RARγ Agonist Promotes Functional Restoration of Severely Injured Skeletal Muscle in Mouse

Min Liu1, Di Rocco Agnese2, Lily Wu1, Masatake Matsuoka1, Suzu Deie3, Motomi-Enomoto-Iwamoto1, Iwamoto Masahiro1 1Division of Orthopedics Surgery, Children Hospital of Philadelphia, Philadelphia, 2Dept of Cancer Biology, Thomas Jefferson University, Philadelphia, and

3Hiroshima University Medical School, Hiroshima Disclosures: Part of this research was supported by the research fund from Clementia Pharmaceuticals to MI. No disclosures for other authors. INTRODUCTION: Skeletal muscles can be damaged or injured by multiple causes including overuse, trauma, infections, or loss of blood circulation. When the damage is not severe, skeletal muscles repair themselves efficiently with major contribution from satellite cells that surround the muscle fibers. In contrast, repair of severely damaged muscles is much more challenging. It takes a long time, and often results in decrease of muscle functionality due to scar formation. We recently showed in mice that selective agonists for nuclear retinoic acid receptor γ (RARγ) blocks genetic or rhBMP-induced forms of heterotopic ossification (HO), a pathology involving ectopic bone formation at the expense of skeletal muscles(1). In the following study, we observed that the agonists reduced muscle degeneration around the HO. Furthermore, muscle injury repair was remarkably delayed in RARγ null mice (2). These observations suggest that activation of RARγ prevents muscle degeneration and/or promotes muscle repair. To follow up this intriguing observation, we investigated the effect of RARγ agonists on functional restoration of experimentally generated critical defect in T-A muscle in mice. METHODS: Animals and Drug treatment: CD1 mice (48-weeks of age) were randomly divided into four groups:1 month control, 1 month treated, 2 month control and 2 month treated groups (n=5). Control mice received vehicle corn oil and the treated mice received oral gavage of palovarotene, a selective RARg agonist (4 mg/kg) every other day during 2-4 weeks after surgery. Surgery: Critical muscle defect injury was generated by thermal cautery as described previously (Figure 1) (2). In situ assessment of TA muscle function: TA muscles were individually attached to the lever arm of a 305B dual-mode servomotor transducer. The sciatic nerve was exposed and all its branches cut except for the common peroneal nerve, which innervates the TA and EDL muscles. TA muscle contractions were then elicited by stimulating the distal part of the deep peroneal nerve via bipolar electrodes. Maximum isometric tetanic force of TA was determined at 150Hz for 500ms duration. The TA specific forces (N/cm2) were calculated by dividing Po by muscle cross-sectional area. Transplantation of mesenchymal stem cells: Muscle derived local mesenchymal cell like population (M-MSC) was obtained by digesting minced muscle tissue of P1-P5 mice with 0.1% collagenase. Bone marrow stroma cells (BMSCs) were isolated as described (1). Human adipose tissue derived MSC (Ad-MSC) was purchased from Zenbio(Cat#ASC-F). Cells were treated with 1mM CD1530 or 300 nM NRX204647 (RARγ agonists) or vehicle for three days before transplantation. Cells (1-2 x104 cells per site) were then transplanted into the muscle defect of 6 weeks old NOD SCID gamma mouse). Tissue was collected 2 weeks after operation and subjected to histological analyses. RESULTS: Histomorphometric analysis revealed that palovarotene treatment significantly increased muscle content and decrease adipose tissue at 1 month after surgery (Figure 1). By 2 months post-surgery, we observed a time-dependent increase in number of the neuromuscular junctions in healing muscle. Mechanical tests of TA muscles revealed that TA muscles of the drug treatment group showed 15-24% higher peak twitch tension, peak tetanic force and muscle specific force. To investigate RARγ target cells during muscle repair, we stained intact and experimentally injured muscle tissue by the antibodies against RARγ and TGM2 (a retinoid target). Interestingly, over 75% cells showed positive to the staining in the interstitial space, but few cells in the muscle fibers, suggesting that the targets of RARγ agonists are resident or circulating MSCs. To test this, we treated MSCs derived from muscle, bone marrow and adipose tissues for 3 days with RARγ agonists or vehicle in culture and transplanted into the muscle injury sites. Transplantation of the RARγ-pretreated BMSCs induced superior muscle healing compared to the non-treated BMSCs transplantation. The pre-treated were retained within or around the muscle defect even 2 weeks after transplantation, and some BMSCs were positive to myosin heavy chain. In contrast, the control BMSCs were barely detectable in the transplanted region. DISCUSSION: Present study shows that the injury model is indeed non-repairable at least for 2 months and that RARγ treatment significantly promoted functional restoration of injured TA-muscle. Histomorphometric and mechanical analysis revealed that functional restoration the critically injured muscle requires 1 month of regeneration phase and at least 1 month of the remodeling phase that are required to reestablish neuromuscular connection. Cell transplantation study suggests that RARγ signaling promotes muscle repair at least indirectly through MSC like cells in the damaged muscle tissue. Thus RARγ agonist might be useful in drug and MSC-based therapies for severely damaged skeletal muscles. SIGNIFICANCE: The research is conducted to explore an effective, easy to employ, and cost-effective therapy for severe muscle injury, one of most common types of traumatic. FUNDING: Supported by Muscular dystrophy association (MDA255541), NIAMS, NIH AR056837 and Clementia Pharmaceuticals. REFERENCES: (1) Shimono K et al., Pacifici M and Iwamoto M: Nat Med 2011, 17:454e460 (2) Di Rocco A et al. and Iwamoto M. Am J Pathol. 2015 Sep;185(9):2495-504.

Fig  2:  RARγ  agonist  treated  BMSC  retains  muscle  defect  injury  site  and  promote  repair. BMSCs  were  prepared  from  RFP  expressing  mouse  and  treated  with  vehicle  or  RARg  agonist  NRX204647  for  3  days.      Cells  were  then  applied  to  the  defect  injury  in  TA-­‐muscle  of  NOG  SCID  mouse. (A-­‐D)  Macro  image  of  DsRed-­‐MSCs.    (E,  F)  Distribution  of  DsRed  positive  cells  in  TA-­‐muscle  sections.    (G,  H)  Merged  images  with  DAPI  (blue)  and  MHC  (green)  staining  (Red).

Fig. 1 Muscle injury model (A)Tibialis anterior (TA) muscle was exposed by skin incision in 7-week old CD1 mice. A round-shape defect (1.5mmX1.5mm) was created by cautery and was closed by suture. (B)The injured muscle was filled with exudate and blood cells 2 days after surgery, and replaced by adipose and fibrous tissues 30 days after surgery.(C)R667(palovarotene) treatment strongly stimulated muscle repair.

ORS 2017 Annual Meeting Poster No.1618