TGFβ1 Improves the Proliferation of Decellularized and Aligned Skeletal Muscle in Extracellular Matrix Hydrogels by m6A Modification-Mediated Integrin Through Inducing ERK Phosphorylation

Abstract Background: The purpose of this study was to illustrate the characteristics of skeletal muscle ECM hydrogels and assess the function and underlying mechanisms of hydrogels combined with TGFβ1 for the myogenesis of skeletal muscle stem cells (SkMSCs). Methods: Six methods were used to obtain extracellular matrix (ECM) from rat skeletal muscle. Hydrogel components and cytocompatibility were assessed by Masson’s trichrome staining, scanning electron microscopy, the sulfated glycosaminoglycan (s-GAG) and bFGF contents and a cell viability assay. Satellite cells were sorted, identified and seeded into skeletal muscle ECM hydrogels with TGFβ1. To determine the function of decellularized aligned skeletal muscle ECM hydrogels with TGFβ1 for the proliferation and differentiation of SkMSCs, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) analysis and immunofluorescence staining were conducted. RNA stability assay and Gene set enrichment analysis (GSEA) were used to further investigated the mechanism underlying the function. Results: Our study established a highly efficient method for the decellularization of skeletal muscle and fabricated an ECM hydrogel without cell components that preserves the rich ECM composition. This hydrogel formed a three-dimensional microstructure and showed good biocompatibility. We found that the decellularized aligned ECM scaffold with TGFβ1 promoted SkMSC proliferation and suppressed SkMSC differentiation through extracellular signal-regulated kinase (ERK) signaling. Our study suggested that TGFβ1 increases the m6A methylation of the integrin mRNA 3’UTR, thereby inducing the phosphorylation of ERK and promoting SkMSCs proliferation and differentiation.Conclusions: Our study explored a highly efficient method for the decellularization of skeletal muscle and fabricated an ECM hydrogel with a three-dimensional microstructure and good biocompatibility. Furthermore, we demonstrated that decellularized aligned ECM scaffolds with TGFβ1 promoted SkMSC proliferation and differentiation through enhancing the m6A methylation of the integrin mRNA 3’UTR, which may serve as a potential therapeutic strategy for the acute and chronic muscle injuries