(A) Northern blot analysis of miR-133a in adult WT mouse tissues. The blot was stripped and reprobed with ³²P-labeled U6 probe as a loading control. Sol, soleus. (B) Expression of miR-133 in skeletal muscle, detected by real-time RT-PCR and expressed relative to U6.

(A) H&E staining of soleus, EDL, G/P, and TA muscles of WT and dKO mice at 12 weeks of age. Scale bars: 40 μm. (B) Immunostaining of TA muscle against laminin. Nuclei are stained with DAPI. dKO TA muscle showed central nuclei. Scale bars: 40 μm. (C) Percentage of centronuclear myofibers in 4 WT mice and 10 dKO mice at 12 weeks of age. For each mouse, more than 500 myofibers were counted for TA and G/P muscles and more than 300 myofibers were counted for soleus and EDL muscles. (D) NADH-TR staining of dKO TA muscle revealed abnormal distribution, radiating intermyofibrillary network (arrows), and ring-like fibers (asterisks). Scale bars: 20 μm. (E) EBD uptake of TA muscles of WT, dKO, and mdx mice. Immunostaining with laminin (green) is shown; EBD is detected as a red signal under fluorescence microscopy. Scale bars: 100 μm. (F) Expression of myogenic genes and of embryonic MHC (Myh3) and perinatal MHC (Myh8) in WT and dKO TA muscle, determined by real-time RT-PCR. n = 3 (WT and dKO).

(A) Expression of mRNA transcripts encoding components of T-tubules and SR was determined by real-time RT-PCR in TA muscles of 12-week-old mice. n = 3 (WT and dKO). (B) Immunostaining of T-tubules and SR in transverse sections of TA muscle from WT and dKO mice at 12 weeks of age. T-tubules were detected by anti-DHPRα, and terminal cisternae of the SR were detected by anti-RyR1. Nuclei were detected by DAPI, and the myofiber perimeter was stained by anti-laminin. Images of multiple levels of the sections were taken and reconstructed to create the 3D effect. Scale bars: 30 μm. (C–J) Electron micrographs of WT and dKO muscle. dKO TA muscle showed accumulation of electron-dense structures (D–F) that were absent in WT TA muscle (C). dKO muscle (H and J) displayed T-tubules (arrows) in abnormal orientations compared with WT muscle (G and I). Scale bars: 2 μm (C and D); 0.5 μm (E–H); 0.2 μm (I and J).

(A) Mitochondria were isolated from red and white gastrocnemius muscle, and oxygen consumption rate (OCR) was measured for RCR, ADP-stimulated state 3 respiration (ADP), and FCCP-stimulated respiration (FCCP). n = 6 (WT and dKO). *P < 0.05 vs. WT. (B) Fatty acid oxidation was measured in isolated mitochondria from red and white gastrocnemius muscle. Citrate synthase enzyme activity was measured in isolated mitochondria from red and white quadriceps muscle. n = 6 (WT and dKO). *P < 0.05 vs. WT.

(A) Position of miR-133a target site in Dnm2 3′ UTR and sequence alignment of miR-133a and the Dnm2 3′ UTR from various species are shown. Conserved miR-133a binding sites in Dnm2 3′ UTR are shown in red. Mutations in Dnm2 3′ UTR were introduced to disrupt base-pairing with miR-133a seed sequences (blue). (B) Luciferase reporter constructs containing WT and mutant Dnm2 3′ UTR sequences were cotransfected into COS-1 cells with a plasmid expressing miR-133a. 48 hours after transfection, luciferase activity was measured and normalized to β-galactosidase activity. (C) Real-time RT-PCR showing expression of Dnm2 mRNA in WT and dKO TA muscle. n = 3 (WT and dKO). (D) Western blot showing expression of dynamin 2 protein in TA muscle of WT and dKO mice. n = 2 (WT and dKO). The blot was stripped and reprobed with an antibody against α-actin as a loading control. Quantification of dynamin 2 protein, determined by densitometry and normalized to α-actin, is also shown.

(A) Western blot analysis of TA muscle from WT and MCK-DNM2 transgenic mouse lines Tg1 and Tg2 using anti–dynamin 2 and anti-myc to show overexpression of transgene. Anti-tubulin was used as a loading control. Protein quantification, determined by densitometry, is also shown. (B) Transverse sections of TA muscles of WT, Tg1, and Tg2 mice at 6 weeks of age were stained with wheat germ agglutinin (WGA) and DAPI to show central nuclei (arrows) in transgenic mice. Scale bars: 100 μm. (C) Percentage of centronuclear myofibers in TA muscle of transgenic mice at 7 weeks of age. (D) Histological analysis of TA and soleus muscles of WT and Tg2 mice at 11 weeks of age. TA muscle sections were stained with H&E, with anti-laminin and DAPI to show central nuclei, and with NADH-TR to reveal abnormal distribution and radiating intermyofibrillary network (arrows). Scale bars: 40 μm. (E) 10-week-old WT and Tg2 mice (n = 3 per group), as well as 3 month-old WT and dKO mice (n = 5 per group), were subjected to forced downhill running on a treadmill. Muscle performance was measured as time to exhaustion. Total running distance is also shown. *P < 0.05; ***P < 0.001.

(A) Immunostaining of TA muscle from WT and dKO mice to detect dynamin 2 and dysferlin. Intracellular accumulation of dysferlin was observed in dKO myofibers. Overlay images indicate localization of dynamin 2 and dysferlin in the intracellular aggregates in dKO muscle. Scale bars: 30 μm. (B) Immunostaining of TA muscle from WT and Tg2 mice to detect dysferlin. Intracellular accumulation of dysferlin was observed in Tg2 myofibers. Scale bars: 30 μm.

(A) Metachromatic ATPase staining and anti–MHC-1 immunostaining of soleus muscle from WT and dKO mice at 12 weeks of age showed an increase in type I myofibers in dKO soleus muscle. H&E staining of the soleus muscles is also shown. Scale bars: 100 μm. (B) Percentage of type I myofibers in soleus muscles, determined by metachromatic ATPase staining. n = 6 (WT and dKO). (C) Expression of transcripts of MHC isoforms in soleus muscle, determined by real-time RT-PCR. n = 3 (WT and dKO). Expression of MHC isoforms from protein extracts of soleus, EDL, and TA muscles from WT and dKO mice was also determined by glycerol gel electrophoresis followed by silver staining.