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Wnt7a treatment ameliorates muscular dystrophy
Associated Data
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.
Duchenne muscular dystrophy (DMD) is a degenerative disorder characterized by muscle weakness and fragility. It is caused by mutations in the X-linked dystrophin gene, affecting 1 in 3,500 newborn males. Dystrophin deficiency results in the disruption of the dystrophin–glycoprotein complex, preventing binding of the actin cytoskeleton to the extracellular matrix. This leads to tearing of the muscle fibers during contraction, resulting in muscle damage, especially in fast fibers (1, 2). The muscle itself cannot compensate for this immense structural damage, ultimately leading to loss of muscle fibers, increased fibrosis, and reduced force generated by the muscle (3).
Satellite stem cells represent a small subpopulation of satellite cells capable of self-renewal and long-term reconstitution of the satellite cell niche after transplantation (4). Previously, we found that the noncanonical Wnt receptor Fzd7 is specifically expressed in satellite stem cells. Recombinant Wnt7a protein dramatically stimulates the symmetric expansion of satellite stem cells, a process requiring both Fzd7 and Vangl2, components of the planar cell polarity signaling pathway. Overexpression of Wnt7a during muscle regeneration results in impressive enhancement of the regeneration process, generating fibers of larger caliber (5). In recent studies, we observed that binding of Wnt7a to Fzd7 directly activates the AKT/mammalian target of rapamycin (mTOR) pathway, thereby inducing myofiber hypertrophy. Notably, association of Fzd7 with PI3kinase is required for Wnt7a to activate the anabolic AKT/mTOR pathway. Wnt7a/Fzd7-mediated induction of this pathway is entirely independent of IGF (Insulin-like growth factor-1) receptor activation (6).
Our experiments have established that Wnt7a/Fzd7 signaling acts at two levels to regulate muscle homeostasis. Wnt7a activity couples muscle growth with stem cell expansion, leading to productive hypertrophy. The ability of Wnt7a to drive these two different pathways in skeletal muscle suggests that Wnt7a may be used as a therapeutic approach to stimulate regeneration of dystrophic muscles in DMD.
Results and Discussion
We first asked whether Wnt7a is able to induce hypertrophy in dystrophic muscles of mdx mice. The mdx mouse strain displays critical hallmarks of the human form of DMD, including high susceptibility to contraction-induced damage and increased muscle degeneration and regeneration (7–9). Extensor digitorum longus (EDL) muscles from mdx mice electroporated with 10 μg of Wnt7a expression plasmid exhibited a 25% increase in muscle wet weight (n = 7; P < 0.01), compared with a 45% increase in WT mice (n = 7; P < 0.004) (Fig. 1A). Concomitant with this increased muscle mass, electroporation with Wnt7a expression plasmid resulted in 44% and 38% increases in the minimal fiber feret of EDL muscles from WT and mdx mice, respectively (n = 4, P < 0.001) (Fig. 1B), whereas electroporation itself had no effect on muscle weight or minimal fiber feret (Fig. S1 C and D).
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.)
Our previous studies have established that Wnt7a induces productive repair and growth by stimulating both satellite cell expansion and true myofiber hypertrophy. An unanswered question was whether or not the Wnt7a-treated muscles generate higher specific forces than control muscles. To address this question, we measured the force generated by EDL muscles from WT mice at the physiological temperature of 37 °C. We measured the specific force in Newtons per square centimeter (i.e., the force per cross-sectional area), because it is independent of muscle mass. We measured the specific tetanic force at 200 Hz, which represents the maximum force that a muscle can generate. The peak tetanic force of WT EDL muscles that had been electroporated with a Wnt7a expression plasmid was 1.2-fold greater than that of control muscles that had been electroporated with a control plasmid (n = 7; P < 0.05) (Fig. S1A). We also measured the specific peak twitch force, which is the response of muscle to a single stimulus. Notably, EDL muscles electroporated with the Wnt7a plasmid also exhibited a 3.4-fold greater force than EDL muscles electroporated with the control plasmid (n = 6; P < 0.01) (Fig. S1B). These results suggest that Wnt7a not only increases muscle mass, but also leads to increased force generated by the muscle independent of muscle mass.
To investigate whether Wnt7a also increases the force generated by muscles from mdx mice, we measured the peak tetanic and twitch forces of EDL muscles from mdx mice after electroporation with a Wnt7a expression plasmid. Given that muscles of mdx mice are unstable at 37 °C, force measurements for these muscles were performed at 25 °C. Measurements using muscles from WT mice were repeated under these conditions to allow comparison of data from mdx and WT muscles. Importantly, when electroporated with the Wnt7a expression plasmid, EDL muscles from mdx mice had a 1.9-fold greater peak tetanic force (n = 6; P < 0.01) (Fig. 1C), which was in fact nearly the same force exhibited by untreated WT EDL muscles. We also detected a 25% greater force in WT EDL muscles after electroporation with the Wnt7a expression plasmid measured under these conditions (n = 6; P < 0.01) (Fig. 1C), whereas electroporation itself had no effect on the peak tetanic or peak twitch force (Fig. S1 E and F). Furthermore, we found that a single 2.5-μg injection of Wnt7a protein led to increased levels of phospho-AKT (pAKT) in adult muscle fibers from mdx mice (Fig. 1E), suggesting that Wnt7a induces hypertrophy in muscles from mdx mice through the AKT/mTOR pathway, as has been reported for WT muscles (6).
Muscles from mdx mice with a higher slow-twitch fiber content are less susceptible to contractile damage (10). Therefore, we investigated whether Wnt7a treatment leads to a shift in fiber types potentially reducing the amount of contraction induced damage in mdx mice. We generated a force-frequency curve by stimulating the EDL muscles with frequencies of 1 Hz up to 200 Hz and plotting the force generated as a percentage of the maximal force generated at 200 Hz. Muscles with a higher content of slow-twitch fibers generally show a leftward shift of the curve. Of note, electroporation of EDL muscles from both WT and mdx mice resulted in a leftward shift in the force-frequency curve (n = 5; P < 0.05) (Fig. 2 A, B), supporting the notion of a shift in fiber types toward an increased amount of slow-twitch fibers after Wnt7a electroporation. Importantly, electroporation itself did not cause a shift in the force-frequency curve in WT or mdx muscles (Fig. S1 G and H). To further investigate the basis of this shift, we performed fiber typing analysis of EDL muscles electroporated with the Wnt7a expression plasmid. We observed a 3-fold increase in the amount of MHC class I fibers and a 1.3-fold increase in the amount of MHC class IIA fibers in WT muscles (Fig. 2 C and E). This is in agreement with the shift of the force-frequency curve toward lower frequencies for muscles treated with Wnt7a. Wnt7a-treated mdx EDL muscles had 1.7-fold more fibers expressing MHC class IIA, with less fibers expressing MHC class IIB (Fig. 2D).
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.
To identify the pathway through which Wnt7a induces the switch in fiber types, we investigated myotubes generated from satellite cell-derived primary myoblasts. Application of Wnt7a recombinant protein at day 3 of differentiation resulted in a significant 1.8-fold increase in the amount of slow MHC-positive myotubes compared with control conditions (Fig. 2F and Fig. S2F). This was accompanied by a decrease in the percentage of fast MHC-positive myotubes. This shift in fiber types was also demonstrated by mRNA level, with increased mRNA levels seen for MHC class I (MYH7; 1.2-fold), MHC class IIa (MYH2; 2.2-fold), and MHC class IIx (1.6-fold) (Fig. S2B), but no change for fetal MHC (MYH8), suggesting that Wnt7a does not lead to premature differentiation, as reported previously (6).
Wnt7a treatment was found to stimulate an increase in the levels of Mef2C protein (Fig. 2G) and Mef2C mRNA (a 1.7-fold increase) (Fig. S1A). Using an shRNA-mediated knockdown of Mef2C, we found that the Wnt7a-mediated fiber-type switch is Mef2C-dependent (Fig. 2H). Knockdown of Mef2C (Fig. S2 C and D) led to a loss of the Wnt7a-dependent increase in the percentage of slow MHC-positive myotubes (Fig. 2H). Notably, Wnt7a-induced hypertrophy was still observed in the absence of Mef2C expression (Fig. S2E). Thus, we conclude that Wnt7a induces a shift in fiber types by acting through Mef2C.
Dystrophic muscles with a higher proportion of slow fibers are less prone to contraction-induced damage (1, 2). After a single injection of recombinant Wnt7a protein into the tibialis anterior (TA) muscle of mdx mice, we quantified the percentage of IgG-positive fibers as a measure of damage (11). In Wnt7a-injected muscles, we found a six-fold decrease in the amount of damaged fibers (n = 4; P < 0.05) (Fig. 3 A–C), suggesting protection of these muscles from contraction-induced injury. Notably, this decrease in damaged fibers was not accompanied by an increase in the amount of revertant fibers (Fig. S3). In addition, Wnt7a injection reduced the number of fibers with centrally located nuclei by half (n = 4; P < 0.01) (Fig. 3D). Remarkably, Wnt7a also stimulated a 2.3-fold increase in satellite cells (n = 4; P < 0.001) (Fig. 3 E–I), providing the increased number of stem cells required for regeneration of damaged fibers, whereas the number of myoblasts remained unchanged after Wnt7a injection (Fig. 3J).
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.
Patients with DMD differ from mdx mice in several respects, including the proliferation potential and hence regenerative capability of satellite cells. Given the significantly decreased satellite cell proliferation in DMD patients compared with mdx mice, we believe that increasing the self-renewal of satellite cells in DMD patients would be beneficial. In this context, it also would be of interest to investigate whether Wnt7a has an effect on the telomere length of satellite cells in DMD patients, in light of the differences in telomere length between mice and humans, which have been attributed to this discrepancy.
To investigate whether the role of the Wnt7a/Fzd7 signaling pathway in skeletal muscle is conserved between human and mouse, we analyzed two independent preparations of human primary myoblasts from healthy male donors for responsiveness to Wnt7a. Application of human Wnt7a to myotubes derived from human primary myoblasts resulted in 2-fold and 1.7-fold increases (P < 0.001) in myotube diameters (Fig. 4A). Concomitant with this increase in myotube size was activation of the AKT/mTOR pathway, indicated by 75% and 65% increases in pAKT and phospho-S6 levels, respectively (Fig. 4 B and C). Furthermore, Wnt7a similarly stimulated a shift in fiber types in human myotubes, resulting in 2-fold and 1.7-fold increases in the degree of slow MHC-positive fibers (Fig. 4 D and E). Thus, we conclude that the function of the Wnt7a/Fzd7 signaling pathway in skeletal muscle is conserved between humans and mice.
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.)
The ultimate goal of therapy for diseases such as DMD is gene replacement; however, this remains a challenging problem, with many obstacles remaining on the path to the clinic (12). The most common cause of death in DMD patients is respiratory failure owing to loss of muscle strength in the muscles essential for respiration, leading to respiratory insufficiency and such complications as pneumonia (13). Thus, increasing muscle strength by treating discrete muscle groups, such as those involved in respiration, is an important therapeutic approach to consider. Our experiments provide compelling evidence that Wnt7a treatment counteracts the significant hallmarks of DMD, including muscle weakness, making Wnt7a a promising candidate for development as an ameliorative treatment for DMD.
Materials and Methods
Electroporation and Protein Injection into Skeletal Muscle.
Electroporation and protein injections were carried out as described previously (6), with the following modifications. For electroporation of EDL muscles, 10 μg of plasmid were used. Protein injections into the TA muscle were performed using 2.5 μg of Wnt7a protein (R&D Systems). Muscles were analyzed at 3 wk after treatment.
Mice.
This study used 10-wk-old male C57/BL10 mice obtained from Charles River Laboratories or 10-wk-old male mdx mice bred in our animal facility. All experiments were performed in accordance with University of Ottawa guidelines for animal handling and animal care determined by the University of Ottawa Animal Care Committee.
Force Measurements.
Force measurements were conducted as described previously (14). In brief, EDL muscles were constantly immersed in physiological saline solution containing 118.5 mM NaCl, 4.7 mM KCl, 2.4 mM CaCl2, 3.1 mM MgCl2, 25 mM NaHCO3, 2 mM NaH2PO4, and 5.5 mM d-glucose. All solutions were continuously bubbled with 95% O2–5% CO2 (vol/vol) and maintained at a pH of 7.4. All experiments were carried out at 37 °C, except dystrophic muscle measurements, which were performed at 25 °C.
Contractions were elicited by passing a current between two platinum electrodes located on opposite sides of the muscle. Twitch contractions were elicited with a single 0.3-ms square pulse of 10 V (supramaximal voltage), whereas tetanic contractions were elicited with a 200-ms train of the same pulse at frequencies of 10–200 Hz. Contractions were elicited every 2 min during the experiment.
Muscle length was adjusted to obtain maximum tetanic force, and a 30-min equilibrium period was allowed before any force-frequency measurements. Force was measured with a dual-mode muscle lever system (Aurora model 300C) and digitized at 5 kHz with a analog-digital board (Keithley model KCPI3104). Peak twitch and tetanic force were calculated as the difference between the maximum force during contraction and the force measured at 5 ms before the contraction.
Cell Culture and Transfection.
Mononucleated muscle-derived cells were isolated from hind-limb muscles of Balb/C mice (4 wk old), cultured, and differentiated as described previously (6). Cells were sorted by FACS and cultured as described previously (5). Recombinant Wnt7a (R&D Systems) was administered at a final concentration of 50 ng/mL. Primary myoblasts were transfected with a lentivirus encoding for shRNA against Mef2C or control shRNA.
Protein and RNA Analyses.
Western blot and immunofluorescence analyses were performed as described previously (15). RNA purification and quantitative RT-PCR were carried out as described previously (6). Antibodies used are listed in Table S1, and primers are listed in Table S2.
Statistical Analyses.
A minimum of three and a maximum of seven replicates were analyzed for each experiment presented. Data are shown as SEM. Statistical significance was assessed by the Student t test, using Microsoft Excel. Differences with a P value < 0.05 were considered significant.
Acknowledgments
We thank C. Florian Bentzinger for amplification of the lentivirus used in this study. M.A.R. holds the Canada Research Chair in Molecular Genetics. This work was supported by grants from the Muscular Dystrophy Association, Canadian Institutes of Health Research, National Institutes of Health, Howard Hughes Medical Institute, Canada Research Chair Program, and Ontario Research Fund, and with support from Fate Therapeutics.
Footnotes
Conflict of interest statement: M.A.R. is a founding scientist with Fate Therapeutics, who is developing Wnt7a as a therapeutic agent.
*This Direct Submission article had a prearranged editor.
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