Wnt7a is required for regeneration of dystrophic skeletal muscle

Abstract

Intramuscular injection of Wnt7a has been shown to accelerate and augment skeletal muscle regeneration and to ameliorate dystrophic progression in mdx muscle, a model for Duchenne muscular dystrophy (DMD). Here, we assessed muscle regeneration and function in wild type (WT) and mdx mice where Wnt7a was deleted in muscle using a conditional Wnt7a floxed allele and a Myf5-Cre driver. We found that both WT and mdx mice lacking Wnt7a in muscle, exhibited marked deficiencies in muscle regeneration at 21 d following cardiotoxin (CTX) induced injury. Unlike WT, deletion of Wnt7a in mdx resulted in decreased force generation prior to CTX injury. However, both WT and mdx muscle lacking Wnt7a displayed decreased force generation following CTX injection. Notably the regeneration deficit in mdx mice was rescued by a single tail vein injection of extracellular vesicles containing Wnt7a (Wnt7a-EVs). Therefore, we conclude that the regenerative capacity of muscle in mdx mice is highly dependant on the upregulation of endogenous Wnt7a following injury, and that systemic delivery of Wnt7a-EVs represents a therapeutic strategy for treating DMD.

Keywords: Duchenne muscular dystrophy; Regeneration, Wnt7a, Extracellular vesicles; Skeletal muscle.

Fig. 1

Lack of Wnt7a expression impairs muscle regeneration. (A-left) H&E stained sections of injured and non-injured TA muscle of mdx mice at 3 dpi. (A-right) Wnt7a immunostained (green) sections of injured and non-injured TA from mdx mice upon 3 dpi. Laminin (red) delineates myofibers. Nuclei are stained with DAPI. (B) Mouse strains used and experimental design. (C and D) Quantification of Pax7-expressing cells in regenerating WT muscle. Scale bar 50 μm. (E and F) Quantification of Pax7-expressing cells in regenerating mdx TA muscle. Scale bar 50 μm. (G and H) Myofiber caliber distribution at 21 d following regeneration in WT and mdx TA muscle with and without Wnt7a deletion. (I and J) H&E stained sections at 21 d following regeneration in WT and mdx TA muscle with and without Wnt7a deletion. Scale bar 100 μm. TA (Tibialis Anterior), dpi (days post injury). n ≥ 4 mice, mean ± s.e.m., p values determined by two-sided Student’s t-test (*p<0.05,**p < 0.01, n.s.=not significant)

Fig. 2

Reduced force generation in regenerated TA muscles lacking Wnt7a. (A) Schematic of mouse strains used and experimental design. (B) Specific force frequency curve in uninjured WT TA muscle with (dark blue) and without (light blue) Wnt7a expression. (C) Specific force frequency curve in uninjured mdx TA muscle with (dark green) and without (light green) Wnt7a expression. (D) Specific force frequency curve of regenerated WT TA muscle, with (dark blue) and without (light blue) Wnt7a expression, at 21 d following injury. (E) Specific force frequency curve of regenerated mdx TA muscle, with (dark green) and without (light green) Wnt7a expression, at 21 d following injury. TA (Tibialis Anterior). n3 mice, mean ± s.e.m., p values determined by two-way ANOVA test (*p<0.05, **p < 0.01, ***p < 0.001, n.s.=not significant)

Fig. 3

Wnt7a-EVs exhibit enhanced bioactivity. (A) Schematic showing the experimental protocol for ex vivo EV treatment of single myofibers isolated from FDB muscles, imaging and analysis using high-content screening. (B) Representative immunofluorescence images of symmetric (top panel) and asymmetric (bottom panel) satellite stem cell divisions. (C) Rate of muscle stem cell division after 42 h in culture is not altered by treatment with either Wnt7a-EVs or with recombinant Wnt7a. (D) Symmetric satellite stem cell divisions are significantly increased by Wnt7a-EV treatment. EVs (extracellular vesicles), FDB (Flexor Digitorum fibers). n5 replicates, mean ± s.e.m., p values determined by two-sided Student’s test (*p<0.05, **p < 0.01, ***p < 0.001, n.s.=not significant)

Fig. 4

Systemic delivery of Wnt7a-EVs rescues muscle regeneration in mdx mice. (A) Schematic of experimental design. (B) Specific Force frequency curve of uninjured mdx TA muscle lacking Wnt7a, comparing untreated (light green), control-EV (dark green) injected, or Wnt7a-EV (red) injected. (C) Specific Force frequency curve of regenerated mdx TA muscle lacking Wnt7a, comparing untreated (light green), control-EV (dark green) injected, or Wnt7a-EV (red) injected. (D) Myofiber caliber distribution of regenerated mdx TA muscle lacking Wnt7a, comparing control-EV (dark green) injected or Wnt7a-EV (red) injected. (E) Wnt7a-EV treatment results in increased numbers of myofibers. (F) H&E stained sections of regenerated TA muscles after 21 dpi injected mice with control-EVs or Wnt7a-EVs. Scale bar 100 μm. (G) Quantification of fibrotic area with WGA staining vs. total area in diaphragms showed in % of injected mice with control-EVs or Wnt7a-EVs. (H) WGA immunostaining (red) of diaphragm sections from CTR-EV or Wnt7a-EV injected mdx mice lacking Wnt7a. Scale bar 500 μm. (I) Myofiber caliber distribution of diaphragms from mdx mice lacking Wnt7a injected with control-EVs or Wnt7a-EVs (J) Minimal fiber feret average in diaphragms of injected mice with control-EVs or Wnt7a-EVs. TA (Tibialis Anterior), dpi (days post injury. n4 mice, mean ± s.e.m., p values determined by two-way ANOVA or two-sided Student’s test (*p<0.05, **p < 0.01, ****p < 0.0001, n.s.=not significant)