Fig 2: Reduction of TGF-ß proteins in decellularized matrices from Sgcg-MRL muscle compared to Sgcg-D2 muscles.Decellularized extracellular (dECM) myoscaffolds were generated from Sgcg-MRL and Sgcg-D2 quadriceps muscles. Myoscaffold sections were stained with (A) Hematoxylin & Eosin and (B) Sirius Red demonstrating removal of cellular content following detergent treatment (1.0% SDS) with maintenance of matrix components. Control samples taken from adjacent sections were incubated in PBS only (0%SDS). (C) Representative IFM images of dECM myoscaffolds co-stained with antibodies to detect TGF-ß1 (red) and LAMA2 (green). Extracellular TGF-ß1 protein expression was dramatically reduced in the MRL background. (D) 100x z-stack images of TGF-ß1 (red) and LAMA2 (green) demonstrate excess TGF-ß1 distributed throughout the matrix in the dystrophic D2 model. (E) Representative IFM images of dECM sections, co-stained with antibodies to detect TGF-ß3 (red) and LAMA2 (green) showing reduction of TGF-ß3 in Sgcg-MRL myoscaffolds. (F) dECM myoscaffolds from Sgcg-MRL (34) and Sgcg-D2 (bottom) showed reduced thrombospondin-4 (THBS4, red) and LAMA2 (green) in the Sgcg-MRL muscle.

Fig 3: The MRL strain increased myofiber size and maximum tetanic force in Sgcg-/- mice.TA muscle was analyzed at 20 weeks. (A) Representative immunofluorescence microscopy (IFM) images showed larger myofibers in the TA of the Sgcg-MRL stained with laminin-a2 (LAMA2). (B) Cross sectional area (CSA) of myofibers was shifted rightward in Sgcg-MRL muscle. (C) CSA was increased in the MRL background (WT-MRL 2544, Sgcg-MRL 2619, WT-D2 2560, Sgcg-D2 2154 µm2). (D) MRL background increased the Minimum Feret Diameter in the Sgcg-/- muscle (WT-MRL 49.1, Sgcg-MRL 49.6, WT-D2 49.6, Sgcg-D2 45.1µm). (E) Force measurements from male TA muscles showing maximum tetanic force was significantly reduced in the Sgcg-D2 strains compared to all other strains (WT-MRL 1085, Sgcg-MRL 828.5, WT-D2 836.3, Sgcg-D2 458.7mN). (F) Physiological Cross-Sectional Area (PCSA) was smaller in D2 compared to MRL muscle for both Sgcg-/- and WT (WT-MRL 7.44, Sgcg-MRL 8.14, WT-D2 5.13, Sgcg-D2 4.85mm2). (G) Specific force was reduced for Sgcg-/- compared to WT in the D2 strain but not the MRL strain (WT-MRL 145, Sgcg-MRL 102.3, WT-D2 163.5, Sgcg-D2 96.0mN/mm2). Graphical quantification of mean ± SD. Two-way ANOVA test with Tukey’s multiple comparison was used to determine statistical significance. * p<0.05, ** p<0.01, *** p<0. 001, **** p< 0.0001.

Fig 4: Decellularized myoscaffolds from Sgcg-MRL muscle promote growth and differentiation of C2C12 myoblasts.(A) C2C12 myoblasts were seeded and proliferated on dECM myoscaffolds from Sgcg-MRL (34) and Sgcg-D2 (bottom) for 24 hours. Then growth media was replaced with low-serum differentiation media for 96 hours. Slides were fixed with PFA for IFM. (B) Imaging of dECM myoscaffolds seeded with C2C12 myoblasts. The left column represents entire myoscaffolds with seeded myoblasts co-stained with LAMA2 (green), desmin (DES, red), and Hoechst (blue) to stain nuclei. The yellow region of interest is magnified in the middle and right columns. DES (red) staining in the right column shows myoblast morphology. (C-H) Three independent biological replicates of the Sgcg-MRL and Sgcg-D2 dECMs were analyzed (labelled as N1 = gray, N2 = black, N3 = white). For each sample, six images of myoblasts were captured at 20x magnification. C shows the percentage area of desmin positivity relative to the number of nuclei in the field (Sgcg-MRL 797.0, Sgcg-D2 197.4). D shows the average cell size (Sgcg-MRL 319.7, Sgcg-D2 120.8 mm2). E shows circularity (Sgcg-MRL .549, Sgcg-D2 .681 AU). F shows myoblast perimeter (Sgcg-MRL 100.5, Sgcg-D2 47.8mm). G shows Feret diameter (Sgcg-MRL 29.6, Sgcg-D2 17.2mm). H shows minimum Feret diameter (Sgcg-MRL 11.5, Sgcg-D2 8.6mm). Graphical quantification of mean ± SD. A Student’s t-test was used to determine significance. **** p<0.0001

Fig 5: Expression of laminins and linkers in control and LAMA2 MD mice. A) Cross-sections of the sciatic nerve from control and dyW/dyW mice stained with antibodies to laminin-a2 and laminin-a4. Note the increase in laminin-a4 immunoreactivity in the endoneurium in dyW/dyW mice. Arrow indicates the perineurium and asterisk the endoneurium. B, C) Cross-sections of the sciatic nerve (B) and TA muscle (C) of 8-week-old mice of the indicated genotype. The linker proteins aLNNd and mag are present in the endoneurium and perineurium of the sciatic nerve (B) and in the muscle fiber basement membrane (C). D) Western blot analysis to detect aLNNd and mag in lysates from triceps brachii muscle of 8-week-old mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. E) Overall phenotype of 14-week-old mice of the indicated genotype. F, G) Body mass (F) and tibia length (G) of 8-week-old mice of the indicated genotype. Data are mean ± SEM. *P < 0.05 and ***P < 0.001, by one-way ANOVA with Bonferroni post hoc test. Scale bars: 50 µm (A, B) and 100 µm (C). N = 5–7 female mice per group.