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. 2012 Dec 11;109(50):20614-9.
doi: 10.1073/pnas.1215765109. Epub 2012 Nov 26.

Wnt7a treatment ameliorates muscular dystrophy

Julia von Maltzahn  1 , Jean-Marc Renaud, Gianni Parise, Michael A Rudnicki
Affiliations

Wnt7a treatment ameliorates muscular dystrophy

Julia von Maltzahn et al. Proc Natl Acad Sci U S A. .

Abstract

Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder of childhood marked by progressive debilitating muscle weakness and wasting, and ultimately death in the second or third decade of life. Wnt7a signaling through its receptor Fzd7 accelerates and augments regeneration by stimulating satellite stem cell expansion through the planar cell polarity pathway, as well as myofiber hypertrophy through the AKT/mammalian target of rapamycin (mTOR) anabolic pathway. We investigated the therapeutic potential of the secreted factor Wnt7a for focal treatment of dystrophic DMD muscles using the mdx mouse model, and found that Wnt7a treatment efficiently induced satellite cell expansion and myofiber hypertrophy in treated mucles in mdx mice. Importantly, Wnt7a treatment resulted in a significant increase in muscle strength, as determined by generation of specific force. Furthermore, Wnt7a reduced the level of contractile damage, likely by inducing a shift in fiber type toward slow-twitch. Finally, we found that Wnt7a similarly induced myotube hypertrophy and a shift in fiber type toward slow-twitch in human primary myotubes. Taken together, our findings suggest that Wnt7a is a promising candidate for development as an ameliorative treatment for DMD.

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Conflict of interest statement

Conflict of interest statement: M.A.R. is a founding scientist with Fate Therapeutics, who is developing Wnt7a as a therapeutic agent.

Figures

Fig. 1.
Fig. 1.
Wnt7a increases the specific force in dystrophic mice. (A) EDL muscles from WT and mdx mice were electroporated with a CMV-Wnt7a-HA expression plasmid. At 3 wk after electroporation, Wnt7a electroporated muscles are significantly heavier than control muscles. n = 7. **P < 0.01. (B) Wnt7a electroporated muscles display significantly larger fiber ferets compared with control electroporated muscles. n = 4. ***P < 0.001. (C) EDL muscles from WT mice electroporated with Wnt7a show a significantly higher peak tetanic force compared with control muscles. Electroporation of EDL muscles from mdx mice also significantly increases the tetanic force, nearly reaching the force of control WT mice. n = 6. **P < 0.01. (D) Electroporation of Wnt7a in EDL muscles of mdx mice significantly increases the maximal twitch force. Healthy animals electroporated with Wnt7a show a tendency toward increased twitch force. n = 5. *P < 0.05. (E) Injection of 2.5 μg of Wnt7a recombinant protein into TA muscles results in increased levels of pAKT (green). Nuclei are counterstained with DAPI (blue). Laminin staining is shown in red. (Scale bar: 50 μm.)
Fig. 2.
Fig. 2.
Wnt7a leads to a switch in fiber types. (A) The force-frequency curve of EDL muscles from WT mice electroporated with Wnt7a shows a significant shift, indicating changes in fiber composition of the electroporated muscles. n = 5. *P < 0.05. (B) Electroporation of EDL muscles from mdx mice results in a shift in the force-frequency curve, suggesting an increase in slow fibers compared with control muscles. n = 5. *P = 0.05. (C) Wnt7a leads to a switch in fiber types in EDL muscles of WT mice. n = 4. *P < 0.05. (D) Wnt7a shifts fiber types in EDL muscles from mdx mice. n = 4. *P < 0.05. (E) Representative images of immunostaining of TA muscles from WT mice stained with antibodies directed to MHC class IIa (green) and laminin (red). Nuclei are counterstained with DAPI (blue). (Scale bar: 100 μm.) (F) Primary myoblasts were differentiated for 3 d, Wnt7a recombinant protein was applied, and cells were differentiated for another 2 d. After immunostaining with antibodies directed to slow MHC or fast MHC, the amount of each fiber type in relation to all myotubes was evaluated. n = 5. ***P < 0.001; *P < 0.05. (G) Wnt7a treatment of primary myotubes results in increased Mef2C protein levels. (H) Knockdown of Mef2C inhibits the shift toward slower fibers mediated by Wnt7a. n = 5. ***P < 0.001; **P < 0.01.
Fig. 3.
Fig. 3.
Wnt7a ameliorates the muscle phenotype in mdx mice. (A) Injection of a single dose of recombinant Wnt7a into TA muscles of mdx mice leads to a decrease in IgG-positive fibers at 3 wk after injection. n = 4. *P < 0.05. (B and C) Representative images of IgG staining (green) and laminin (red) of TA muscles from mdx mice injected with recombinant Wnt7a (B) or BSA as a control (C). Nuclei are counterstained with DAPI (blue). (Scale bar: 250 μm.) (D) Wnt7a reduces the number of fibers with centrally located nuclei. n = 4. **P < 0.01. (E–H) Representative images of mdx muscles electroporated with a Wnt7a expression plasmid (E and F) or a control plasmid (G and H). Pax7 staining is shown in green (E and G); laminin staining, in red (F and H). Nuclei are counterstained with DAPI (blue). (Scale bar: 100 μm.) (I) Wnt7a increases the number of satellite cells (marked by Pax7 expression) of TA muscles from mdx mice. n = 4. ***P < 0.001. (J) Wnt7a increases the number of satellite cells marked by the expression of Pax7 (MyoD-positive and -negative), but does not increase the numbers of myoblasts (Pax7-negative, MyoD-positive). n = 4.
Fig. 4.
Fig. 4.
Wnt7a induces hypertrophy and leads to a switch in fiber types in human primary myotubes. (A) Application of Wnt7a recombinant protein at day 3 of differentiation results in hypertrophy of treated myotubes. Analysis of the myotubes from two independent healthy male donors was carried out at day 5 of differentiation. n = 4. ***P < 0.001. (B) Wnt7a activates the AKT/mTOR pathway in human primary myotubes. Shown are representative blots of myotubes generated from donor 1. (C) Densitometric analysis of immunoblots from primary myotubes treated with Wnt7a reveal a significant increase in pAKT, pS6, and slow MHC compared with control myotubes. Wnt7a did not change the levels of total MHC, suggesting that Wnt7a does not lead to precocious differentiation in human primary myotubes. n = 3. *P < 0.05; **P < 0.01. (D) Application of Wnt7a at day 3 of differentiation results in an increased number of slow MHC-positive myotubes compared with control conditions. The increase in the number of slow MHC-positive myotubes is concomitant with a decrease in the number of fast MHC-positive myotubes. Total numbers of myotubes were similar in Wnt7a-treated and control mice. n = 3. *P < 0.05; ***P < 0.001. (E) Representative images of immunostainings of Wnt7a-treated (Upper) and control (Lower) human primary myotubes. Myotubes were generated from primary myoblasts isolated from donor 1. Fast MHC (Left) and slow MHC (Right) are shown in green. Fetal MHC (red) served as a marker for myotubes in general. Nuclei are counterstained with DAPI (blue). (Scale bar: 100 μm.)
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