Fig. 3: Prolonged MYTHO depletion in skeletal muscle results in excessive growth, impairs muscle contractility, and induces several severe myopathy features.

A Upper panel: schematic representation of the experimental design. Lower left panel: quantification of Mytho mRNA expression in the TA at 3, 6, 12, or 20 weeks post AAV-mediated transduction (sh-RNA scramble or sh-RNA MYTHO). Lower middle panel: TA muscle mass at 3, 6, 12, or 20 weeks post AAV-mediated transduction. Lower right panel: representative image of TA muscle at 12 weeks post-AAV-mediated transduction. Scale bars = 0.5 cm. B Muscle-specific force measured in situ at 3, 6, 12, and 20 weeks post-injection of AAV sh-RNA scramble or AAV sh-RNA MYTHO. C Representative images of succinate dehydrogenase (SDH) histochemistry (upper panel), HE staining (second panel), Masson’s trichrome staining (third panel), and Evans blue staining of TA muscles at 6 weeks post AAV-mediated transduction. Arrows indicate degenerative fibers. Scale bars = 40 μm. D Quantification of Evans blue positive fibers in TA 6 weeks post AAV-injection E, F F4/80⁺ immunostaining and quantification of F4/80⁺ positive fibers area in TA 6 and 12 weeks post-injection of AAV sh-RNA scramble or sh-RNA MYTHO. Scale bars = 40 μm. G Quantification of IgG-positive fibers at 6 weeks and 12 weeks post-injection of AAV sh-RNA scramble or AAV sh-RNA MYTHO H Representative images of Laminin/DAPI staining (upper image) and analysis of fiber diameter, number of fibers/animal and % of centronuclear fibers (lower panels) of TA muscles transfected for 12 weeks with AAV sh-RNA scramble or AAV sh-RNA MYTHO. Scale bars = 40 μm. I Representative myosin heavy chain (MHC) immunolabeling and analysis of fiber type proportion and fiber diameter in TA injected with either AAV sh-RNA scramble or AAV sh-RNA MYTHO. Black scale bar = 500 µm, white scale bars = 40 µm. J–N RT-qPCR quantification of the mRNA expression of MyoG, Myh8, Myh2, Myh1, and Myh4 in TA muscles transfected with AAV sh-RNA scramble or AAV sh-RNA MYTHO for 3, 6, 12, or 20 weeks. Sample sizes indicated in each graph represent biological replicates. Data in A, B, D and I were analyzed with paired two-tailed t tests (*p < 0.05). Data in F, G, J–N were analyzed with two-way ANOVA, and corrections for multiple comparisons were performed with the two-stage step-up method of Benjamini, Krieger, and Yekutieli (∗p < 0.05 and q < 0.1). The min ferret diameter distribution in H was analyzed with two-way ANOVA and corrections for multiple comparisons were performed with the two-stage step-up method of Benjamini, Krieger, and Yekutieli (∗p < 0.05 and q < 0.1). All other comparisons in H were analyzed with paired two-tailed t tests (*p < 0.05). Data are presented as mean ± SEM (with individual data points). Detailed information on raw data, statistical tests, p values, and q values are provided in the Source Data file. The drawing in A was created with BioRender.com.