Genetic and Pharmacologic Inhibition of Myostatin Restores Muscle Mass in a Dynamin 2 ‐Related Centronuclear Myopathy Mouse Model

Figures

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  FIGURE 1. Postnatal skeletal muscle growth is impaired in KI mice. (a) Body weights (left) were similar in WT (n = 7–9) and KI (n = 7–11) mice at 1, 2 and 8 months (mo) of age. Tibia length (right) indicates similar growth rates between WT (n = 7–8) and KI (n = 8) mice. (b) Representative magnetic resonance images of leg muscle cross‐sectional area in 1‐, 2‐ and 8‐month‐old WT and KI mice. Scale bar equals 1 mm. (c) Quantification of magnetic resonance images (left) showed impaired skeletal muscle growth between 1 and 2 months of age in KI mice (n = 4–6) compared to WT mice (n = 5–6). Fold change in leg muscle cross‐sectional area (right) between 1 and 2 months of age (2 mo/1 mo) and 2 and 8 months of age (8 mo/2 mo). (d) Tibialis anterior muscle weight (left) in WT (n = 8) and KI (n = 8) mice at 1, 2 and 8 months of age. Fold change in tibialis anterior muscle weight (right) between 1 and 2 months of age (2 mo/1 mo) and 2 and 8 months of age (8 mo/2 mo). (e) The time spent on the rotarod was lower in KI mice (n = 9) compared to WT mice (n = 10). (f) Absolute (left) and normalized (right) muscle grip strength in KI mice (n = 9–10) and WT mice (n = 9–10). Data are means ± SD. (a left, c, e…

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  FIGURE 2. Histological abnormalities are associated with worsening muscle growth defect. (a) Representative images of a cross‐section of tibialis anterior muscle immuno‐stained with laminin (red) in WT and KI mice at the age of 1 and 2 months (mo). Scale bar equals 50 μm. (b) Quantification of muscle fibre cross‐sectional area in WT (n = 4–5) and KI (n = 4) mice. A mean of 537 ± 60 fibres per muscle was analysed. (c) Representative nicotinamide adenine dinucleotide tetrazolium reductase (NADH‐TR) staining and laminin immunostaining (red) of tibialis anterior muscle sections of 1‐ and 2‐month‐old KI mice. Scale bar equals 50 μm. (d) The proportion of fibres displaying NADH‐TR staining abnormalities increases between 1 and 2 months of age in KI mice (n = 5–6). No NADH‐TR staining abnormality was observed in WT mice. An average of 550 ± 129 positive fibres and 2534 ± 280 negative fibres were counted per muscle. (e) The presence of NADH‐TR staining abnormalities is associated with a lower min Feret diameter in 2‐month‐old KI mice (n = 6). A total of 18 507 fibres were analysed including 3302 positive fibres and 15 205 negative fibres. (f) Representative western blot images of oxidative…

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  FIGURE 3. Mechanisms involved in skeletal muscle growth defect of KI mice. (a) Representative laminin (red)/Pax7 (green) immunolabelling of tibialis anterior muscle cross‐section in WT and KI mice aged of 1 and 2 months (mo). Scale bars equal 50 μm. (b) The number of Pax7‐positive cells decreases in KI mice (n = 7–8) when compared to WT mice (n = 7–9). An average of 825 ± 277 fibres was analysed per muscle. (c) MyoD mRNA level in the tibialis anterior muscle of WT (n = 5–7) and KI (n = 7) mice. (d, left) Transcript levels of Eif4ebp1, Foxo3, Trim63, Fbxo32 and Fbxo30 in the tibialis anterior muscle of WT (n = 5–7) and KI (n = 6–10) mice. (d, right) Representative immunoblot of FOXO3 and stain‐free gel used for protein normalization. Quantification of FOXO3 protein content in 2‐month‐old WT (n = 9) and KI (n = 7) mice. (e) Transcripts levels of autophagy‐lysosome pathway related genes in the tibialis anterior muscle of WT (n = 4–7) and KI (n = 7) mice. (f) Representative immunoblots P62 and LC3B‐I and stain‐free gels used for protein normalization. Quantification of P62 and LC3B‐I protein content in 2‐month‐old WT (n = 9) and KI (n = 6–7) mice. Data are means ± SD (b) Data were analysed by…

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  FIGURE 4. Myostatin gene invalidation in KI mice increases skeletal muscle mass and restores muscle function. (a) Schematic view of the breeding strategy to generate KI mice lacking exon 3 of the myostatin (Mstn) gene (KIKO mice). (b) Myostatin mRNA level in WT (n = 5–7), KI (n = 7) and KIKO (n = 7–9) mice at the age of 1, 2 and 8 months (mo). Transcript was undetectable in KIKO mice. (c) Body weight of WT (n = 7–9), KI (n = 7–11) and KIKO (n = 7) mice. (d) Myostatin gene invalidation increases tibialis anterior muscle weight (n = 8). (e) Myostatin gene invalidation restores muscle grip strength (WT, KI, KIKO: n = 9–10). (f) Myostatin gene invalidation restores rotarod performance (WT, KI, KIKO: n = 9–10). Data of WT and KI mice have been previously presented in Figure 1. Data are means ± SD (b) Data were analysed with a one‐way ANOVA followed by Tukey's multiple comparison test. (c, e and f) Data were analysed by a two‐way ANOVA with repeated measures followed by Sidak's multiple comparison test. (d) Data were analysed by a two‐way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. a, significantly different from corresponding…

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  FIGURE 5. Pharmacological inhibition of myostatin/activin A restores muscle mass in KI mice. (a) Schematic view of the myostatin (Mstn) signalling pathway highlighting the inhibitory effect of soluble activin type IIB receptor (sActRIIB‐Fc). (b) Schematic view of the experimental design. KI mice were treated twice a week by intraperitoneal injections of sActRIIB‐Fc at the dosage of 5 mg kg−1 starting at 4 weeks of age for four consecutive weeks. Mice were then maintained in the animal facility five more weeks without any treatment (washout). Tissues were removed either at the end of the treatment (8 weeks of age) or 5 weeks later (13 weeks of age). (c) Myostatin plasma concentration in WT‐PBS (n = 8–11), KI‐PBS (n = 7–11) and KI‐5 mg kg−1 (n = 4–7) mice. (d) Myostatin mRNA level in the tibialis anterior muscle of WT‐PBS (n = 10), KI‐PBS (n = 7–11) and KI‐5 mg kg−1 (n = 7–8) mice. (e) Body weight of WT‐PBS (n = 8–13), KI‐PBS (n = 8–11) and KI‐5 mg kg−1 (n = 9–14) mice. (f) Tibialis anterior muscle weight of WT‐PBS (n = 8–12), KI‐PBS (n = 7–11) and KI‐5 mg kg−1 mice (n = 13–14) mice. Data are means ± SD (c and f) Data were analysed with a two‐way ANOVA followed by Tukey's multiple comparison…

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  FIGURE 6. Mechanisms underlying the hypertrophic effect of soluble activin type IIB receptor. (a) Immunostaining (left) representative of pericentriolar material 1 (PCM1) immunostaining (green) counter‐stained with laminin (red) in WT‐PBS, KI‐PBS and KI‐5 mg kg−1 mice at 8 and 13 weeks and number of PCM1‐positive nuclei per fibre in the tibialis anterior muscle (right) of WT‐PBS mice (n = 5), KI‐PBS mice (n = 3–5) and KI‐5 mg kg−1 mice (n = 3–4). A mean of 736 ± 321 fibres per muscle was counted. (b) Actr2, Actr3 and Wasl mRNA level in the tibialis anterior muscle of WT‐PBS (n = 10), KI‐PBS (n = 7–11) and KI‐5 mg.kg−1 (n = 6–8) mice. (c, left) Transcripts levels of components of autophagy‐lysosome pathway in the tibialis anterior muscle (left) of WT‐PBS (n = 9–10), KI‐PBS (n = 7–11) and KI‐5 mg.kg−1 (n = 7–8) mice and for Trim63 mRNA level in tibialis anterior muscle (right) of WT‐PBS mice (n = 10), KI‐PBS mice (n = 7–11) and KI‐5 mg kg−1 mice (n = 7). (c, right) Representative immunoblots of P62 and LC3B‐I and stain‐free gels used for protein normalization. Quantification of P62 and LC3B‐I protein content in 2‐month‐old WT (n = 8–9), KI‐PBS (n = 8–9), and KI‐5 mg.kg−1 (n = 7) mice. (d)…

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  FIGURE 7. Soluble activin type IIB receptor treatment does not restore skeletal muscle force of KI mice. (a) Representative nicotinamide adenine dinucleotide tetrazolium reductase (NADH‐TR) staining of tibialis anterior muscle cross‐sections (left) of KI‐PBS and KI‐5 mg kg−1 mice at 8 and 13 weeks were asterisks show fibres with abnormal NADH‐TR staining and the number of fibres with NADH‐TR staining abnormalities (right) in KI‐5 mg kg−1 (n = 7–8) compared to KI‐PBS (n = 7–9); a mean of 2804 ± 317 fibres was analysed per muscle. Tibialis anterior muscle fibres from WT mice did not show any NADH‐TR staining abnormalities and were therefore not represented. (b) Muscle force‐frequency relationship in WT‐PBS (n = 7–11), KI‐PBS (n = 6) and KI‐5 mg kg−1 (n = 3–5) mice at 8 and 13 weeks. (c) Maximal absolute isometric force (Fmax), specific isometric force (Fs), and f50 (stimulation frequency at which 50% of Fmax is achieved) of tibialis anterior muscle in WT‐PBS (n = 7–11), KI‐PBS (n = 6) and KI‐5 mg kg−1 (n = 3–5) mice at 8 and 13 weeks. (d) Schematic view of the molecular targets analysed. Chrna1 encodes the alpha1 subunit of the nicotinic acetylcholine (Ach) receptor (nAchR). Aqp4 encodes…