5-hydroxytryptamine 1B receptor-stimulating agent for use as a promoter of satellite cells self-renewal and/or differentiation

Abstract

The present invention relates to the field of muscle regeneration, and more particularly to the replenishment of the in vivo muscle stem cells pool. It more specifically relates to a 5-hydroxytryptamine B1 receptor-stimulating agent, and to a composition comprising said agent, for use as i) a promoter of satellite cells self-renewal and/or differentiation, and/or ii) an agent preventing and/or inhibiting the satellite cells pool exhaustion. The invention further encompasses therapeutic and screening methods.

Claims

1. A method, comprising: contacting satellite cells with an effective amount of a direct 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent selected from a triptan, a vortioxetine, and an ergotamine, to thereby promote satellite cell proliferation by increasing division rate.

2. The method of claim 1, wherein the satellite cells are contacted in vitro.

3. The method of claim 2, wherein the satellite cells are contacted by administering an effective amount of the direct 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent to an isolated biological sample comprising satellite cells.

4. The method of claim 1, wherein the satellite cells are contacted by administering the direct 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent to a subject affected by a natural or by a pathological loss and/or damage and/or impairment of skeletal muscle tissue to thereby provide a therapeutic benefit to the subject.

5. The method of claim 4, wherein the subject has sarcopenia.

6. The method of claim 5, wherein the subject has cancer-induced sarcopenia.

7. The method of claim 1, wherein the agent is vortioxetine.

8. The method of claim 1, wherein the agent is a triptan.

9. The method of claim 8, wherein the triptan is selected from sumatriptan, rizatriptan, zolmitriptan, eletriptan, almotriptan, frovatriptan, naratriptan, avitriptan, and donitriptan.

10. The method of claim 1, wherein the agent is modified to comprise at least one positively charged chemical moiety.

11. The method of claim 10, wherein the positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.

12. The method of claim 10, wherein the agent is a positively charged vortioxetine selected from the group consisting of salts of vortioxetine, vortioxetine coupled to a positively charged amino acid, pyrrolidinium-vortioxetine, pyperazinium-vortioxetine, dimethylammonium-vortioxetine, sulfonium-vortioxetine, N-oxide-vortioxetine, sulfoxide-vortioxetine, and phosphonium-vortioxetine.

13. The method of claim 10, wherein the agent is histidine-vortioxetine or pyrrolidinium-vortioxetine.

14. A method of promoting satellite cell proliferation by increasing division rate, comprising administering a pharmaceutical composition comprising at least one direct 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent selected from a triptan, a vortioxetine, and an ergotamine and at least one pharmaceutically acceptable excipient to the subject.

15. The method of claim 14, wherein the agent is vortioxetine.

16. The method of claim 14, wherein the agent is a triptan.

17. The method of claim 16, wherein the triptan is selected from sumatriptan, rizatriptan, zolmitriptan, eletriptan, almotriptan, frovatriptan, naratriptan, avitriptan, and donitriptan.

18. The method of claim 14, wherein the agent is modified to comprise at least one positively charged chemical moiety.

19. The method of claim 18, wherein the positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.

20. The method of claim 18, wherein the agent is a positively charged vortioxetine selected from the group consisting of salts of vortioxetine, vortioxetine coupled to a positively charged amino acid, pyrrolidinium-vortioxetine, pyperazinium-vortioxetine, dimethylammonium-vortioxetine, sulfonium-vortioxetine, N-oxide-vortioxetine, sulfoxide-vortioxetine, and phosphonium-vortioxetine.

21. The method of claim 18, wherein the agent is histidine-vortioxetine or pyrrolidinium-vortioxetine.

22. A method of promoting muscle regeneration and/or delaying progression of at least one of natural or pathological loss, damage and impairment of skeletal muscle in a subject by increasing satellite cell division rate, comprising administering a pharmaceutical composition comprising at least one direct 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent selected from a triptan, a vortioxetine, and an ergotamine and at least one pharmaceutically acceptable excipient to the subject.

23. The method of claim 22, wherein the subject has a pathological loss of skeletal muscle tissue(s).

24. The method of claim 22, wherein the subject has a natural loss of skeletal muscle tissue(s).

25. The method of claim 22, wherein the subject has damage and/or impairment of skeletal muscle tissue(s).

26. The method of claim 22, wherein the subject has sarcopenia.

27. The method of claim 22, wherein the triptan is selected from sumatriptan, rizatriptan, zolmitriptan, eletriptan, almotriptan, frovatriptan, naratriptan, avitriptan, and donitriptan.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Key gene markers expressed in satellite cells (MSC) and progenitor cells (MPC) of adult skeletal muscle (extract from Shi et al., 2006).

(2) FIG. 2. Fluoxetine increases the vessel number and the satellite cell (SC) number in skeletal muscle

(3) (a) Schematic representation of fluoxetine delivery and sacrifice time points. (b) Number of satellite cells per mm.sup.2 on section. (c-d) Histological section of tibialis anterior of a placebo treated mouse (c) and fluoxetine treated TgPax7nGFP mouse (d). The arrows point to the satellite cells. (e) Number of vessels per mm.sup.2 after intra peritoneal (IP) or per os administration of fluoxetine at different time points. (f-g) Histological section of tibialis anterior of a placebo (f) and fluoxetine treated (g) Flk1.sup.GFP/+ mouse. The picture displays endogenous GFP. (h) Number of CD31+ cells in Matrigel plugs subcutaneously grafted in placebo and fluoxetine treated mice. (i-j) Representative image of a Matrigel plug after 6 weeks in a C57Bl/6 mouse treated with placebo (i) or fluoxetine (j). (k-l) Representative image of a vessel (arrows) in the Matrigel plug detected by HE staining in placebo (k) or fluoxetine (l) treated mice. (m) Vessel length 4 days post-plating of Cytodex® beads covered with HUVEC. (n) Representative image of a Cytodex® bead 4 days-post plating in presence of placebo plasma. (o) Representative image of a Cytodex® bead 4 days-post plating in presence of fluoxetine treated mouse plasma.

(4) Using immunostainings and FACS cell sorting in Figures (p) to (v):

(5) (p) Number of Pax7GFP expressing cells per digested TA (expressed as an absolute number) counted by FACS. (q) Representative FACS profile of digested TA of TgPax7nGFP mouse. (r) Cumulative number of BrdU+ SC.Tg:Pax7nGFP mice (n=3 per time point) received BrdU in drinking water together with fluoxetine or placebo from the beginning of the treatment to the end. TA muscle was digested and cells were isolated by FACS, spined on a slide and immuno-stained against BrdU. (s) Percentage of BrdU+ cells in Tg:Pax7nGFP mice (n=3 per time point). Tg:Pax7nGFP mice received the fluoxetine treatment per os and were injected with BrdU twice (12 h and 4 h before death) TA muscle was digested and cells were isolated by FACS, spined on a slide and immuno-stained against BrdU. (t) Number of vessels per mm.sup.2 counted on histological section using CD31 immuno-labeling in placebo and fluoxetine treated animals. (u-v) Representative histological sections of laminin and CD31+ immunostaining in placebo (u) and fluoxetine (v) treated mice

(6) For Figures (a) to (o): n=8 mice used per condition, except for the in vivo experiments cells where n=6. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(7) For Figures (p) to (v): n=7 mice used per condition, except for BrdU experiments n=3 per time point. Data are represented as mean±s.d. **P<0.01. Scale bar represents 100 μm.

(8) FIG. 3. Fluoxetine improves muscle regeneration by increasing the number of satellite cells

(9) (a) Schematic representation of fluoxetine delivery, muscle injuries, BrdU injections and sacrifice time points. (b) Number of Pax7GFP+ cells per mm.sup.2 4 days post-injury. (c-d) Immunostaining of Pax7GFP+ cells on section after placebo (c) and fluoxetine (d) treatment. (e) Number of differentiating (Myogenin+) cells 4 days post-injury. (f-g) Representative pictures of Myogenin and GFP cells in placebo (f) and fluoxetine (g) treated animals. (h-i) Haematoxylin and eosin staining of cryo-sectioned TA 14 days post-injury in the placebo (h) and fluoxetine (i) treated animals. (j) Fibre size in μm.sup.2 in placebo and fluoxetine 14 days post-injury. (k) Percentage of fibrotic area 14 days post injury in placebo and fluoxetine treated mice. (l) Haematoxylin and eosin staining of cryo-sectioned TA after serial injuries. n=7 mice used per condition, except for the count of GFP+ cells where n=9. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(10) FIG. 4. Muscle regeneration is faster and the number of self-renewing satellite cells is higher after fluoxetine treatment.

(11) (a) Number of immune Gr1 (granulocytes) and F4/80 (macrophages) in the placebo and fluoxetine treated mice, 4 and 14 days post-injury. (b-c) Histological section of staining with sirius red (fibrosis) 14 days post injury in placebo (b) and fluoxetine (c) treated animals. (d) Calcium deposit 14 days post-injury in the placebo vs. fluoxetine treated animals. (e) Number of fibres in the placebo vs. fluoxetine treated animals 14 days post-injury. (f) Number of vessels in a Flk1.sup.GFP/+ mouse 28 days post-injury in the placebo vs. fluoxetine treated animals. (g) Number of SC in a TgPax7nGFP mouse 28 days post-injury in the placebo vs. fluoxetine treated animals. (h) Number of SC after several rounds of injury in the placebo vs. fluoxetine treated animals. (i) First division assessed by live videomicroscopy in vitro. Cells were plated either with plasma coming from placebo treated C57Bl/6 or fluoxetine treated C57Bl/6 animals. (j) Division rate assessed by live videomicroscopy in vitro. Cells were plated either with plasma coming from placebo treated C57Bl/6 or fluoxetine treated C57Bl/6 animals. (k) Percentage of differentiating (myogenin+) cells 4 days post-plating. Cells were plated either with plasma coming from placebo treated C57Bl/6 or fluoxetine treated C57Bl/6 animals.

(12) n=7 mice used per condition. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(13) FIG. 5. The effect of fluoxetine on endothelial and satellite cells is achieved via 5-HT1 B receptors

(14) (a) Scheme of fluoxetine and inhibitor delivery. (b) Quantification by RT-qPCR of the different serotoninergic receptors displayed as a fold increased/placebo in endothelial and satellite cells. (c) Number of differentiating cells per mm.sup.2 (myogenin+) in the placebo, fluoxetine and fluoxetine plus GR127935 5-HT1 B antagonist 4 days post injury. (d) Fibre size 14 days post-injury in μm.sup.2 in the placebo, fluoxetine and fluoxetine plus GR127935 5-HT1 B antagonist. (e) Percentage of fibrotic area 14 days post injury. (f) Number of SC per TA of a Tg:Pax7nGFP mouse in fluoxetine, fluoxetine and inhibitor GR127935, inhibitor GR127935 alone. (g-h) representative pictures of the number of GFP+ cells on section in the fluoxetine (g) and fluoxetine with GR127935 5-HT1 B inhibitor (h). The picture displays endogenous GFP. (i) Number of vessels in Flk1.sup.GFP/+ mice after fluoxetine treatment and GR127935 5-HT1 B antagonist. (j-k) Representative histological section of the number of vessels (counted with endogenous GFP from Flk1.sup.GFP/+ mouse) in fluoxetine (j), fluoxetine and inhibitor (k) per mm.sup.2. n=7 mice used per condition (n=5 for the controls). Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(15) FIG. 6. 5-HT1 BR, and not 5-HT2 BR, is involved in fluoxetine effect.

(16) (a) Number of SC 4 days post-injury in the placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonist and MDL100907 5-HT2 BR antagonist. (b) Calcium deposit 14 days post-injury in the placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonist. (c) Number of immune Gr1 (granulocytes) and F4/80 (macrophages) 4 days post-injury in the placebo, fluoxetine, fluoxetine and GR127935 5-HT1 B antagonist and MDL100907 5-HT2 BR antagonist. (d) Number of immune Gr1 (granulocytes) and F4/80 (macrophages) 14 days post-injury in the placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonist and MDL100907 5-HT2 BR antagonist. (e) Number of differentiating cells (myogenin+) 4 days post-injury with fluoxetine and fluoxetine and 5-HT2 BR antagonist. (f) Number of SC from TgPax7nGFP after 5-HT2 BR inhibition by MDL100907 antagonist. (g) Number of vessels from Flk1.sup.GFP/+ after 5-HT2 BR inhibition by MDL100907 antagonist.

(17) n=6 mice used per condition (n=5 for the controls). Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(18) FIG. 7. In vitro plasma from fluoxetine treated mice accelerates differentiation at early stages and increases self-renewal at later stages of both murine and human satellite cells.

(19) (a) Scheme of fluoxetine and inhibitor delivery in vitro. (b) Percentage of Pax7+ cells among the total number of FACS sorted SC through time in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (c) Number of self-renewed (also called reserve cells)SC (Pax7+/EdU−) 14 days post plating of FACS sorted SC in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (d) Percentage of MyoD+ cells among the total number of FACS sorted SC through time in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (e) Percentage of Myogenin+ cells among the total number of FACS sorted SC through time in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (f) First cell division assessed by live videomicroscopy in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (g) Division rate assessed by live videomicroscopy in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (h) Percentage of Pax7+ cells investigated by immunofluorescence in vitro co-cultured with CP94253 5HT1B specific agonist. (i) Percentage of Myogenin+ cells investigated by immunofluorescence in vitro co cultured with CP94253 5HT1BR specific agonist. (j) Differentiation (Myogenin+) cells coming from primary human SC obtained through pre-plating technique 4 days post-plating in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor. (k) Self-renewal (Pax7+/EdU−) cells coming from primary human SC obtained through pre-plating technique 14 days post-plating in placebo plasma, fluoxetine plasma, fluoxetine plasma with GR127935 inhibitor in vitro, fluoxetine plasma with MDL100907 inhibitor.

(20) n=6 mice used per condition, except for human myoblasts where n=4 per condition. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001. Scale bar represents 100 μm.

(21) FIG. 8. Fluoxetine improves the phenotype of dystrophic mice

(22) (a) Quantification of the necrotic area (in mm.sup.2) in placebo and fluoxetine treated animals. (b) Fibre size (area) of placebo and fluoxetine-treated Mdx mice. (c-d) Haematoxylin and eosin staining of cryo-sectioned TA of Mdx mice treated with either placebo (c) or fluoxetine (d). (e) Number of vessels in Mdx mouse either treated with placebo or with fluoxetine counted by immunostaining with CD31 and expressed in number of cells per mm.sup.2. (f) Number of cycling cells (Pax7+ and BrdU+ cells) in Mdx mouse either treated with placebo or with fluoxetine. (g) Example of Luminex® on Interleukin 6 (IL6) representing the level of protein expression in picogram/gram of plasma on both placebo and fluoxetine treated mice. (h) Example of Luminex® on Interleukin 10 (IL10) representing the level of protein expression in picogram/gram of plasma on both placebo and fluoxetine treated mice. (i) Number of Gr1+ cells in cryo-sectioned TA of placebo treated and fluoxetine treated Mdx mice. Numbers are displayed in absolute number per cross sections.

(23) FIG. 9. Antagonising 5HT1B-R eliminates the beneficial effects of fluoxetine on dystrophic phenotype

(24) (a) Quantification of the necrotic area (in mm.sup.2) in placebo, fluoxetine and fluoxetine with GR127935 5HT1BR inhibitor treated animals. (b) Fibre size (area) of placebo, fluoxetine and fluoxetine with GR127935 5HT1BR inhibitor treated Mdx mice. (c-e) Haematoxylin and eosin staining of cryo-sectioned TA of Mdx mice treated with either placebo (c) or fluoxetine (d) or fluoxetine and GR127935 inhibitor (e). (f) Number of vessels in Mdx mouse either treated with placebo, fluoxetine or fluoxetine and GR127935 5HT1BR inhibitor counted by immunostaining with CD31 and expressed in number of cells per mm.sup.2. (g) Number of cycling cells (Pax7+ and EdU+ cells) in Mdx either treated with placebo, with fluoxetine or fluoxetine and GR127935 5HT1BR inhibitor. (h) Number of Gr1+ cells in cryo-sectioned TA of placebo treated and fluoxetine treated Mdx mice. Numbers are displayed in absolute number per cross sections. n=9 mice per condition. Data are represented as mean±s.d. *P<0.05; ***P<0.001; ns: not significant. Scale bar represents 100 μm. (i) Force grip test. The total force of animals was measured (4 limbs) in fluoxetine and placebo treated mice. (j) Maximal tension (6V) of edl muscle fibers of fluoxetine or placebo treated mdx mice. (k) Tension of isolated edl through different voltage in placebo and fluoxetine treated mdx mice.

(25) FIG. 10. Vortioxetine increases the number of vessels and the number of satellite cells in vivo. (a) Schematic representation of vortioxetine delivery and timing of sacrifice. (b) Number of vessels counted on section after CD31 immunostaining per mm.sup.2 after I.P treatment. (c) Number of vessels counted on section after CD31 immunostaining per mm.sup.2 after P.O treatment. (d) Number of satellite cells counted by FACS in placebo, 12 days and 3 weeks vortioxetine I.P treated Tg:Pax7nGFP mice. (e) Number of satellite cells counted by FACS in placebo, 12 days and 3 weeks vortioxetine P.O treated Tg:Pax7nGFP mice.

(26) FIG. 11. Vortioxetine increases the number of vessels and the number of satellite cells in vivo and in vitro via the 5-HT1 B receptor. (a) Number of vessels counted on section per mm.sup.2 with CD31 immunostaining after vortioxetine treatment of TgPax7nGFP mouse I.P treatment for 12 days at 20 mg/Kg. (b) Number of satellite cells counted by FACS per tibialis anterior after vortioxetine treatment of Tg:Pax7nGFP I.P treatment for 12 days at 20 mg/Kg. (c-e) Cells were sorted by FACS from Tg:Pax7nGFP mice and plated at 2000 cells per cm.sup.2. The following day vortioxetine was added at 10 μM. At the indicated time points cells were fixed and stained for Pax7 (c), Myod (d), MyoG (e).

(27) FIG. 12. Vortioxetine derivatives Histidine-vortioxetine and Pyrrolidinium-vortioxetine increases the number of vessels and satellite cells: (a-b) Cells were sorted by FACS from Tg:Pax7nGFP mice and plated at 2000 cells per cm.sup.2. The following day Histidine-vortioxetine or Pyrrolidinium-vortioxetine were added at 10 μM (a) Percentage of Pax7+ cells at different time points in vitro with PBS or vortioxetine or Histidine-vortioxetine or Pyrrolidinium-vortioxetine. (b) Percentage of MyoG+ cells at different time points in vitro with PBS or vortioxetine or Histidine-vortioxetine or Pyrrolidinium-vortioxetine (c-d) PBS (n=4) or Histidine-vortioxetine (n=5) or Pyrrolidinium-vortioxetine (n=5) was injected IP to Tg:Pax7nGFP mice for 12 days at 20 mg/kg. Muscle (TA) were digested and counted by cytometry. (c) displays histidine-vortioxetine vs PBS injections (d) displays pyrrolidinium-vortioxetine vs PBS. Data are represented as mean±s.d. **P<0.01.

EXAMPLES

Fluoxetine, Vortioxetine and Derivatives Thereof Increase Muscle Stem Cell Number and Improve Regenerative Capacity of the Muscle

1. Material and Methods

(28) 1.1. Mice Injection and Injury

(29) All procedures in this study were approved by the Animal Care and Use committee at the Institut Pasteur (CETEA 2014-004). Unless specified 8 weeks old male mice were used in this study and housed on a 12:12 light/dark cycle in a pathogen free facility with controlled temperature and humidity. Food and drink were given ad libidum.

(30) Animals were anesthetized with ketamine (Imalgene1000 100 mg/Kg Merial) and Xylazine (Rompun2% 20 mg/Kg Bayer) prior to injury. Animals were hydrated and treated with analgesic (Buprenorphin Axience 0.3 mg/kg) twice a day for 4 d following injury. For the injury, mice were anesthetized as previously described and 10μl of 12.5 μg/ml Notexin (Lotaxan) was injected in the Tibialis anterior. All protocols were reviewed by the Institut Pasteur, the competent authority, for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations. This project has been reviewed and approved (#2013-0044) by the Institut Pasteur ethic committee (C2EA 89-CETEA).

(31) Among the mice tested, Flk1.sup.GFP/+ mice, in which green fluorescent protein (GFP) is targeted in VEGF-receptor-2 gene locus, and which exhibits a bright GFP signal in all endothelial cells, were kindly provided by Alexander Medvinsky (Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK).

(32) 1.2. Histological Analysis

(33) Tibialis anterior (TA) was carefully dissected and snap frozen in liquid-nitrogen-cooled isopentane for a few minutes and stored at −80° C. prior to cryosectioning (10 μm sections). Sections were kept at room temperature overnight before staining. Sections were then rehydrated in PBS for 10 minutes and fixed in 10% formalin for 3 minutes. The sections were then routinely stained with haematoxylin and eosin (HE) using an automated stain machine or manually with red sirius.

(34) The slides were assessed by double blinding and automated when possible (fibre diameter, cell count, infarcted area).

(35) 1.3. Immunostainings

(36) Immunostaining was performed on cryosections fixed with 4% paraformaldehyde (PFA EMS #15710) in cold PBS, permeabilized with 0.5% Triton X-100 20 min at room temperature, washed, and blocked with 10% BSA for 30 min. Sections were incubated with primary antibodies overnight at 4° C. (see Table 1 below) and with Alexa-conjugated secondary antibodies 1/250 and Hoechst for 45 minutes. Sections were then analysed using an automated axioscan (Zeiss) or inverted Observer.Z1 Apotome (Zeiss). For apoptosis assessment, cells were collected in 2% serum, spun on polyD-lysine (Sigma-Aldrich #P6407), and immediately fixed with PFA 4%.

(37) TABLE-US-00001 TABLE 1 List of antibodies used in the study. Antigen Host Concentration References Ly-6C (Gr1) Rat 0.5 μg/ml Caltag LabRM3030 CD31 Rat  15 μg/ml BD Pharmingen 550274 Pax7 Mouse  12 μg/ml DSHB Laminin Rabbit 0.69 μg/ml  Sigma-Aldrich L9393 Secondary Variable 0.5 μg/ml JacksonImmuno Donkey anti according to #711486152 (Rabbit) Rabbit (IgG the primary #200162037 (Mouse) Fraction Ab host Monoclonal
1.4. Cell Sorting, Count and Culture

(38) Muscle dissection was done as previously described in cold DMEM. Muscles were then chopped with small scissors and put in a 50 ml Falcon tube with collagenase 0.1% and trypsin 0.25% at 37° C. with gentle agitation. After 20 minutes, the supernatant was collected in 20% serum placed on ice, and the collagenase/trypsin solution was added to continue the digestion. Once muscle was completely digested, the solution was filtrated using 40 μm cell strainers. Satellite cells were cultured in 1:1 DMEM-Glutamax (Gibco #41965-039):MCDB201 (Sigma #M6770) containing 20% serum FBS (Biowest S1860). Medium was filtered using 0.22 μm filters. Cells were plated on Matrigel coating (BD Biosciences #354234) and kept in an incubator (37° C., 5% CO.sub.2). For some in vitro experiments, plasma was extracted from fluoxetine or vortioxetine treated animals after 6 weeks by heart puncture followed by centrifugation at 1500 g for 15 min. The thus-obtained supernatant replaced FBS in the culture medium; the rest of the medium was unchanged.

(39) For satellite cell counting, only the tibia/is anterior muscle was dissected and digested as described earlier, and the totality of the tube was analysed to assess the number of satellite cells per muscle. FACS analysis was done using a FACSasia (Beckman). All analyses and quantitation were performed using Summit v4.3 software from DakoCytomation and FloJo software. Cells were labelled with propidium Iodide 10 μg/ml (Sigma-Aldrich #P4170) to exclude dead cells and displayed using the PE (Phycoerythrin, Red) channel on the FACS profile.

(40) 1.5. Live Video Microscopy

(41) Cells isolated by FACS were plated overnight on a 24-well glass bottom plate (P24G-0-10-F; MatTek) coated with matrigel (BD Biosciences #354234) and placed in an incubator in pre-equilibrated medium (1:1 DMEM Glutamax: MCDB [Sigma-Aldrich], 20% FCS (Biowest S1860). The plate was then incubated at 37° C., 5% CO.sub.2 (Zeiss, Pecon). A Zeiss Observer.Z1 connected with a LCI PInN 10×/0.8 W phasell objective and AxioCam camera piloted with AxioVision was used. Cells were filmed for up to 5 days, and images were taken every 30 min with brightfield and phase filters and MozaiX 3×3 (Zeiss). Raw data were transformed and presented as a video.

(42) 1.6. Image Analysis

(43) For image analysis (fibrosis quantification), ImageJ 1.46r software was using between 10 different photos randomly taken per section and 3 sections minimum per experimental group. The pictures were converted in a binary image and the pixel values then collected. For fibre size, the sections were immunostained with rabbit anti Laminin (Sigma-Aldrich #L9393) diluted at 1/200, overnight at 4° C. Secondary Donkey ant Rabbit 488 (DL488 JacksonImmuno #711486152) were used at 1/200 45 minutes at room temperature. The fibre perimeter was done automatically by using Pixcavator® software.

(44) 1.7. Luminex®(Multiplex Immunoassay) Snap frozen plasma samples (n=6 per condition) were thawed out, and supernatant was processed for Luminex® multiple cytokine and chemokine analysis (Bio-Plex® Pro™ Mouse Cytokine Standard 23-Plex, Group I and Standard 9-Plex, Group II). Normalization was done by sample weight of frozen muscle.
1.8. RT-qPCR

(45) Total RNA was isolated from cells using the RNAeasy Micro kit (Qiagen). The total RNA was reverse-transcribed using Superscript® III Reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed using Power Sybr Green PCR Master Mix (Applied Biosystems) and the rate of dye incorporation was monitored using the StepOne™ Plus RealTime PCR system (Applied Biosystems). At least three biological replicates were used for each condition. Data were analyzed by StepOne Plus RT PCR software v2.1 and Microsoft excel. GAPDH transcript levels were used for normalisation of each target (=ΔCT). Real-time PCR C.sub.T values were analyzed using the 2-(ΔΔCt) method to calculate the fold expression (ΔΔCTmethod, Livak et al., 2001).

(46) 1.9. Force Measurement

(47) The grip strength test is a non-invasive method designed to evaluate mouse muscle force in vivo. A grip meter (Bio-GT3, BIOSEB), attached to a force transducer, measures the peak force generated. Placebo and fluoxetine (6 weeks treatment) mdx mice were placed with the four paws on a grid and gently pulled backward until they released the grip. Five trials were conducted and, before statistical analysis, a mean value was calculated for each mouse using the tree median data. Results are expressed as the result of tree peak forces (in g), normalized to the body weight (in g).

(48) 1.10. Skinned Fibres Experiments

(49) TA muscles were dissected from placebo and fluoxetine treated mice (6 weeks of treatment). Small bundles of two to five fibers were manually isolated from the muscles as previously described. Chemical skinning was carried out using Triton X-100. Skinned fibers were mounted in the Displacement Measuring System KD 2300 (model 0.5 SU, Kaman Instrumentation, Colorado Springs, Colo., USA). To perform force measurements, skinned fiber preparations were incubated for 1 h in relaxing solution (pCa 9.0, low calcium content) containing 1% Triton X-100 (v/v) to solubilize the sarcolemma and the sarcoplasmic reticulum membranes, and were subsequently washed several times in relaxing solution without detergent. Fibres were adjusted to slack length and then stretched progressively until the tension developed became maximal. Isometric tension was recorded continuously using a chart recorder (model 1200, Linear, Reno, Nev., USA). The tension obtained was normalized to fibre cross-sectional area.

(50) 1.11. Statistical Analysis

(51) Statistical analysis was performed using GraphPad Prism software using appropriate tests (non-parametric Mann-Whitney unless specified) and a minimum of 95% confidence interval for significance; p values indicated on figures are <0.05 (*), <0.01 (**), and <0.001 (***). Figures display average values of all animals tested±SD or ±SEM for RT-qPCR, or as indicated.

2. Results

(52) 2.1. Fluoxetine

(53) 2.1.1. Fluoxetine Increases Vessel Number and Satellite Cells Number in the Skeletal Muscle

(54) In order to investigate the effect of fluoxetine on vessels number, intra peritoneal (I.P) or per os (P.O) administration of 18 mg/kg of fluoxetine was performed for either 3 or 6 weeks to Flk1.sup.GFP/+ or TgPax7nGFP mouse (FIG. 2a), allowing the direct visualisation of endothelial cells and satellite cells (SC) respectively. Throughout the study, focus was made on the tibialis anterior (TA). In skeletal muscle, SC are central for muscle repair. By histological count, it was observed that the number of SC was higher when treated with fluoxetine (9.7±3.1SC per mm.sup.2 in the placebo vs. 16.3±5.4SC in the treated, p=0.04) (FIGS. 2b-d and p-q). These results were confirmed by digesting the TA and counting the number of SC directly by cytometry using the Tg:Pax7nGFP mouse (Sambasivan et al., 2009) (p=0.02) (FIGS. 2p,q). SC are localized close to capillaries and angiogenesis is known to be crucial for muscle repair SC survival with cellular interplays between vessel cells and SC. The number of vessels was therefore quantified. In control Flk1.sup.GFP/+ mouse (placebo), the number of vessels was of 870±213.1 vessels per mm.sup.2 and increased after 3 weeks of I.P treatment (1103±110.2 vessels per mm.sup.2), and even more after 6 weeks (1384±175.3 vessels per mm.sup.2, p=0.008) (FIG. 2e). Neovessels presented with normal histological appearance associating a basal lamina and a permeable lumen sometimes containing red blood cells. The daily administration of fluoxetine I.P might cause unwanted peritoneal inflammation that could interfere with angiogenesis since inflammation and neovascularization are intertwined in some models (Sadat et al., 2014). Therefore, fluoxetine was administered P.O for 6 weeks: a similar increase in the number of vessels was observed (1256±62.2 vessels per mm.sup.2 p=0.008, FIG. 2e-g). Those results were confirmed in another model, by administrating fluoxetine to C57Bl/6 mice P.O for 6 weeks and performing immuno-labelling of CD31+ cells on cryo-sectioned TA. 841±137 CD31+ cells per mm.sup.2 were counted in the placebo-treated mice vs. 2260±361 CD31+ cells per mm.sup.2 in the fluoxetine-treated mice, p=0.008 (FIG. 2t-v), confirming the previous observation. After delivery of fluoxetine 6 weeks in vivo the levels of IL-la, IL-1b, IL-2, and eotaxin are downregulated whereas IL-4 and IL-13 are upregulated (p=0.02). The other tested cytokines did not show any significant changes (Table 2).

(55) TABLE-US-00002 TABLE 2 Fluoxetine decreases the basal levels of cytokines. The table below represents the plasmatic levels of cytokines in picograms per μl of plasma measured by Luminex ® assay in the placebo and fluoxetine treated mice. Quantity in Quantity in Placebo Fluoxetine Statistically p Cytokine (pg/μm) treated (pg/μm) significant value IL-1a 234.2 ± 33   150.8 ± 22.8 * 0.02 IL-1b 1398 ± 380 756.2 ± 205  * 0.02 IL-2 48.30 ± 8.1  82.53 ± 11.7 * 0.02 IL-4 52.71 ± 6.7  93.76 ± 9.9  * 0.02 IL-5 21.13 ± 16.7  7.2 ± 3.2 ns 0.06 IL-6 20.54 ± 8.4  10.68 ± 1.8  ns 0.11 IL-9 89.02 ± 15.5 72.54 ± 14.8 ns 0.11 IL-10 16.10 ± 7.8  36.60 ± 27.9 ns 0.34 IL-12(p40) 43.7 ± 43  59.7 ± 36  ns 0.48 IL-12(p70) 130.1 ± 53.7 118.2 ± 40   ns 0.48 IL-13 626.6 ± 69.8 875.1 ± 147  * 0.03 IL-17  48.3 ± 22.2  29.8 ± 15.3 ns 0.34 Eotaxin 44.10 ± 19   18.7 ± 4.6 * 0.02 G-CSF  3884 ± 1657 3066 ± 288 ns 0.8 GM-CSF  29189 ± 39901  8800 ± 1438 ns 0.34 IFN-g 32.78 ± 6.1  28.5 ± 8.9 ns 0.2 KC  8290 ± 10202  717 ± 502 ns 0.11 MCP-1  73.5 ± 45.9 26.6 ± 20  ns 0.06 MIP-1a 64.6 ± 39   34.6 ± 17.3 ns 0.48 MIP-1b 570.4 ± 154  442.1 ± 49   ns 0.11 RANTES  120 ± 111  53.6 ± 27.1 ns 0.34 TNF-a 8318 ± 865  7846 ± 2171 ns 0.34 FGF-basic   1057 ± 141.3 1276 ± 618 ns 0.88 MIG  100 ± 8.1 83.4 ± 11  * 0.05 PDGF-bb  653 ± 256 401 ± 74 ns 0.34 VEGF 313.44 ± 91    2554 ± 1041 * 0.02 The p value is calculated using Mann-Whitney test. n = 7 animals per condition. *: p ≤ 0.05; ns: non-statistically significant.

(56) To further confirm the above results, an ex vivo Matrigel angiogenesis assay was used. To do so, a cold Matrigel was introduced subcutaneously, which solidifies and allows the penetration by host cells and the formation of new blood vessels in C57Bl/6 treated P.O for 6 weeks. The number of CD31 expressing cells was higher in the fluoxetine treated plugs (72.9±22.6 CD31+ cells per mm.sup.2) compared with the placebo (25.20±13.8 CD31+ cells per mm.sup.2, p=0.002) (FIGS. 2h-l); confirming the previous observation.

(57) These data were further confirmed by in vitro HUVEC assay (human endothelial cells). C57Bl/6 mice were treated P.O for 6 weeks and plasma was extracted from blood. HUVEC cells were then plated on cytodex beads and cultured in either of the plasma. The growth of HUVEC incubated with plasma coming from treated animals was faster 4 days post plating (p≤0.0001) (FIGS. 2m-o).

(58) Cell division was quantified after BrdU administration during the entire per os treatment with fluoxetine, and the results showed that 90% of the SC population was dividing (FIG. 2r). A short pulse of BrdU 16 h before death showed that most of the SCs were dividing between the third and the fifth week (FIG. 2s).

(59) 2.1.2. Fluoxetine Improves Muscle Regeneration Potential

(60) To investigate if fluoxetine had a functional impact on muscles, notexin injury was performed after fluoxetine treatment (FIG. 3a) and the muscle ability to regenerate on Tg:Pax7nGFP mice was investigated (Sambasivan et al., 2009). The comparison 4 days and 14 days post injury of the muscles showed in both cases a better regeneration (FIG. 3b-j). Indeed, major differences were observed in the muscle regeneration features between placebo and fluoxetine treated mice. At 4 days post-injury treated animals showed a higher number of SC (459±283 in the treated vs. 177.2±49 in the placebo, p=0.04) (FIG. 3b-d) and more cells were already differentiating (p=0.0006) (FIG. 3e-g). 14 days post-injury in the placebo group, regenerating centro-nucleated fibers showed variable size (anisocytosis, 81±28.4 μm.sup.2), multifocal endomysial infiltration by mononuclear inflammatory cells (9.14±3.9 Gr1+ cells and 12.6±3.9 F4/80+ cells per section), the presence of multiple large basophilic foci of calcium deposition (19±4 per mm.sup.2), and mild fibrosis of the endomysium (6.4±2% of total muscle area) (FIGS. 3h-k). By contrast, in fluoxetine treated mice, regenerating fibers were bigger and showed less variable size (129±12.6 μm.sup.2, p=0.005), less inflammatory cells (4.1±2.6 Gr1+ cells; p=0.018 and 4.7±2.7 F4/80 cells; p=0.0017), less calcium deposits (3.2±3 per mm.sup.2 p=0.0006), and less endomysial fibrosis (2.2±0.7 of total muscle area p=0.007) (FIGS. 3h-k, and FIGS. 4a-d)). The number of fibers however remained the same (p=0.4, FIG. 4e). Twenty-eight days post-injury, the muscle was fully regenerated; no differences were observed between the two groups, except for a higher number of vessels (p=0.036) and SC (p=0.076) in fluoxetine treated animals (FIGS. 4f,g). These data were confirmed in vitro by plating SC from Tg:Pax7nGFP mouse in 20% plasma originating from C57Bl/6 treated mice or placebo. By live-videomicroscopy, it was observed that the first satellite cells division occurred faster (26.45 h±1.18 post-plating in fluoxetine treated plasma vs. 29.08 h±0.37 in control plasma p=0.02, with higher division rate (12.5±2.1 h in placebo vs. 8.7±0.7 h in fluoxetine; p=0.047) (FIGS. 4i,j). A higher number of myogenine positive cells 4 days post-plating was also observed (12±5.5% of total plated cells in the placebo vs. 34.75±6.3% in the fluoxetine treated; p=0.02, (FIG. 4k) as well as faster forming myofibers; confirming the observations made in vivo of a faster differentiation and repair of muscle fibers. Interestingly, together with this improved regeneration, a decrease in the inflammation of the muscle after injury was observed as previously (Table 3). For example, the pro-inflammatory cytokine IL-6 dropped from 4258±665pg/μl in the injured vs. 2459±920 pg/μl in the injured with fluoxetine treatment (p=0.02).

(61) To further challenge the muscle, several rounds of injury were performed after fluoxetine treatment, in order to exacerbate the phenotype and insure that the satellite cells (SC) pool was not exhausted. The number of SC remained constant per TA even after 3 rounds of injury (FIG. 4h). At the histological level the muscle was well regenerated in the injured and re-injured cases (FIG. 31), showing that the SC were still functional stem cells after fluoxetine treatment.

(62) TABLE-US-00003 TABLE 3 Fluoxetine decreases the levels of cytokines after injury. The table represents the plasmatic levels of cytokines in picograms per μl of plasma measured by Luminex ® assay in the placebo and fluoxetine treated and notexin injured mice. Quantity in Quantity in placebo fluoxetine 4 days 4 days PI treated Statisificantly P Cytokine PI (pg/μm) (pg/μm) significant value IL-1a 307.6 ± 180   107.6 ± 33.2  * 0.05 IL-1b 3698 ± 2202 2039 ± 435  ns 0.34 IL-2 206.5 ± 187   47.33 ± 15   * 0.03 IL-4 156.1 ± 52   303.9 ± 5    * 0.02 IL-5 566 ± 82  265 ± 41  * 0.02 IL-6 4258 ± 665  2459 ± 920  * 0.02 IL-9 426.3 ± 89   241 ± 56  * 0.02 IL-10 342 ± 36  176 ± 39  * 0.03 IL-12(p40) 336 ± 179 171 ± 84  ns 0.2 IL-12(p70) 987.2 ± 693   359 ± 240 ns 0.11 IL-13 2028 ± 1242 691 ± 268 * 0.02 IL-17 1800 ± 1075 1558 ± 525  ns 0.8 Eotaxin 10155 ± 6873  3692 ± 934  ns 0.11 G-CSF 7014 ± 7628 2689 ± 364  ns 0.34 GM-CSF 30928 ± 49630 3962 ± 2289 ns 0.2 IFN-g 149.2 ± 35.72 68.84 ± 44.6  * 0.02 KC  8290 ± 10202 717 ± 502 * 0.05 MCP-1 21380 ± 21143 6560 ± 7168 * 0.05 MIP-1a 2492 ± 784  4214 ± 4861 ns 0.68 MIP-1b 1770 ± 1034 1337 ± 540  ns 0.68 RANTES 4658 ± 1870 2804 ± 826  * 0.05 TNF-a 7846 ± 2171 10386 ± 1266  * 0.02 FGF-basic 1115 ± 117  1618 ± 232  * 0.02 MIG 45730 ± 41234 28105 ± 38006 ns 0.2 PDGF-bb  2965 ± 317.6  1777 ± 148.5 * 0.02 VEGF 653.4 ± 203   3334 ± 59  * 0.02 The p value is calculated using Mann-Whitney test. n = 4 animals used per condition. PI: post-injury; *: p ≤ 0.05 ns: non-statistically significant.
2.1.3. Effects of Fluoxetine on Vessels and Satellite Cells is Obtained Through Stimulation of the 5-HT1 B Serotonin Receptor

(63) In order to understand how vessels were activated, endothelial cells (CD34+, CD31+, Sca-1+, CD45−) from digested muscle were FACS cell-sorted after 6 weeks P.O fluoxetine treatment (FIG. 5a). RT-qPCR was then performed on serotonin receptors subtypes to further characterize by which the endothelial cells could be activated (FIG. 5b). A 90±35 fold increase was observed in the 5-HT1 BR subtype in the treated vs. placebo mice (p=0.0035), and 30±13 (p=0.015) fold increase in 5-HT2 BR subtype in the fluoxetine treated animals (FIG. 5b). The other tested subtypes (5-HT1 AR, 5-HT1 DR, 5-HT1 FR, 5-HT2 AR, 5-HT2 CR) showed non-statistically significant increase (FIG. 5b). These data were confirmed using endothelial markers from digested muscle after 6 weeks P.O fluoxetine treatment (data not shown). The same observation was made in FACS cell-sorted SC from Tg:Pax7nGFP mice (FIG. 5b).

(64) To investigate the role of the 5-HT1 BR, the GR127935 hydrochloride inhibitor (a 5-HT1 BR antagonist) was delivered in osmotic pump together with fluoxetine treatment P.O for 6 weeks and vessel count in Flk1.sup.GFP/+ mice and SC count in Tg:Pax7nGFP mice were performed. The number of vessels was lower in fluoxetine and inhibitor treated Flk1.sup.GFP/+ mice (1028±173 vessels per mm.sup.2) compared with fluoxetine and PBS treated Flk1.sup.GFP/+ mice (1949±576 vessels per mm.sup.2, p=0.0159) (FIGS. 5i-k). The number of SC was also lower in fluoxetine and inhibitor treated Tg:Pax7nGFP mice (4674±1414 SC per TA) compared with fluoxetine and PBS treated Tg:Pax7nGFP mice (7283±2325 SC per TA p=0.04) (FIGS. 5f-h). Although a 30 fold increase of 5-HT2 B receptor was detected after fluoxetine treatment, no inhibition of fluoxetine effects were observed when antagonizing it with MDL100907 (a 5-HT2 BR antagonist) (FIGS. 6f,g).

(65) After injury, the inhibition of 5-HT1 BR by GR127935 suppressed the beneficial effects of fluoxetine treatment with a lower number of SC (FIG. 6a) and differentiating cells (FIG. 5c), a lower fiber size (FIG. 5D) and higher calcium deposits (FIG. 6B) 14 days post-injury, a higher percentage of fibrosis (FIG. 5e) and a higher infiltration of immune Gr1 and F4/80 cells (FIG. 6c,d). After injury, the inhibition of 5-HT2 BR by MDL100907 did not suppress the beneficial effects of fluoxetine (FIG. 6e).

(66) Those results were confirmed in vitro (FIG. 7a). The number of Pax7 positive cells decreased faster 4 days post-plating in plasma from fluoxetine treated animals (24%±6 in fluoxetine treated vs. 39.25%±8 in placebo p=0.02) (FIG. 7b) and was close to the one of the placebo when incubated with GR127935 (35.25%±6.2 p=0.32) (FIG. 7b). 14 days post-plating, a higher number of reserve cells (Pax7 positive cells that are quiescent in vitro) was detected in the plasma from fluoxetine-treated animals (9.4%±0.7) than in the placebo treated animals (4.4%±0.7, p=0.01), and when GR127935 was added in vitro the number of reserve cells dropped (4.5%±0.5, p=0.9) (FIGS. 7b,c). MyoD (an activation marker) expression did differ at any time investigated in the 3 tested conditions (FIG. 7d), however Myogenin (MyoG), a differentiation marker displayed a 3 times increase in expression when plated with plasma coming from fluoxetine-treated mice (p=0.0017) (FIG. 7e). This difference was lost when SC were plated with GR127935 (p=0.4) (FIG. 7e). The faster exit of quiescence and higher division rates were also lost when adding GR127935 in vitro (FIGS. 7f,g). The effects of the plasma from fluoxetine-treated animals were persistent when adding MDL100907 (FIGS. 7b-g). Importantly, in vitro, the direct addition of a 5-HT1B agonist in the culture media triggered the same effect as the addition of plasma from fluoxetine-treated mice (FIG. 7 h,i); we observed faster differentiation at early time points post plating and a higher rate of self-renewal at later time points post plating.

(67) The same results were obtained when primary human myoblasts were plated in plasma from fluoxetine-treated mice, with a faster differentiation 4 days post-plating (p=0.05) (FIG. 7j) and a higher self-renewal of the cells 14 days post-plating (p=0.02) (FIG. 7k). Those effects were lost when antagonising 5-HT1 BR but not 5-HT2 BR (FIG. 7j,k).

(68) 2.1.4. Fluoxetine Improves the Mdx Phenotype

(69) Fluoxetine was delivered P.O. for 6 weeks to Mdx mice (Bulfield G et al., 1983), a Duchenne muscular dystrophy mouse model. The fluoxetine treated Mdx mice exhibited less foci of necrotic fiber (2893±803 mm.sup.2 in average) compared with non-treated Mdx control (5041±1629 mm.sup.2 in average, p=0.04) (FIG. 8a). The fiber size was also overall bigger in Mdx treated-animals (4.241±0.9 pixels in treated vs. 3.192±0.3 in placebo p=0.051) (FIG. 8b). Accordingly, the number of regenerating foci decreased in the Mdx mice treated with fluoxetine (FIG. 8c-d). The number of vessels increased in the treated mice (1459±327 vessels per mm.sup.2) when compared to the placebo (942.4±113 vessels per section, p=0.008) (FIG. 8e). Interestingly the number of cycling satellite cells also decreased, correlating with the decreased number of regenerative foci previously observed (FIG. 8f). The treated mice also displayed a lower level of cytokines (34.52±21 pg/ml of IL6, Table 4) compared with placebo (580±158 pg/ml of IL6, p=0.004) (FIG. 8g, Table 4). The same decrease was observed for other pro-inflammatory cytokines (data not shown). Of note, the level of IL10 (an anti-inflammatory cytokine) did not change in the treated animals (99.6±56 pg/ml of MO) compared with placebo (64.1±50, p=0.4) (FIG. 8h, Table 4). This observation is in line with the decreased infiltration of inflammatory cells observed in the muscle (9.6±3.7 Gr1+ cells observed per section in the placebo vs. 4±2.4 Gr1+ cells in the treated mice p=0.04) (FIG. 8i). Notably, when Mdx mice were treated with fluoxetine and a 5-HT1B inhibitor, the beneficial effects were lost (FIG. 9a-h).

(70) At the functional level the total muscle force of fluoxetine treated mdx mice was increased by 56% (FIG. 91) and isolated single fibers of the extensor digitorum longus (edl) force was also increased by 45% (FIG. 9J-K). This was confirmed on soleus muscle (data not shown).

(71) TABLE-US-00004 TABLE 4 Fluoxetine decreases the levels of cytokines in a dystrophin mouse model Mdx. The table represents the plasmatic levels of cytokines in picograms per μl of plasma measured by Luminex ® assay in the placebo and fluoxetine treated Mdx mice. Quantity in Quantity in Placebo Fluoxetine Mdxl Statistically Cytokine Mdx (pg/μm) treated (pg/μm) significant P value IL-1a 119.8 ± 54    28.8 ± 10.6 *** 0.0006 IL-1b 1174 ± 252 436 ± 86 *** 0.0006 IL-2 50.11 ± 27    22.02 ± 15.13 * 0.04 IL-3 63.8 ± 14   21.4 ± 23.7 * 0.01 IL-4 56.46 ± 94    29.5 ± 27.3 ns 0.9 IL-5  101.6 ± 101.8 10.59 ± 5.5  *** 0.0006 IL-6  324 ± 343  12.9 ± 10.24 ** 0.0012 IL-10 407.9 ± 167  176.5 ± 96   ns 0.06 IL-12(p40) 2015 ± 874  922 ± 414 ** 0.007 IL-12(p70) 2557 ± 839 182 ± 90 ** 0.002 IL-13 1942 ± 459 1065 ± 380 ** 0.007 IL-17 91.6 ± 39  21.2 ± 21  ** 0.001 Eotaxin 2398 ± 521 850.4 ± 565  ** 0.002 G-CSF 1369 ± 880 65.20 ± 43.6 *** 0.0006 GM-CSF 1272 ± 284  447 ± 199 ** 0.002 IFN-g 428.3 ± 193  24.7 ± 16  *** 0.0006 KC 2606 ± 898 30.68 ± 10.2 *** 0.0006 MCP-1 2704 ± 522  339 ± 172 *** 0.0006 MIP-1a  415 ± 175  75.5 ± 12.6 *** 0.0006 MIP-1b  1978 ± 1219 91.48 ± 84   *** 0.0006 RANTES 223.3 ± 151  19.15 ± 6.3  *** 0.0006 TNF-a  9927 ± 1702 2355 ± 782 *** 0.0006 FGF-basic 139 ± 37 118.2 ± 85   ns 0.25 MIG  4320 ± 3003 1649 ± 624 * 0.01 PDGF 2005 ± 721  2984 ± 1573 ns 0.25 VEGF  88.2 ± 18.9  32.1 ± 22.3 ** 0.0041 The p value is calculated using Mann-Whitney test. n = 7 animals used per condition. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns: non-statistically significant.
2.2. Vortioxetine
2.2.1. Vortioxetine Increases Vessel Number in the Tibialis Anterior

(72) In order to investigate the effect of vortioxetine on vessels number, intra peritoneal (I.P) administration of 20 mg/kg of vortioxetine was performed for either 12 days, 3 or 6 weeks to C57Bl/6 mice (FIG. 10a). The number of CD31+ cells was counted by immunostaining. The number of vessels in the placebo is (863.5±115.6 vessels per mm.sup.2) and increases after 12 days of I.P treatment (2274±926 vessels per mm.sup.2, p=0.03), and even more after 3 weeks (2847±705 vessels per mm.sup.2, p=0.02) (FIG. 10b). However, there was no statistically significant differences between 12 days and 3 weeks of treatment (p=0.49). Neovessels display a normal histological appearance associated with a basal lamina and a permeable lumen sometimes containing red blood cells. The daily administration of vortioxetine IP might cause unwanted peritoneal inflammation that could interfere with angiogenesis since inflammation and neovascularization are intertwined in some models. Vortioxetine was therefore administered per os (P.O) for 12 days: a similar increase in the number of vessels was observed (2451±595 vessels per mm.sup.2 p=0.028, FIG. 10c). To further confirm those results, vortioxetine will be administered to Flk1.sup.GFP/+ mice P.O and I.P for 12 days, and an ex vivo Matrigel angiogenesis assay and plasma from vortioxetine treated mice will be used on HUVEC (endothelial cells).

(73) 2.2.2. Vortioxetine Increases the Number of Satellite Cells

(74) In order to count the number of satellite cells, the tibialis anterior (TA) of Tg:Pax7nGFP mice were digested and analysed by FACS in placebo and vortioxetine (12 days, 20 mg/Kg) treated mice. Cells were then counted by cytometry (FIG. 10d) per TA and a clear increase in SC number was observed in treated (8660 satellite cells±699) vs. non-treated (4444 satellite cells±802 p=0.008) mice (FIG. 10a). These data were confirmed by histological count on sections, where a two-fold increase was found on TA sections. After 3 weeks of vortioxetine treatment, the number of satellite cells was even higher (9351 satellite cells±706, p=0.0079) but no statistically significant differences were found between 12 days and 3 weeks of treatment (p=0.15). Vortioxetine was also administered P.O at 20 mg/kg: a similar increase in SC number has been observed, thereby confirming the I.P results (FIGS. 10d,e).

(75) 2.2.3. Vortioxetine Increases the Number of Vessels and Satellite Cells Via the Stimulation of the 5-HT1 B Receptor

(76) To investigate the role of the 5-HT1 BR, GR127935 hydrochloride inhibitor, a specific 5-HT1 BR antagonist, was delivered in osmotic pump together with vortioxetine treatment I.P for 12 days. The number of vessels was lower in vortioxetine and inhibitor treated Flk1.sup.GFP/+ mice (928±207 vessels per mm.sup.2) compared with vortioxetine and PBS treated Flk1.sup.GFP/+ mice (2274±926 vessels per mm.sup.2, p=0.028 (FIG. 11a). The number of SC was also lower in vortioxetine and inhibitor treated Tg:Pax7nGFP mice (5540±411 SC per TA) compared to vortioxetine only (FIG. 11b). No inhibition of vortioxetine effects was observed when antagonizing it with MDL100907, an inhibitor of the 5-HT2 B receptor (FIGS. 11a,b), neither for the vessels nor the SC number.

(77) After injury, the inhibition of 5-HT1 BR by GR127935 repressed the beneficial effects of the vortioxetine 12 days-treatment: indeed, one could observe a lower number of SC and differentiating cells, a lower fiber size and higher calcium deposits 14 days post-injury, a higher percentage of fibrosis as well as a higher infiltration of immune Gr1 and F4/80 cells.

(78) Those results were confirmed in vitro as shown in FIGS. 11c-e. Satellite cells were isolated from Tg:Pax7nGFP mice and cells were plated at 2.000 cells per cm.sup.2. After overnight, vortioxetine was added at 10 μM. The number of Pax7 positive cells decreased faster 4 days post-plating (23%±2 in vortioxetine treated vs. 38.25%±6 in PBS p=0.02) (FIG. 11c) and was close to the control (PBS) when incubated with the GR127935 inhibitor (25.5%±4.2 p=0.32) (FIG. 11c). 14 days post-plating, a higher number of reserve cells (Pax7 positive cells that are quiescent in vitro) were detected in the vortioxetine-treated cells (9%±0.7) than in the PBS (3.5%±0.9, p=0.01), and when GR127935 inhibitor was added in vitro the number of reserve cells dropped (3.5%±0.9, p=0.9) (FIG. 11c). The expression of MyoD (an activation marker) did not differ at any time investigated in the 3 tested conditions (FIG. 11d). However, Myogenin (MyoG), a differentiation marker displayed a 2 times increase in expression, 4 days post-plating with vortioxetine (p=0.0017) (FIG. 11e). This difference in expression was lost when SC were plated with the GR127935 5-HT1 BR inhibitor (p=0.4) (FIGS. 11c-e). The positive effects of vortioxetine were lost when GR127935 was added in vitro together with vortioxetine (FIGS. 11c-e). These effects were however persistent when adding the 5-HT2 BR antagonist (FIGS. 11c-e). This means that the positive effects observed when plating vortioxetine in vitro with satellite cells is mediated via the 5HT1B receptor and not 5HT2B.

(79) These in vitro experiments clearly demonstrate that, similarly to the observations made in vivo, vortioxetine directly acts on satellite cells and stimulate their differentiation, at a faster regeneration rate than fluoxetine. Most importantly, an increase in the number of self-renewing satellite cells is observed after 14 days in vitro, which highlights the capacity of vortioxetine to increase the pool of muscle stem cells.

(80) 2.3. Vortioxetine Derivatives

(81) 2.3.1. Histidine-Vortioxetine and Pyrrolidinium-Vortioxetine Increases the Number of Vessels and Satellite Cells

(82) To test whether, 2 derivatives of vortioxetine, namely histidine-vortioxetine and pyrrolidinium-vortioxetine, could have similar or improved effect compared to the unmodified vortioxetine, the in vitro approach as described in section 2.2.3 above was used and the differentiation cascade of satellite cells was investigated by assessing the expression pattern of Pax7 (a marker of stemness and quiescence) and Myogenin (MyoG, a marker of differentiation) as described above in section 2.2.3. To do so, satellite cells from Tg:Pax7-nGFP were isolated by FACS and plated at 2.000 cells per cm.sup.2 (seeded overnight). The following day, the vortioxetine, its derivatives, or the control PBS was added at 10 μM and the cells were fixed at the indicated time points.

(83) The differentiation rate of the cells was faster with vortioxetine, pyrrolidinium-vortioxetine and histidine-vortioxetine than with PBS (control). Indeed, after 4 days in PBS 38.2±6.7% of cells were still Pax7+, against 23.7±2.78% when cells were plated with vortioxetine (p≤0.001), 25.25±4% with pyrrolidinium-vortioxetine (p≤0.001) and 22.5±3.3% with histidine-vortioxetine (p≤0.0001) (FIG. 12a). The Myogenin staining data further confirmed this observation with 13.25±2% of cells MyoG+ in the PBS control against 34.5±2.5% with vortioxetine (p≤0.0001), 19.75±0.47% with pyrrolidinium-vortioxetine (p≤0.05) and 30±1.7% with histidine-vortioxetine (p≤0.0001) (FIG. 12b).

(84) Taken together, these results demonstrate that, in vitro, the pyrrolidinium-vortioxetine and histidine-vortioxetine trigger a fast differentiation of satellite cells similar to vortioxetine.

(85) Importantly, when assessing the self-renewal of satellite cells, it was observed that both pyrrolidinium-vortioxetine and histidine-vortioxetine displayed more Pax7+ cells 14 days post-plating (with medium changed every 4 days). Indeed, at the end of the differentiation process in vitro, 2±0.4% of cells were Pax7+ with PBS against 10.5±0.65% with vortioxetine, 12.5±1.9% with pyrrolidinium-vortioxetine (0.0.01), and 12.25±1.3% with histidine-vortioxetine (p≤0.05). This indicates that the number of self-renewing cells in vitro is higher with pyrrolidinium-vortioxetine and histidine-vortioxetine when compared with vortioxetine alone (FIGS. 12a,b; 20% increase). 20 mg/Kg Histidine-vortioxetine or Pyrrolidinium-vortioxetine was injected IP for 12 days in TgPax7nGFP mice: a doubling in the number of SCs was observed in the TA. These results indicates that those 2 derivatives have the same effect compared with vortioxetine (FIG. 12c,d).

3. Discussion

(86) Antidepressant drugs of the selective serotonin reuptake inhibitor (SSRI) class (e.g., fluoxetine) are commonly used to treat a wide spectrum of mood disorders (Marsella et al., 1975). Interestingly, the use of fluoxetine to improve regeneration of other organs than brain has never been assessed.

(87) The present results show that the administration of not only fluoxetine, but also of vortioxetine (a well-known atypical antidepressant), increased the number of vessels and, most importantly, of satellite cells in skeletal muscle. These results were confirmed using different approaches (genetics and immunostaining), routes of administration (intraperitoneal and per os) and different concentrations. These data were further confirmed ex vivo with more vessels invading the Matrigel plugs in fluoxetine treated mice. In vitro, the human endothelial cells HUVEC displayed more divisions when plated in plasma of mice treated with fluoxetine, when compared with plasma coming from placebo treated mice. Primary human satellite cells also displayed differentiation at early time points and higher self-renewal potential at late time points post-plating.

(88) Crucially, fluoxetine or vortioxetine treated animals displayed a faster regeneration when injured, and this was sustainable through multiple rounds of injury. The faster regeneration was assessed both at the genetic (Myogenin expression) and histological level. Besides, the regeneration was faster with vortioxetine compared to fluoxetine (2 weeks instead of 6 weeks). When looking for the mechanism of action that could be involved in this improved regeneration, the speed of activation did not seem to bet involved, as MyoD was detected at the same time points both in vivo and in vitro in the treated and control mice. Instead, the initial number of satellite cells was almost doubled in vivo. In vitro, the satellite cells displayed more cell divisions and a faster differentiation when plated in fluoxetine treated mice plasma.

(89) When assessing the self-renewal of satellite cells, it was further observed that both pyrrolidinium-vortioxetine and histidine-vortioxetine displayed better effect in vitro than vortioxetine alone 14 days post-plating. At early time points post-plating (4 days), it was observed that both pyrrolidinium-vortioxetine and histidine-vortioxetine displayed faster differentiation.

(90) It is also shown that the delivery of fluoxetine or vortioxetine was triggering an exit of quiescence of the satellite cells. Indeed, more cells were detected after 6 weeks treatment and as these were BrdU+, it meant that those extra cells come from division of the existing cells (self-renewal). This could be at the root of the faster regeneration of muscle after an injury. Indeed, it is possible that the higher number of satellite cells explains the faster differentiation however their faster activation and division rate upon injury in vitro is more likely to explain the faster differentiation. This could mean that satellite cells, although quiescent, exit their dormant state and activate faster upon injury or when in need of activation.

(91) The in vivo and in vitro data displayed herein are thus showing a 2 steps mode of action that could have different mechanisms depending on the cellular, physiological or pathological state. When dormant, the satellite cells could be activated by fluoxetine or vortioxetine or any 5-HT1B stimulation that would trigger their self-renewal in a controlled and limited manner. After the cellular activation, at one point in the cascade of differentiation the 5-HT1BR activation could trigger faster differentiation (either because the cell density is higher, or because, as showed, the direct activation of the 5-HT1B receptor triggers differentiation (not exclusive hypothesis)). Thus, the 5-HT1BR stimulation triggers exit of dormancy of satellite cells and increases cell rate division, self-renewal in normal physiological conditions but also differentiation when needed.

(92) Delivery of fluoxetine also increased the levels serotonin receptors 5-HT1 BR by 90 fold in the endothelial and SC cells, and pharmaceutical delivery of 5-HT1 BR inhibitor cancelled the effects of fluoxetine, both on the increase of vessels and satellite cells number. In vitro, the fluoxetine increased the speed of differentiation and the number of reserve cells, while the addition of the inhibitor canceled those effects. This is a key point, as it shows that fluoxetine, or metabolites found in plasma generated in vivo by fluoxetine administration, can directly act on the SC by targeting the 5-HT1 B receptor.

(93) The inhibition of 5-HT2 BR did not affect the results, indicating that the effect of fluoxetine from plasma treated mice or directly mediated by vortioxetine acts specifically on the 5-HT1 B receptor.

(94) Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein (Emery, 2002). The structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress, and together with important ischemia, it generates myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Due to these characteristics targeting both vessels (ischemia) and satellite cells (muscle regeneration) could be of main interest.

(95) After delivery of fluoxetine to dystrophic mice (Mdx), great variations in the muscle fiber size and centronucleated fibers were still observed but the number of necrotic foci dramatically decreased and the overall diameter of fibers was higher. By Luminex® assay, it was also showed that the global levels of cytokines, a well-known readout of inflammation in Mdx mouse, decreased. This was associated with a diminution of the number of infiltrated inflammatory cells.

(96) Taken together, those results indicate that the delivery of fluoxetine, vortioxetine, or derivatives thereof, to dystrophic patients has the potential to decrease the progression of the dystrophy, by increasing muscle regeneration, more particularly the number of muscle stem cells (in addition to vessels). The present data show that this effect is directly mediated via the 5-HT1 B receptor which is expressed both on endothelial cells and muscle stem cells.

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