Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2/mdx Mice

Figure 1. Characterization of Cardiac Cross-Sections from 21-Week-Old D2/WT, D2/mdx, BL10/WT, and BL10/mdx Mice Representative images of (A) muscle architecture (assessed by H&E), (B) calcification (red; assessed by alizarin red; AR), (C) fibrosis (assessed by Masson’s trichrome; MT), (D) IgG staining (green), (E) β-sarcoglycan (β-SG), and (F) β-dystroglycan (β-DG) immunofluorescence in cardiac cross-sections from 21-week-old mice. Quantification of (G) AR and (H) MT, expressed as percentage area. Representative images and quantification of protein expression of (I) phosphorylated and total GSK-3β (GSK) and (J) phosphorylated and total AKT assessed by western blot. Scale bars, 100 μm. Data represented as mean ± SEM, *p < 0.05 versus vehicle control mice, n = 6/group.

Figure 2. Utrophin Expression in 21-Week-Old D2/mdx Mice Administered AAV-μ-Utro Compared to Vehicle Control Mice Localization assessed by immunofluorescence and expression of full-length and micro-utrophin (Utro and μ-Utro, respectively) in cross-sections from (A) heart, (B) extensor digitorum longus (EDL), (C) diaphragm, (D) soleus, and (E) tibialis anterior (TA) muscles. Utrophin IF was also assessed in longitudinal section of TA muscles (F). Expression was assessed by western blot and shown relative to loading control (total protein stain; TPS). Scale bars, 100 μm, n = 8/group.

Figure 3. Skeletal Muscle Functional Testing and Muscle Architecture of D2/mdx Mice Administered AAV-μ-Utro Compared to Vehicle Control Mice (A) Muscle architecture assessed by H&E staining of cross-sections of EDL, tibialis anterior (TA), diaphragm, and soleus muscles from treated mice compared to vehicle control mice. (B) Specific force and (C) percentage force drop following 15% eccentric contraction in the extensor digitorum longus (EDL) muscles and (D) specific force and (E) percentage force drop following 15% eccentric contraction in diaphragm muscle strips from D2/mdx mice administered AAV-μ-Utro compared to vehicle control mice. Scale bar, 100 μm. Data represented as mean ± SEM, *p < 0.05 versus vehicle control mice, n = 8/group.

Figure 4. Assessment of Cardiac Pathology in D2/mdx Mice Administered AAV-μ-Utro Compared to Vehicle Control Mice Representative images of (A) muscle architecture (assessed by H&E), (B) calcification (red; assessed by alizarin red; AR), (C) fibrosis (assessed by Masson’s trichrome; MT; indicated in blue) of cardiac cross-sections from treated mice compared to vehicle control mice. Quantification of (F) AR and (G) MT, expressed as percentage area. Representative images and quantification of (H) β-dystroglycan (β-DG) and (J) phosphorylated and total GSK-3β (GSK) assessed by western blot. β-DG was normalized to loading control (total protein; TPS). Scale bar, 100 μm. Data represented as mean ± SEM, *p < 0.05 versus vehicle control mice, n = 8/group.

Figure 5. Cardiac Cine-MRI Assessment of D2/mdx Mice Administered AAV-μ-Utro Compared to Vehicle Control Mice (A) Representative cardiac cine-MRI short axis mid-papillary slices at end systole and diastole. Quantification of (B) left ventricle (LV) mass, LV stroke volume, LV ejection fraction, and right ventricle (RV) ejection fraction of D2/mdx mice administered AAV-μ-Utro compared to vehicle control mice. Data represented as mean ± SEM, *p < 0.05 versus vehicle control mice, n = 8/group. Left ventricle, LV; right ventricle, RV.