Fig 1.
Experimental design and characterization of mdx/utrn-/- mice.
(A) Experimental timeline of the study: Whole-body plethysmography (WBP) and grip strength testing were performed weekly starting at 6 weeks of age until disease end-stage. (B) Kaplan-Meier survival curve of male WT (n = 8) and mdx/utrn-/- (n = 8) mice. (C) The weight of the male WT (n = 8) and mdx/utrn-/- mice (n = 8) at indicated ages. (D) Scores for kyphosis in WT (n = 8) and mdx/utrn-/- mice (n = 8). (E) Grip strength of WT (n = 8) and mdx/utrn-/- mice (n = 8). Data are presented as mean±SEM. Statistical significance was determined using a mixed-model 2-way ANOVA; post hoc analysis using uncorrected Fisher’s LSD * P < 0.05; **P < 0.01; ***P < 0.001, **** P < 0.0001.
Fig 2.
Respiratory insufficiency in mdx/utrn-/- mice starting at 6 weeks of age.
(A) Whole-body plethysmography chambers are used to study respiratory parameters. (B-F) Measures of respiratory function in both WT (n = 8) and mdx/utrn-/- (n = 8) mice- frequency (B), tidal volume (C), minute ventilation (D), peak inspiratory flow (E) and peak expiratory flow (F) are analyzed during normoxia (“Baseline”) and at a respiratory challenge with hypoxia and hypercapnia at the indicated ages. Data are presented as mean±SEM. Statistical significance was determined using a 2-way mixed-model ANOVA; post hoc uncorrected Fisher’s LSD test. #P < 0.05; ##P < 0.01; ###P < 0.001, #### P < 0.0001 during normoxia and * P < 0.05; **P < 0.01; ***P < 0.001, **** P < 0.0001 during respiratory challenge.
Fig 3.
Hypoventilation in the mdx/utrn-/- mice.
(A-D) Measures of respiratory metabolism in both WT (n = 5) and mdx/utrn-/- (n = 5) mice at 6–7 weeks of age and disease end stage- ventilatory equivalent for carbon dioxide (V E/VCO2) (A), ventilatory equivalent for oxygen (V E/VO2) (B), carbon dioxide production (Vco2) (C), and oxygen consumption (Vo2) (D) are analyzed during normoxia (“Baseline”) and at a maximal respiratory challenge with hypoxia and hypercapnia. Data are presented as mean±SEM. Statistical significance was determined using a 2-way mixed-model ANOVA; post-hoc uncorrected Fisher’s LSD test * P < 0.05; **P < 0.01; ***P < 0.001, **** P < 0.0001.
Fig 4.
Diaphragm muscle weakness in the mdx/utrn-/- mice.
(A) The representative traces of ex vivo diaphragm muscle force-frequency relationship for wild-type and mdx/utrn-/- mice. (B-C) Diaphragm function at 6 weeks of age in both WT (n = 5) and mdx/utrn-/- (n = 5) mice- diaphragm- force-frequency relationship across a broad stimulus range (50–300 Hz) (B) and power of diaphragm at 35% of maximal tetanic force (C). Data are presented as mean±SEM. Statistical significance was determined using a two-way RM ANOVA; post-hoc uncorrected Fisher’s LSD test (B) and unpaired Student’s t-test (C) * P < 0.05; **P < 0.01; ***P < 0.001, **** P < 0.0001.
Fig 5.
Histopathology in the mdx/utrn-/- mice.
(A-C) Representative images of hematoxylin and eosin stained diaphragm (A) and tongue (B) and tibialis anterior (C) of the end-stage mdx/utrn-/- and same-aged WT control mice. The black arrow indicates the infiltration of inflammatory cells, and centralized nuclei are indicated by the white arrow. Scale bars: 90 μm.
Fig 6.
Muscle fibrosis in the mdx/utrn-/- mice.
(A-C) Picro-Sirius red staining to measure fibrosis in the diaphragm (A), tongue (B) and tibialis anterior (C) of end-stage mdx/utrn-/- and age matched WT control mice. Scale bars: 90 μm.
Fig 7.
Absence of dystrophin in the mdx/utrn-/- mice.
(A-C) Dystrophin staining of the diaphragm (A), tongue (B) and tibialis anterior (C) of end-stage mdx/utrn-/- and age matched WT control mice. Dystrophin (green) DAPI (blue). Scale bar: 90um.
Fig 8.
Fragmented neuromuscular junction morphology in mdx/utrn-/- mice.
(A-B) The motor end plate of the diaphragm from WT (A) and mdx/utrn-/- (B) mice stained with α-bungarotoxin. (C-D) Expression of postsynaptic markers- Chrna7 (C) and Musk (D) on NMJ in the diaphragm of the end-stage mdx/utrn-/- mice and age-matched WT control mice. Scale bars: 50 μm. Statistical significance was determined using unpaired Student’s t-test * P < 0.05; **P < 0.01; ***P < 0.001, **** P < 0.0001.