Hip region muscular dystrophy and emergence of motor deficits in dysferlin‐deficient Bla/J mice

Figure 1. Locomotor deficits in Bla/J mice tested in an open field. Data shown are means ± SEM for each group for a 1‐h test session. *P < 0.05.

Figure 2. Reduced rearing movements in Bla/J mice tested in an open field. Data shown are means ± SEM for each group for a 1‐h test session. *P < 0.05.

Figure 3. Impaired motor coordination in Bla/J mice tested on an accelerating rotarod. Mice were given two trials (T1 and T2) at each time point. Maximum trial length was 5 min. Data shown are means + SEM. *P < 0.05.

Figure 4. Emergence of motor deficits in Bla/J mice. Data are means ± SEM for (A) grip strength in a 1‐min wire hang task, (B) digging in a 30‐min marble‐burying task, (C) latency to a visible platform in a Morris water maze, and (D) swimming speed during a visible platform task (weeks 13, 18, 67, and 72) and during a 5‐min or 6‐min free swim (weeks 38 and 48). Data for the visible platform task are the mean + SEM) of four 1‐min trials per day. One Bla/J male mouse was not tested in the 6‐min free swim because of a skin lesion. *P < 0.05.

Figure 5. Longitudinal evaluation of total mouse body mass and select wet muscle mass in Bla/J mice. Recorded weekly live mouse body mass for C57BL/6J compared to Bla/J mice separated by gender showed BLA/J males did not differ in body mass from C57BL/6J control mice (A), while BLA/J females (B) body mass was significantly higher than C57BL/6J genetic background control mice over almost all time points, with means increasingly differing past 50 weeks of age. Initial number of mice (weeks 9–72) were six male and eight female C57BL/6J controls and eight male and eight female Bla/J. Data shown are means + SEM for each group. *P < 0.05. (C) Muscle mass for specific muscles were compared in a separate cohort of male mice (N = 16) for each genotype. Mice were sacrificed at 12 months of age.

Figure 6. Select gait indices invoking neuromuscular function. Mice were evaluated at 25 weeks. (A) Forepaw placement angle was significantly more open in Bla/J mice compared to WT mice, differences that were sustained during uphill and downhill walking. (B) Hind paw placement angle during full stance was more open during horizontal walking at 24 cm/s and 40 cm/s. Hind paw placement angle was less adaptive in Bla/J mice to changes in walking conditions. (C) Hind limb shared stance was significantly briefer in Bla/J mice than in WT mice, suggesting muscle weakness. This characteristic was more apparent in downhill walking than during uphill walking.

Figure 7. Kaplan–Meier survival curve. There were no significant differences in survival between Bla/J and C57B6 mice up to sacrifice at 82 weeks of age by log‐rank (Mantel–Cox) test analysis (P = 0.53) or Grehan–Breslow–Wilcoxon test analysis (P = 0.48).

Figure 8. Circulating creatine kinase activity in WT and Bla/J mice. Blood samples were taken at the indicated time points from the indicated mouse models and analyzed for creatine kinase activity (N = 16 per genotype and equally divided by gender). Data are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001.

Figure 9. MRI evaluation. (A) Longitudinal high‐resolution MRI images of single Bla/J female mouse over 6–12 months observation period (gray scale images) and fused water (blue color) and fat (red) images. Yellow arrows point to the regions where muscle wasting and fat accumulation occurs. Black bands in gray scale images are assigned as fat, which is confirmed by water/fat MR imaging experiment (same regions seen in fat images, red). (B) Quantified muscle volumes for hip region; gluteus and psoas muscles, and lower leg muscles; gastrocnemius and tibialis anterior. Data (N = 16 for both groups) are shown as mean + SEM, unpaired nonparametric Mann–Whitney test comparing ranks, ***P < 0.0005.

Figure 10. Magnetic resonance spectroscopy of Bla/J muscle. (A) Location of a MR spectroscopic voxel in gluteus maximus muscle and stacked 1H‐MR spectra from 6 and 9 months old C57BL/6J and Bla/J mice showing increased and progressive levels of fatty acids in Bla/J mice (red box; assignment of extramyocellular lipids [EMCL] and intramyocellular lipids [IMCL] lipids, different lipid resonance assigned as per protons in a fatty acid chain). (B) Increase of quantified EMCL 1.5 ppm resonance in the gluteus muscle and (C) quantified EMCL 1.5 ppm resonance in gastrocnemius muscle. Data (N = 16 for both groups) are shown as mean + SEM, unpaired nonparametric Mann–Whitney test comparing ranks, ***P < 0.0005.

Figure 11. Extramyocellular lipid accumulation in Bla/J muscle over time. (A) Quantified levels of IMCL 0.9 and 1.3 ppm, and EMCL 1.1 and 1.5 ppm IMCL signal intensities in the gastrocnemius muscle. (B) Quantified levels of IMCL 0.9 and 1.3 ppm, and EMCL 1.1 and 1.5 ppm IMCL signal intensities in the gluteus muscle. Data (N = 16 for both groups) are shown as mean + SEM, unpaired nonparametric Mann–Whitney test comparing ranks, ***P < 0.0005.

Figure 12. Magnetic resonance analysis of metabolites. (A) Creatine and choline were found to be significantly lower in Bla/J mice at 6 months of age, while taurine was significantly lower in Bla/J mice at 6 and 9 months.