Pathological remodeling of the extracellular matrix (ECM) underlies many fibrotic disorders, including muscular dystrophy. In fibrotic disorders, large matricellular proteins like proteoglycans and glycoproteins, and smaller secreted factors are progressively deposited into the matrix. Tissue decellularization uses detergent treatment to remove the cellular component, leaving behind intact the matrix components. Decellularized ECMs (or dECMs) have most commonly been studied in intact organs, but more recently, this method has been adapted to tissue sections on slides. We generated dECM from genetically different mouse models of muscular dystrophies including dystrophin-deficient, gamma-sarcoglycan-deficient, and dysferlin-deficient mice, as well as analyzing distinct background mouse strains known to alter the ECM. Formalin fixed samples were assessed for complete decellularization, preservation of ECM architecture, and protein retention using histological staining and immunofluorescence microscopy. Excess collagen deposition correlated with fibrosis severity across the different dystrophic models. Each muscular dystrophy subtype demonstrated excess decorin deposition in the matrix. Dystrophin- and gamma-sarcoglycan-deficient muscles displayed excess thrombospondin 4, while dysferlin-deficient muscle had excess decorin but not excess thrombospondin 4. Annexins A2 and A6 were increased across all dystrophic dECMs, but annexin deposition was greatest in dysferlin-deficient muscular dystrophy. In this model, annexin A2 was diffusely increased but annexin A6 was found in discrete regions of marked excess protein within the matrix. These studies highlight the differential nature of the core matrisome and its associated proteins in different forms of muscular dystrophy, where these proteins are positioned to influence the surrounding myofibers and inflammatory infiltrate.
Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphys Roth Large (MRL) strain has super-healing properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-b signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-b1 and TGF-b3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.
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