Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Sep;185(9):2495-504.
doi: 10.1016/j.ajpath.2015.05.007. Epub 2015 Jul 21.

Selective Retinoic Acid Receptor γ Agonists Promote Repair of Injured Skeletal Muscle in Mouse

Agnese Di Rocco  1 , Kenta Uchibe  1 , Colleen Larmour  1 , Rebecca Berger  1 , Min Liu  2 , Elisabeth R Barton  3 , Masahiro Iwamoto  4
Affiliations

Selective Retinoic Acid Receptor γ Agonists Promote Repair of Injured Skeletal Muscle in Mouse

Agnese Di Rocco et al. Am J Pathol. 2015 Sep.

Abstract

Retinoic acid signaling regulates several biological events, including myogenesis. We previously found that retinoic acid receptor γ (RARγ) agonist blocks heterotopic ossification, a pathological bone formation that mostly occurs in the skeletal muscle. Interestingly, RARγ agonist also weakened deterioration of muscle architecture adjacent to the heterotopic ossification lesion, suggesting that RARγ agonist may oppose skeletal muscle damage. To test this hypothesis, we generated a critical defect in the tibialis anterior muscle of 7-week-old mice with a cautery, treated them with RARγ agonist or vehicle corn oil, and examined the effects of RARγ agonist on muscle repair. The muscle defects were partially repaired with newly regenerating muscle cells, but also filled with adipose and fibrous scar tissue in both RARγ-treated and control groups. The fibrous or adipose area was smaller in RARγ agonist-treated mice than in the control. In addition, muscle repair was remarkably delayed in RARγ-null mice in both critical defect and cardiotoxin injury models. Furthermore, we found a rapid increase in retinoid signaling in lacerated muscle, as monitored by retinoid signaling reporter mice. Together, our results indicate that endogenous RARγ signaling is involved in muscle repair and that selective RARγ agonists may be beneficial to promote repair in various types of muscle injuries.

PubMed Disclaimer

Figures

Supplemental Figure S1
Supplemental Figure S1
Chemical structures of retinoids used in this study.
Supplemental Figure S2
Supplemental Figure S2
Retinoic acid receptor γ (RARγ) agonists block fibrodysplasia ossificans progressiva (FOP)–like heterotopic ossification and skeletal muscle deterioration. A: X-ray images of hind legs of FOP-like model mice 1 month after induction of mutant activin receptor (ALK2Q207D) treated with vehicle or 4 mg/kg per gavage of RARγ agonist CD1530. Massive heterotopic ossification (HO) forms in control vehicle–treated mice but not in RARγ-treated mice. B: Transverse sections at the indicated level (yellow lines in A) in the X-ray images were stained with Masson's trichrome (MT). Skeletal muscle tissue severely distorts around HO in the vehicle-treated control mice, whereas deterioration of muscle tissues is much milder in the RARγ-treated group. Parallel sections (OC/Lami/DAPI) were stained for osteocalcin (OC; green), laminin (Lami; red), and nucleus (DAPI; blue). Scale bars: 5 mm (B, top panels); 250 μm (B, middle and bottom panels).
Supplemental Figure S3
Supplemental Figure S3
Histology of longitudinal sections of injured muscles after critical defect muscle injury. Critical defect muscle injury surgery was performed in 8-week-old CD1 mice. Injured muscles were harvested 30 days (A–D) or 60 days (E) after surgery. A–D: Longitudinal sections of injured muscles were stained with Masson's trichrome. Muscle, adipose, and fibrous tissues were stained in red, white, and blue, respectively. Muscle (B), adipose (C), and fibrous (D) tissues were separately selected using Image-Pro software version 7.0. Each tissue was highlighted in green. E: Longitudinal sections stained with hematoxylin and eosin. The initial defect regions are not fully repaired even after 2 months in three independent samples (controls 1 to 3). Scale bars: 0.1 mm (A–E).
Supplemental Figure S4
Supplemental Figure S4
The effects of various retinoic acid receptor γ (RARγ) agonists on muscle repair. Critical defect muscle injury surgery was performed in 7-week-old female CD1 mice. Mice were then randomly divided and treated with vehicle or selective RARγ agonists (BMS270394; 4 mg/kg per gavage), R667 (palovarotene; 4 mg/kg per gavage), and CD437 (4 mg/kg per gavage) on days 8, 10, and 12 after surgery. Muscle tissues were collected from vehicle (control), R667-treated, and CD1530-treated mice 30 days after critical defect injury surgery. Macro views of three independent samples (top panels) and representative Masson's trichome staining image of the transverse section (bottom panels) are shown in each group. The muscle injury sites are clearly visible as whitish spots in control, whereas the defect sites of RARγ agonist–treated samples are less apparent. n = 3.
Supplemental Figure S5
Supplemental Figure S5
Changes in distribution of phosphorylated Smad1/5/8 during muscle repair in the critical defect muscle injury model. Critical defect muscle injury surgery was performed in 8-week-old CD1 mice. Transverse sections of uninjured muscles (A and F) and injured muscles harvested 2 (B, G, and K), 5 (C, H, and L), 14 (D, I, and M), and 30 (E, J, and N) days after surgery were stained with anti–phospho-Smad1/5/8. F–N are magnified images of the indicated areas in A–E. Arrowheads in F indicate phospho-Smad1/5/8–positive satellite cells. Arrows in G indicate coagulated area. Arrowheads in M and N indicate phospho-Smad1/5/8–positive immature muscle fibers with central nuclei. Scale bars: 100 mm (A–E); 50 mm (F–N).
Figure 1
Figure 1
Critical defect muscle injury model. A: Macro images of surgical procedure for critical muscle defect injury. Tibialis anterior (TA) muscle was exposed after removal of hair and skin incision. A round defect (1.5 × 1.5 mm) was then generated by a thermal cautery, and the skin was closed by suture. B–M: Histology of injured muscle after surgery. Transverse sections of injured TA muscles were prepared 2 (B, F, and J), 5 (C, G, and K), 14 (D, H, and L), and 30 (E, I, and M) days after surgery and stained with hematoxylin and eosin. F–M: Magnified images of the boxed areas in B–E. Arrows in F indicate coagulated area. Scale bars: 100 μm (B–E); 50 μm (F–M).
Figure 2
Figure 2
The effects of retinoic acid receptor γ (RARγ) agonists on muscle repair in a critical defect muscle injury model. Critical defect muscle injury surgery was performed in 7-week-old female CD1 mice (six mice per group). Mice were then randomly divided and treated with vehicle, 4 mg/kg per gavage CD1530, or 4 mg/kg per gavage R667 on days 5, 7, and 9 after surgery. Muscle tissues were collected from vehicle (Control)-, R667-, and CD1530-treated mice 30 days after critical defect injury surgery. A: Representative images of transverse sections of muscles. Images that contain the largest defect were determined by visual inspection of serial sections. Three independent samples from three separated mice per group are shown. B: Representative serial transverse sections (every five slides) of muscles. The number in the upper corner indicates slide number. The interval of every five slides is approximately 1 mm. The muscle defects were mostly replaced with the mixture of fibrous and adipose tissues. The area occupied with fibrous or adipose tissue was smaller in the RARγ agonist-treated sample than that of control. We repeated the experiments three times and obtained similar results. Scale bar = 1 mm (B).
Figure 3
Figure 3
Histological analysis of injured muscles treated with retinoic acid receptor γ agonists. Muscles collected from vehicle (Control)-, R667-, and CD1530-treated mice in Figure 2 were subjected to Masson's trichrome staining, followed by histological analysis. A: Image of Masson's trichrome (MT) staining. Muscle, adipose, and fibrous tissues were stained in red, white, and blue, respectively. B–D: Muscle (B), adipose (C), and fibrous (D) tissues were separately selected using Image-Pro software version 7.0. Each tissue was highlighted in green. E: Composition of adipose, muscle, and fibrous tissues in uninjured muscle (Control) or injured muscles treated with vehicle (Vehicle), CD1530, or R667. We analyzed three transverse sections covering the most severely damaged region (200-μm distance) and obtained an average value of composition for each sample. Uninjured control muscle (five mice per group) and injured muscles (six mice per group) were analyzed. Values are means ± SD (E). ∗P < 0.05. Scale bar = 0.5 mm (A–D).
Figure 4
Figure 4
The effects of retinoic acid (RA) and RA receptor α (RARα) agonist on muscle repair. A and B: Critical defect muscle injury surgery was performed in 7-week-old female CD1 mice (four mice per group). Mice were then randomly divided and treated with vehicle, 4 mg/kg per gavage RA, or 12 mg/kg per gavage RARα agonist (NRX195183) on days 5, 7, and 9 after surgery. A: Injured tibialis anterior (TA) muscles of vehicle, RA, and RARα agonist groups were harvested 30 days after injury and stained with Masson's trichrome. B: Compositions of adipose, muscle, and fibrous tissues were analyzed. C: Critical defect muscle injury surgery was performed in wild-type (WT) and RARγ-null mice. Sections of injured muscles were prepared 30 days after surgery and stained with Masson's trichrome. Similar results were obtained in two independent experiments. D: WT and RARγ-null mice (three mice per group) received cardiotoxin injection in TA muscles. Sections of TA muscles were prepared 2 weeks after injection and stained with Masson's trichrome. Muscle repair process was remarkably delayed in RARγ-null mice. Similar results were obtained in two independent experiments. Values are means ± SD of four samples (B). n = 3 (C). Scale bars: 0.5 mm (A, C, and D).
Figure 5
Figure 5
Up-regulation of local retinoid signaling after muscle injury. Lacerated muscle injury surgery (injured muscle) or sham surgery (only skin incision, sham operation) was performed in RARE-LacZ mice (three mice per group), as described previously. Muscles were collected 1, 4, and 7 days after surgery and subjected to LacZ staining. Similar results were obtained in all three mice.
Figure 6
Figure 6
Gene expression of retinoic acid metabolism–related molecules and receptors in injured and intact skeletal muscle. Lacerated muscle injury surgery (injury+, black column) or sham surgery (only skin incision, injury−, white column) was performed in 7-week-old CD1 mice (four mice per group). Muscles were harvested 8 hours and 2, 4, 11, 18, or 25 days after surgery (A) or 4 days after surgery (B and C). Total RNAs were prepared from the muscles, reverse transcribed, and subjected to real-time PCR (A) or conventional PCR (B and C). A: Changes of gene expression of retinoic acid (RA) metabolism–related enzymes after muscle injury. Graph shows relative expression levels of indicated genes to sham-operated on sample: Aldh1a1, Aldh1a2, and Aldh1a3 encode enzymes for RA biosynthesis; Crabp1 encodes an RA-binding protein; Cyp26a1 and Cyp26b1 encode enzymes that metabolize RA. B: PCR products generated with the primers that were used for real-time PCR. The results indicate specificity of these primers. C: PCR analysis of gene expression of RA receptors (RARs) in uninjured (injury−) and injured muscle (injury+). Values are means ± SD of four samples (A). d, days; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hours; RXR, retinoid X receptor.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Close G.L., Kayani A., Vasilaki A., McArdle A. Skeletal muscle damage with exercise and aging. Sports Med. 2005;35:413–427. - PubMed
    1. Aarimaa V., Rantanen J., Heikkila J., Helttula I., Orava S. Rupture of the pectoralis major muscle. Am J Sports Med. 2004;32:1256–1262. - PubMed
    1. McCarthy E.F., Sundaram M. Heterotopic ossification: a review. Skeletal Radiol. 2005;34:609–619. - PubMed
    1. Rantanen J., Thorsson O., Wollmer P., Hurme T., Kalimo H. Effects of therapeutic ultrasound on the regeneration of skeletal myofibers after experimental muscle injury. Am J Sports Med. 1999;27:54–59. - PubMed
    1. Almekinders L.C. Anti-inflammatory treatment of muscular injuries in sport: an update of recent studies. Sports Med. 1999;28:383–388. - PubMed

Publication types

MeSH terms