Fig. 4

Multicore myopathy in mice carrying exon-15 mutations. a Top panel: H-E staining of vastus lateralis cryosections. Arrows designate central nulei and the arrowhead in delACAG indicates three centralized nuclei. Middle panel: NADH-TR histochemistry of vastus cryosections showing multiple areas with no enzymatic activity (cores) in delACAG mice and very few in dupA mice. Arrows depict examples of cores. For both panels: n = 7(wt), 9(delACAG), 3(dupA) and scale bars 50 µm. Lower panel: ATPase (pH 4.3) histochemistry in whole soleus cryosections revealing increased type 1 fibers in both mutants, n = 5(wt), 6(delACAG), 3(dupA). Scale bar 0.5 mm. b Quantification of fiber density (number of fibers/microscope field of view). Graph represents mean ± s.d. c Minimal Feret’s diameter (MFD, µm) showing relative frequency (%) of different fiber sizes with respect to the total number of fibers analyzed. Differences in mean MFD between wt and homozygous mutants were statistically significant (wt: 49.8 µm ± 12.9 s.d., delACAG: 29.1 µm ± 9.9 s.d., dupA: 38.3 µm ± 9.7 s.d.). d Percentage of fibers with central nuclei (% CNFs) in wt, delACAG and dupA mice. Graph corresponds to mean ± s.d. For b–d H-E cryosections of vastus lateralis were used and a total number of n = 455(wt), 1726(delACAG), 465(dupA) fibers were analyzed from 7(wt), 9(delACAG) and 3(dupA) mice. e Percentage (%) of type I fibers calculated in soleus sections expressed as mean ± s.d. n = 5(wt), 5(delACAG), 3(dupA) mice. One-way ANOVA (***) with Tukey post-hoc test was used in b and e and Kruskal-Wallis (***) with Dunn’s multiple comparisons test in c and d. f Representative TEM images of gastrocnemius from wt, delACAG and dupA mice, n = 3(wt), 4(delACAG), 2(dupA) mice. Scale bar 10 µm. Numbers identify different fibers. Note the variable disorganization of delACAG fibers. Arrows indicate cores. g TEM images of gastrocnemius showing different defects in delACAG fibers including: large cores (left and middle image. Scale bars 10 µm), central nuclei and abnormal mitochondrial accumulations (middle image), and Z-line abnormalities including Z-streaming (arrows, right image. Scale bar 2 µm). Arrows in the central picture point to ring-shaped granules shown in Fig. 7a