The pro-fibrotic connective tissue growth factor (CTGF/CCN2) correlates with the number of necrotic-regenerative foci in dystrophic muscle

Abstract

Connective tissue growth factor (CTGF/CCN2) has strong inflammatory and profibrotic activities. Its expression is enhanced in skeletal muscular dystrophies such as Duchenne muscular dystrophy (DMD), a myopathy characterized by exacerbated inflammation and fibrosis. In dystrophic tissue, necrotic-regenerative foci, myofibroblasts, newly-regenerated muscle fibers and necrosis all occur simultaneously. To determine if CCN2 is involved in the appearance of the foci, we studied their presence and characteristics in mdx mice (DMD mouse model) compared to mdx mice hemizygous for CCN2 (mdx-Ccn2+/-). We used laser capture microdissection followed by gene expression and immunofluorescence analyses to investigate fibrotic, inflammation and regeneration markers in damaged and non-damaged areas in mdx and mdx-Ccn2+/- skeletal muscle. Mdx mice foci express elevated mRNAs levels of transforming growth factor type beta, collagen, fibronectin, the myofribroblast marker α-SMA, and the myogenic transcription factor myogenin. Mdx foci also show elevated levels of MCP-1 and CD-68 positive cells, indicating that CCN2 could be inducing an inflammatory response. We found a significant reduction in the number of foci in mdx-Ccn2+/- mice muscle. Fibrotic and inflammatory markers were also decreased in these foci. We did not observe any difference in Pax7 mRNA levels, a marker for satellite cells, in mdx mice compared to mdx-Ccn2+/- mice. Thus, CCN2 appears to be involved in the fibrotic response as well as in the inflammatory response in the dystrophic skeletal muscle.

Keywords: CTGF/CCN2; Fibrosis; Inflammation; Muscular dystrophy; Necrotic-regenerative focus.

Fig. 1

Features of necrotic-regenerative foci in mdx mice. a Upper panels: H&E staining of gastrocnemius crossections from wild type and mdx mice. Magnifications: upper panel bar: 200 μm; lower panel bar: 50 μm. The necrotic-regenerative foci are shown delimeted by black lines. Lower panels: Immunostaining for the fibrotic markers fibronectin and collagen III. Bar 50 μm. b CCN2 immunostaining of gastrocnemius crossections from wild type and mdx mice. Arrows indicate the necrotic-regenerative foci of mdx muscle where CCN2 staining is more intense. Bar 100 μm

Fig. 2

mdx-Ccn2+/− muscle shows reduced necrotic-regenerative foci and is correlated with reduced CCN2 levels. a Left panel: H&E staining of gastrocnemius crossections from mdx-Ccn2+/+ and mdx-Ccn2+/− mice at different magnifications. Left bar: 100 μm, right bar: 50 μm. The necrotic-regenerative foci are shown delimited by black lines. Right panel: Quantitation of the degree of damage: % damaged area relative to the total area of mdx-Ccn2+/+ and mdx-Ccn2+/− crossections. # T-test p < 0.05 mdx-Ccn2+/+ vs mdx-Ccn2+/−. b Quantitative qPCR of CCN2 from LMD samples of damaged (black bars) and non-damaged (gray bars) areas of gastrocnemius from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. White bars correspond to wild type (Ccn2+/+) or wild type hemizygous for CCN2 (Ccn2+/−). # One way ANOVA p < 0.05 vs non-damaged areas. & p < 0.05 vs Ccn2+/+. c CCN2 immunostaining of gastrocnemius crossections from Ccn2+/+, Ccn2+/−, mdx-Ccn2+/+ and mdx-Ccn2+/− mice. Bar 100 μm

Fig. 3

Fibrotic markers expression levels are reduced in the damaged areas of mdx-Ccn2+/− skeletal muscle. Quantitative qPCR of a TGF-β, b Fibronectin and c α-SMA, from LMD samples of damaged (black bars) and non-damaged (gray bars) areas in gastrocnemius muscle from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. White bars correspond to wild type (Ccn2+/+) or wild type hemizygous for CCN2 (Ccn2+/−). # One way ANOVA p < 0.05 vs non-damaged areas, & p < 0.05 vs Ccn2+/+

Fig. 4

Myogenic markers expression levels are reduced in the damaged areas of mdx-Ccn2+/− skeletal muscle. Quantitative qPCR of a Pax7 and b Myogenin, from LMD samples of damaged (black bars) and non-damaged (gray bars) areas in gastrocnemius muscle from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. White bars correspond to wild type (Ccn2+/+) or wild type hemizygous for CCN2 (Ccn2+/−). # One way ANOVA p < 0.05 vs non-damaged areas, & p < 0.05 vs Ccn2+/+

Fig. 5

There are fewer regenerating fibers in the muscle of mdx-Ccn2+/− mice. a Embryonic myosin (EM), and Laminin (LAM) immunostaining to evaluate regenerating fibers and basement membrane respectively, in gastrocnemius crossections from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. Nuclei were visualized with Hoechst. Bar: 100 μm. b The percentage of EM positive fibers was quantified by counting 5 different fields for three mdx-Ccn2+/− and mdx-Ccn2+/+ mice (a mean of 600 fibers per animal were counted). Data corresponds to mean ± standard deviation. *p < 0.05 T-test mdx-Ccn2+/− vs mdx-Ccn2+/+

Fig. 6

Inflammation markers are reduced in the damaged areas of mdx-Ccn2+/− mice. a Quantitative qPCR of MCP1, from LMD samples of damaged (black bars) and non-damaged (gray bars) areas of gastrocnemius muscle from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. White bars correspond to wild type (Ccn2+/+) or wild type hemizygous for CCN2 (Ccn2+/−). # One way ANOVA p < 0.05 vs non-damaged areas, & p < 0.05 vs Ccn2+/+. b CD68 immunostaining to asses monocytes/macrophages in gastrocnemius crossections from mdx-Ccn2+/+ and mdx-Ccn2+/− mice. Bar: 50 μm