Osteopontin-stimulated expression of matrix metalloproteinase-9 causes cardiomyopathy in the mdx model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is a common and lethal form of muscular dystrophy. With progressive disease, most patients succumb to death from respiratory or heart failure, or both. However, the mechanisms, especially those governing cardiac inflammation and fibrosis in DMD, remain less understood. Matrix metalloproteinase (MMPs) are a group of extracellular matrix proteases involved in tissue remodeling in both physiologic and pathophysiologic conditions. Previous studies have shown that MMP-9 exacerbates myopathy in dystrophin-deficient mdx mice. However, the role and the mechanisms of action of MMP-9 in cardiac tissue and the biochemical mechanisms leading to increased levels of MMP-9 in mdx mice remain unknown. Our results demonstrate that the levels of MMP-9 are increased in the heart of mdx mice. Genetic ablation of MMP-9 attenuated cardiac injury, left ventricle dilation, and fibrosis in 1-y-old mdx mice. Echocardiography measurements showed improved heart function in Mmp9-deficient mdx mice. Deletion of the Mmp9 gene diminished the activation of ERK1/2 and Akt kinase in the heart of mdx mice. Ablation of MMP-9 also suppressed the expression of MMP-3 and MMP-12 in the heart of mdx mice. Finally, our experiments have revealed that osteopontin, an important immunomodulator, contributes to the increased amounts of MMP-9 in cardiac and skeletal muscle of mdx mice. This study provides a novel mechanism for development of cardiac dysfunction and suggests that MMP-9 and OPN are important therapeutic targets to mitigating cardiac abnormalities in patients with DMD.

FIGURE 1. Expression of MMP-9 in left ventricle (LV) of mdx mice

LV muscle from 12-month old wild-type, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice was isolated and processed to study the level of MMP-9 protein. A) Gelatin zymography gel and quantification of MMP-9 bands in zymograms, and B) Western blot along with densitometric quantification presented here demonstrate that the levels of MMP-9 protein are increased in LV of mdx mice. MMP-9 protein was undetectable in heart of mdx/Mmp9^−/− mice and the levels of an unrelated protein tubulin remained comparable between WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice. Black line indicates that intervening lanes have been spliced out. N=3 in each group. *p<0.01, values significantly different from WT mice.

FIGURE 2. Ablation of MMP-9 reduces cardiac injury in mdx mice

A). Frozen cross-sections made from heart (at the center of ventricles) of 1 year old wild-type (WT), mdx/Mmp9^+/+ and mdx/Mmp9^−/− mice were stained with H&E and photomicrograph. Data presented here demonstrate that the deletion of Mmp9 gene in mdx mice improves cardiac structure. Arrows point to damaged/necrotic area. Scale bar: 50 μm. B). Quantification of necrotic area in H&E-stained images. *p<0.01, values significantly different from WT mice. ^#p<0.01, values significantly different from mdx/Mmp9^+/+ mice. C). Representative photomicrographs of cardiac sections stained with Cy3-labelled goat anti-mouse IgG demonstrating that cardiac injury is considerably reduced in mdx/Mmp9^−/− mice compared with mdx/Mmp9^+/+ mice. Scale bar: 50 μm. N=6 in each group.

FIGURE 3. Role of MMP-9 in accumulation of macrophages and expression of inflammatory cytokines in heart of mdx mice

A). Frozen heart sections prepared from 1-year old wild-type (WT), mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice were immunostained with anti-F4/80 using a method as described in “Materials and Methods”. Representative photomicrographs presented here demonstrate that inhibition of MMP-9 reduces the concentration of macrophages in cardiac muscle of mdx mice. Scale bar: 50 μm. B). Quantification of number of F4/80-positive cells in heart sections. C). The mRNA levels of CD68 in heart of WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice measured by QRT-PCR assay. D). Fold changes in mRNA levels of TNF-α and IL-1β in heart of WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice. N=6 in each group. *p<0.01, values significantly different from WT mice. ^#p<0.01, values significantly different from mdx/Mmp9^+/+ mice.

FIGURE 4. Deletion of Mmp9 gene diminishes fibrosis in cardiac muscle of mdx mice

A). Hearts isolated from 1-year old wild-type (WT), mdx/Mmp9^+/+ and mdx/Mmp9^−/− mice were sectioned transversely at 5 μm thickness and Masson’s Trichrome staining was performed. Representative photomicrographs presented here demonstrate that the levels of collagen (blue colored) is considerably reduced in mdx/Mmp9^−/− mice compared to littermate mdx/Mmp9^+/+. B). Quantification of area under fibrosis in heart sections of WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice. N=6 in each group. C). Left ventricle muscle from 1-year old WT, mdx/Mmp9^+/+ and mdx/Mmp9^−/− were analyzed for the expression of Col1a1 and Col3a1 by QRT-PCR. *p<0.01, values significantly different from mdx/Mmp9^+/+ mice.

FIGURE 5. Effects of ablation of MMP-9 on cardiac function in mdx mice

(A). M- mode echocardiograms of 1-year old wild-type, mdx/Mmp9^+/+ and Mdx/Mmp9^−/− mice obtained with two-dimensional echocardiography from short-axis midventricle view of hearts. Arrows represent LVPWd. Fold change in B). LVIDd, and C). LVPWd values between WT, mdx/Mmp9^+/+ and Mdx/Mmp9^−/− mice. D) Fractional shortening (FS) gave higher values for mdx/Mmp9^−/− mice compared to littermate mdx/Mmp9^+/+ providing an evidence of improvement in heart function after inhibition of MMP-9 in mdx mice. N=6 in each group. *p<0.01, values significantly different from WT mice. ^#p<0.01, values significantly different from mdx/Mmp9^+/+ mice.

FIGURE 6. Role of MMP-9 on the expression of various MMPs and TIMPs in heart of one-year old mdx mice

Total mRNA was isolated from heart of one year old WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice and the mRNA levels of various MMPs and TIMPs were measured by QRT-PCR. N=4 in each group. *p<0.01, values significantly different from wild-type mice. ^#p<0.01, values significantly different from mdx/Mmp9^+/+ mice.

FIGURE 7. Effect of ablation of MMP-9 on the activation of MAPKs and Akt in heart of 1-year old mdx mice

A) Protein extracts prepared from heart of 1-year old WT, mdx/Mmp9^+/+, and mdx/Mmp9^−/− mice, were immunoblotted using antibody against phosphorylated or total ERK1/2, JNK1/2, p38MAPK, and Akt protein. Representative immunoblots presented here demonstrate that the level of phosphorylation of ERK1/2 and Akt was reduced in LV of mdx/mmp9^−/− mice compared to mdx/Mmp9^+/+ mice. B). Densitometric quantification of immunoblots. N=4 in each group. *p<0.05, values significantly different from mdx/Mmp9^+/+ mice. ^#p<0.05, values significantly different from mdx/Mmp9^+/+ mice.

FIGURE 8. Involvement of OPN in expression of MMP-9 in heart and skeletal muscle of mdx mice

3-week old wild-type mice were given a single retro-orbital injection of OPN protein (100 μg/kg body weight). After 12h, the levels of MMP-9 protein in left ventricle (LV) and tibial anterior (TA) muscle were measured by Western blot. Representative immunoblots and quantification of bands (bar diagrams) presented here demonstrate that OPN increases the levels of MMP-9 protein in (A) Heart and (B) TA muscle of mice. *p<0.05, values significantly different from mice treated with PBS alone. 3–4 week old mdx mice were given three intraperitoneal injections of anti-OPN or isotype control every 3rd day for a total of 10 days. LV and TA muscle isolated were used to measure the level of MMP-9 protein by Western blot. Data presented here demonstrate that treatment with OPN-neutralizing antibody significantly reduces the levels of MMP-9 protein in both (C) Heart and (D) TA muscle. N=3 in each group. ^#p<0.05, values significantly different from mdx mice treated with isotype control.