Generation of miR-206–KO;mdx mice.
mice in a 129SvEv-C57BL/6 mixed background were described previously (23
mice were purchased from the Jackson Laboratory. miR-206–/–
male mice were bred to homozygous Dmdmdx/mdx
female mice to generate miR-206+/–;Dmdmdx/+
female mice or miR-206+/–;Dmdmdx/Y
male mice. miR-206+/–;Dmdmdx/Y
male mice were then bred to Dmdmdx/mdx
female mice to generate miR-206+/–;Dmdmdx/mdx
female mice were then intercrossed with miR-206+/–;Dmdmdx/Y
male mice to generate miR-206–/–;Dmdmdx/mdx
mice and miR-206–/–;Dmdmdx/Y
mice, which are referred to as miR-206–KO;mdx
mice. Mice used in this study were all on a 129SvEv-C57BL/6 mixed background.
CTX from Naja mossambica mossambica (Sigma-Aldrich) was dissolved in sterile saline to a final concentration of 10 μM and aliquoted and stored at –20°C. Mice were anesthetized by intraperitoneal injection of 2.5% Avertin at (15 μl/g). Mouse legs were shaved and cleaned with alcohol. TA muscles were injected with 50 μl of CTX with a 26-gauge needle. After injection, animals were kept under a warming lamp until recovery.
Northern blot analysis.
Total RNA was isolated from cultured cells by Trizol
(Invitrogen) or from mouse skeletal muscle tissues by the miRNeasy Mini Kit (QIAGEN). Northern blots to detect miR-206, miR-1, and miR-133 and U6 were performed as described previously (52
P-labeled Star-Fire oligonucleotide probes (IDT) against mature miRNAs and U6 were used for hybridization.
RT-PCR and real-time RT-PCR analysis.
RNA was treated with Turbo RNase-free DNase (Ambion Inc.) prior to the reverse transcription step. RT-PCR was performed using random hexamer primers (Invitrogen). Real-time RT-PCR on miRNA was performed using the TaqMan microRNA assay kits (ABI) according to the manufacturer’s protocol. Real-time RT-PCR was performed using TaqMan probes (ABI) or SYBR green probes. SYBR green primers used were as follow: Gja1-F: 5′-GGACCTTGTCCAGCAGCTT-3′, Gja1-R: 5′-TCCAAGGAGTTCCACCACTT-3′; Notch3-F: 5′-AAGCGTCTCCTGGATGCTG-3′, Notch3-R: 5′-GAATCTGGAAGACACCCTGG-3′; pre-133b-RT-F: 5′CTGGTCAAACGGAACCAAGT-3′, pre-133b-RT-R: 5′-TGATGGCAAAACCAGCATTA-3′.
For the microarray, total RNA was isolated from TA muscle of mdx and miR-206–KO;mdx mice at 3 months of age (n = 3 for each). Microarray analysis was performed by the University of Texas Southwestern Microarray Core Facility using the Mouse Genome Illumina Mouse-6 V2 BeadChip. Data have been deposited in NCBI’s Gene Expression Omnibus (GEO GSE36077).
Histological analysis of skeletal muscle.
Skeletal muscle groups were harvested and flash frozen in embedding medium containing a 3:1 mixture of Tissue Freezing Medium (Triangle Biomedical Sciences) and gum tragacanth (Sigma-Aldrich) or fixed in 4% paraformaldehyde and processed for routine paraffin histology. Frozen sections were cut on a cryotome and stained with H&E as previously described (53
). Masson’s trichrome, von Kossa staining, and oil red O staining were performed using standard procedures. Evans blue dye uptake was performed as described (53
Frozen sections were fixed in freshly prepared 4% paraformaldehyde for 20 minutes on ice and then treated with 0.3% Triton X-100/PBS at room temperature for 20 minutes. Sections were incubated with mouse IgG-blocking solution from the M.O.M. kit (Vector Lab) diluted in 0.01% Triton X-100/PBS at room temperature for 1 hour. Sections were then incubated with 5% goat serum (Sigma-Aldrich) in M.O.M. protein diluent for 30 minutes. Sections were incubated with primary antibodies diluted in M.O.M. protein diluent at 4°C overnight. The next morning, slides were washed with PBS and incubated with secondary antibodies diluted in M.O.M. protein diluent at room temperature for 45 minutes. Sections were then washed and mounted with VectaShield Mounting Medium with DAPI. Pictures were taken with a Zeiss confocal microscope. Primary and secondary antibodies were as follows: Desmin (1:100, Dako), Laminin (1:200, Sigma-Aldrich), MF20 (DSHB), Pax7 (1:100, DSHB), Alexa Fluor 594 goat anti-mouse IgG1 (1:400, Invitrogen), and Alexa Fluor 488 goat anti-rabbit IgG (1:400, Invitrogen). Wheat germ agglutinin (WGA) staining was performed using Alexa Fluor 555–conjugated WGA (Invitrogen) as previously described (53
). Immunostaining of NMJs was done as previously described (23
). PECAM1 staining was performed using anti-mouse CD31 antibody (1:40, Dianova) and a biotinylated secondary antibody coupled with streptavidin–horseradish peroxidase, followed by DAB chromagen reaction.
Isolation of SCs by FACS.
CTX was injected into hind limb muscles of WT and miR-206–KO mice, and activated SCs were isolated 3 days after injection as previously described (7
). Briefly, hind limb muscles were pooled, minced, and digested with 0.2% Collagenase II (Gibco; Invitrogen), followed by trituration. SCs were then isolated by further digestion of myofibers with 0.1% Dispase (Gibco; Invitrogen) and 0.05% Collagenase II (Gibco; Invitrogen). Cell suspension was filtered through a 40-μm cell strainer, and SCs were pelleted after centrifugation.
For FACS, SCs were counted and resuspended in PBS/3% BSA at 1 × 106 cells/50 μl. Cells were then incubated with the following antibodies for 1 hour on ice: Alexa Fluor 488–conjugated rat anti-mouse CD34 (1:50) (AbD Serotec), PE-labeled rat anti-mouse CD45 (1:100), PE-labeled rat anti-mouse CD31 (1:100), PE-labeled anti-mouse Sca-1 (1:3000) (all from BD Biosciences — Pharmingen). After incubation, cells were washed twice, filtered through a 40-μm cell strainer, and resuspended in PBS/3% BSA at a concentration of 2 × 107 cells/ml. Cells were separated on a MoFlo Cytometer (Beckman Coulter). Sorting gates were strictly defined by the forward scatter and side scatter patterns of SCs as well as the positive control cells labeled with Alexa Fluor 488–CD34 and the negative control cells labeled with PE-CD45, PE-CD31, and PE–Sca-1. Cells positive for Alexa Fluor 488–CD34 and negative for PE-CD45, PE-CD31, and PE–Sca-1 were sorted to enrich for activated SCs.
SCs isolated by FACS were cultured on Matrigel-coated (BD Biosciences) plates in growth medium consisting of HAM’s F-10 medium, 20% bovine calf serum (BCS), and 5 ng/ml bFGF (Gibco; Invitrogen). Medium was changed daily. Cells were passaged at 70% confluency to prevent spontaneous differentiation. To induce differentiation, cells at 80% confluency were switched to differentiation medium containing DMEM and 2% horse serum.
For immunostaining, cells were grown on Matrigel-coated cover slips in either growth medium or differentiation medium. Cells were fixed on cover slips with 4% paraformaldehyde for 15 minutes, washed with PBS, and permeabilized with 0.3% Triton X-100/PBS for 5 minutes. Cells were blocked with 5% goat serum/PBS/0.1% Triton X-100 for 3 minutes, followed by incubation with primary antibodies (diluted in 5% goat serum/PBS/0.1% Triton X-100) for 2 hours. Cells were then washed in PBS and incubated with secondary antibodies for 1 hour. After washing with PBS, cover slips were mounted on glass slides with VectoLab Mounting Medium (with DAPI). The following antibodies were used: anti-Pax7 (1:100, DSHB), MF20 (DSHB), Alexa Fluor 594 goat anti-mouse IgG1 (for Pax7) (1:400, Invitrogen), and Alexa Fluor 555 goat anti-mouse IgG (for MF20) (1:400, Invitrogen).
Western blot analysis.
Total protein was extracted from cultured cells using RIPA buffer containing protease inhibitor cocktail (Roche) and 1 mM PMSF. Protein concentrations were measured by BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein from different samples were resolved on 4%–20% SDS-PAGE. Western blotting was performed by standard protocol. The following antibodies were used: Pax7 (1:100, DSHB), Notch3 (1:1000, Abcam), IGFBP5 (1:500, Santa Cruz Biotechnology Inc.), connexin43 (1:500, Cell Signaling), and α-tubulin (1:5000, Sigma-Aldrich). For detection of Pax7, HRP-conjugated anti-mouse IgG1 secondary antibody (Santa Cruz Biotechnology Inc.) was used.
Transfection and luciferase assays.
3′ UTR fragments of Notch3, Igfbp5, and Pax7 containing miR-206 binding sites were cloned into pMIR-REPORT Vector (Ambion), respectively. Mutagenesis of the miR-206–binding sites, cell culture, and luciferase assay were performed as previously described (52
The treadmill test was performed using the Exer-6M (Columbus Instruments) at 15 degrees downhill. Mice were warmed up at 5 m/min for 5 minutes before the test. For the test, mice ran on the treadmill at 5 m/min for 2 minutes, 7 m/min for 2 minutes, 8 m/min for 2 minutes, and 10 m/min for 5 minutes. Afterwards, speed was increased 1 m/min to a final speed of 20 m/min. Exhaustion was defined by the inability of the animal to remain on the treadmill despite electrical prodding.
Serum CK measurement.
Four-week-old and 5-month-old animals were anesthetized by Avertin, and blood was obtained from the periorbital vascular plexus directly into microhematocrit tubes (70 μl, Fisher Scientific). Serum was obtained by allowing the blood to clot at room temperature for 30 minutes and then centrifuging at 1,700 × g for 10 minutes. Serum CK was measured by the University of Texas Southwestern Metabolic Core Facility.
All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Texas Southwestern Medical Center at Dallas.
Data are presented as mean ± SEM. Differences between groups were tested for statistical significance using the unpaired 2-tailed Student’s t test. P < 0.05 was considered significant.