Characterization and comparison of muscle fibrosis in two mouse models and In Vivo test of an anti-fibrotic molecule

Muscle fibrosis, a hallmark of severe muscular dystrophies, is a mechanism not completely understood, characterized by marked deposition of collagens and other extracellular matrix (ECM) components, progressively replacing muscle fibres. Fibrosis also occurs in other human conditions affecting different organs/tissues including liver, heart, kidney and lung. In skeletal and cardiac muscle, dystrophin is associated with a large complex of sarcolemmal and cytoskeletal proteins, the dystrophin-glycoprotein complex (DGC), that confers a structural link between the laminin-α2 in the ECM and the cytoplasmic actin. Mutations in genes encoding several DGC components are associated with severe muscular dystrophies. In particular, Duchenne muscular dystrophy (DMD), due to lack of dystrophin protein, is the most frequent of such conditions in childhood. Moreover mutations in sarcoglycan genes cause autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F), that have similar clinical and histopathological features to DMD, although severity is variable. The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in human LGMD2E. This model exhibits disruption of DGC in skeletal, cardiac, and smooth muscle that causes severe muscular dystrophy, cardiomyopathy and vascular abnormalities. The mdx mouse, is the most-used model for DMD, however, differently from patients, the mdx mouse has mild clinical features and shows little endomysial fibrosis in limb muscles. In order to elucidate mechanisms leading to ECM protein deposition and characterize the progression of fibrosis, we have evaluated histopathological and molecular features in Sgcb-null mice at different ages, and compared them (at selected significant ages) with age-matched mdx mice. In particular, in Sgcb-null mouse quadriceps and diaphragms we determined extent of fibrosis, numbers of necrotic, regenerating and centronucleated fibres evaluating as well collagen (I, III and VI), decorin, and TGF-β1 transcript and protein levels. Then we compared Sgcb-null and mdx mice measuring the extent of fibrosis, protein levels of collagens, decorin and TGF-β1, and at the age of maximum tissue rearrangement (12 weeks), we assessed macrophage numbers and osteopontin and TGF-β1 transcript levels. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears a more promising model for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments compared to mdx mice. MicroRNAs (miRNAs) are small non-coding RNA molecules, whose main function seems to be the downregulation of gene expression by various mechanisms. Recent evidences indicate that miRNAs play fundamental roles in pathological processes; aberrant expression of miRNAs has been related to fibrosis through regulation of anti- and pro-fibrotic genes. In particular, miR-21 is one of the most highly upregulated miRNAs during tissue injury, and its persistent overexpression disrupts tissue repair and contributes to fibrosis in various tissues. With in vitro study we assessed that miR-21 expression was significantly increased both in DMD muscle biopsies and DMD muscle-derived fibroblasts. To assess the therapeutic potential of miR-21 inhibition, we performed a pilot study in which we treated mdx mice with antagomiR-21. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and restored PTEN and SPRY-1 expression. These effects were evident only a month after treatment at lowest dose reported effective in the literature. These findings indicate that miR-21 could represent a therapeutic target to reduce fibrosis.