 and 850 myofibers were counted for three mice treated with PBS or THI, with or without CTX injury. For functional analysis outlined in Figure 4B, 4.75- to 5-MO male mdx on a C57BL/10 background (C57BL/10ScSn-Dmdmdx/J) were used for the 14-day treatment of THI or vehicle. Following the same dose and treatment regimen, mdx were treated with THI (n = 10) or vehicle (n = 9) for 14 days following CTX injury to left TAs and quadriceps. The same mdx strain was compared to wt C57BL/10 animals in Figure 4C and for exogenous S1P treatment depicted in Figure 4D. Animals used to evaluate the degree of CTX injury in EDL (Additional file 1: Figure S8) were 4-MO female mdx (n = 4, C57BL/10ScSn-Dmdmdx/J background), injected in left TAs with CTX and with approximately 3 μl India ink, added to the tip of the needle to mark injection penetration. Following CTX injections, mice were immediately injected IP with 1% EBD. Both left (injured) and contralateral uninjured TA and EDL muscles were harvested and frozen in OCT compound 12 hours post injury.


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)

S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmdmdx/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-Dmdmdx/J) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured and untreated mdx (C57BL/10ScSn-Dmdmdx/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.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: S1P promotes functional improvement of mdx (C57BL/10ScSn-Dmdmdx/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-Dmdmdx/J) indicate specific force improved in injured but not uninjured THI-treated EDL muscles. (D) Incubation of uninjured and untreated mdx (C57BL/10ScSn-Dmdmdx/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.
Mentions: Peripheral blood cells from 1.5-month-old (MO) wild type (wt) C57BL/k6 and mdx mice on a C57BL/k6 background (B6Ros.Cg-Dmdmdx-4Cv/J, herein referred to as mdx4cv) were analyzed (Figure 1A). Blood was collected before and 12 hours following the last of two 250 μl intraperitoneal (IP) injections of 0.15 mg/ml THI in PBS. Injections were 6 hours apart. This injection regimen and dose was repeated for all subsequent experiments involving THI, but for longer-treatment durations as outlined. Six 5-MO mdx4cv males were used for the experiments in Figure 1B, and Additional file 1: Figure S1 and S2. For Figures 2 and 3, and Additional file 1: Figures S3 to S7, six 11-MO females and seven 16-MO males mdx4cv were used for these experiments. In these mice, the left tibialis anterior (TA) and quadriceps femoris (quads) were injured with 10 nM CTX (Calbiochem, Darmstadt, Germany) from Naja nigricollis. Once more, THI-treated mice were injected IP with 250 μl 0.15 mg/ml THI in PBS, twice daily (injections 6 hours apart) immediately after injury and for the first 3 days following injury. The vehicle controls were injected IP with PBS. On day 4 post injury, 5-MO mdx4cv animals were euthanized for S1P and creatine kinase (CK) analysis. On day 17 post CTX, 11-MO and 16-MO mdx4cv mice were also injected IP with 1% Evans Blue dye (EBD) to label persistently damaged (dye permeable) muscle fibers [12], and euthanized on day 18 post injury for histopathology analysis. Muscles for S1P and expression analysis (from 5-MO mdx4cv) were frozen directly in liquid nitrogen, while muscles taken for histopathology were frozen under liquid nitrogen cooled isopentane in optimal cutting temperature (OCT) compound. All myofibers were measured for the minimum diameters on the cross-sections of mouse quadriceps muscle using ImageJ software (Bethesda, MD, USA). Between 750 and 850 myofibers were counted for three mice treated with PBS or THI, with or without CTX injury. For functional analysis outlined in Figure 4B, 4.75- to 5-MO male mdx on a C57BL/10 background (C57BL/10ScSn-Dmdmdx/J) were used for the 14-day treatment of THI or vehicle. Following the same dose and treatment regimen, mdx were treated with THI (n = 10) or vehicle (n = 9) for 14 days following CTX injury to left TAs and quadriceps. The same mdx strain was compared to wt C57BL/10 animals in Figure 4C and for exogenous S1P treatment depicted in Figure 4D. Animals used to evaluate the degree of CTX injury in EDL (Additional file 1: Figure S8) were 4-MO female mdx (n = 4, C57BL/10ScSn-Dmdmdx/J background), injected in left TAs with CTX and with approximately 3 μl India ink, added to the tip of the needle to mark injection penetration. Following CTX injections, mice were immediately injected IP with 1% EBD. Both left (injured) and contralateral uninjured TA and EDL muscles were harvested and frozen in OCT compound 12 hours post injury.

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