Figure 1.
MMP-9 is not involved in the initial onset of muscle pathology in mdx mice.
(A) Representative photomicrographs of GA muscle sections of 4-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice displaying hallmarks of mdx pathology. Area under necrosis and centronucleated myofibers evaluated by H&E staining (top panel). Muscle sections were immunostained with Cy3-labeled goat anti-mouse IgG to detect permeable/damaged fibers (middle panel). Formation of new myofibers was assessed by staining with embryonic myosin heavy chain (eMyHC) antibody (lower panel). Scale bar: 20 µm. Quantification of (B) percentage of centronucleated fibers; (C) Cy3-labelled IgG filled fibers per field; and (D) number of eMyHC-positive fibers per field of mdx;Mmp9+/+ and mdx,MMP9+/− mice. N = 4 in each group. Error bars represent SD.
Figure 2.
Inhibition of MMP-9 enhances satellite cell population in skeletal muscle of mdx mice.
(A) Representative photomicrograph of TA muscle section from 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− stained for Pax7 and DAPI. Arrows point to Pax7+ satellite cells. Scale bar: 50 µm. (B) Quantification of relative number of Pax7+ cells per myofiber in TA muscle sections from 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice. (C) Representative FACS dot plots showing the percentage of satellite cells in GA muscle of 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice. Negative selection antibodies (CD45, CD31, Ter119) are in upper box whereas positive selection antibody (α7-integrin) is in lower box. (D) Quantification of satellite cells by FACS in GA muscles of 8-week mdx;Mmp9+/+ and mdx;Mmp9+/− mice. N = 4 in each group Error bars represent SD. *p<0.05 values significantly different from that of mdx;Mmp9+/+ mice.
Figure 3.
Inhibition of MMP-9 promotes M2 macrophage phenotype in skeletal muscle of mdx mice.
GA muscle of 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice were isolated and processed for FACS analysis for M1 and M2 macrophages in the F4/80+ population after gating out CD45 and Sca1 cells. (A) FACS dot plots displaying the percentage of CD11c+ M1-macrophages (upper left quadrant). (B) Representative dot plots for the percentage of CD206+ M2 macrophages (lower right quadrant). (C) Quantification of relative proportion of M1 and M2 macrophages by FACS technique in GA muscle of mdx;Mmp9+/+ and mdx;Mmp9+/− mice. (D) Fold change in relative mRNA levels of CD163, TNF-α, IL-1β, IL-6, IFN-γ, Il-10, and IL-4 measured by QRT-PCR assay in GA muscle of 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice. N = 3 or 4 in each group. Error bars represent SD. *p<0.05, values significantly different from mdx;Mmp9+/+ mice.
Figure 4.
Inhibition of MMP-9 increases Notch signaling in skeletal muscle of mdx mice.
GA muscles were isolated from 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice and processed for QRT-PCR analysis. Fold change in expression of (A) Notch receptors- Notch1, Notch2 and Notch3; (B) Notch ligands- Jagged1, Jagged2, DLL1 and DLL4; and (C) Notch target genes- Hes1, Hes6, HeyL, and Hey1. N = 3–4 in each group. Error bars represent SD. *p<0.05, values significantly different from littermate mdx;Mmp9+/+ mice.
Figure 5.
Inhibition of MMP9 improves canonical Wnt pathway but suppresses non-canonical Wnt signaling.
GA muscles isolated from 8-week old mdx;Mmp9+/+ and mdx;Mmp9+/− mice and processed for QRT-PCR or Western blotting. (A) Relative mRNA levels of Wnt3a, Wnt4, Wnt5a, Wnt7a and Wnt11. (B) Relative mRNA levels Frizzled (Fzd)1, Fzd2, Fzd4 and Fzd6. (C) Relative mRNA levels of Axin2. N = 3 (for Western) and N = 3 (for QRT-PCR) in each group. (D) Representative immunoblots showing Wnt3a and Axin2 and unrelated protein tubulin in GA muscle. Error bars represent SD. *p<0.01, values significantly different from littermate mdx;Mmp9+/+ mice.
Figure 6.
Inhibition of MMP-9 improves engraftment of transplanted cells in skeletal muscle of mdx mice.
After 24 h of cardiotoxin injection, TA muscle of mdx;Mmp9+/+ and mdx;Mmp9+/− were injected with 5×105 myoblasts that were prepared from mTmG mice. After 28 days, TA muscle was isolated and engraftment of transplanted myoblasts into dystrophic muscle was analyzed directly or by staining with dystrophin antibody. (A) Representative images show mT protein florescence (top panel) and dystrophin (lower panel) in TA muscle section of mdx;Mmp9+/+ and mdx;Mmp9+/− mice. Scale bar: 50 µm. (B) Quantification of the extent of myoblast engraftment. Average number of engrafted myofibers per cluster (area where implanted myoblasts are stained positive) is reported. N = 8 in each group. Error bars represent SD. *p<0.05 values significantly different from mdx;Mmp9+/+ mice.