HDAC inhibitors modulate microRNA content of fibroadipogenic progenitor-derived exosomes to promote regeneration and Inhibit fibrosis of dystrophic muscles

This thesis aims to identify the extra-cellular mediators of the functional cross-talk between Fibro-Adipogenic Progenitors (FAPs) and Muscle Stem Cells (MuSCs) in Duchenne Muscular Dystrophy (DMD). For this purpose, we analyzed the role of extracellular vesicles (EVs) released by dystrophic FAPs to MuSCs, and performed a functional characterization of their microRNA content in response to HDAC inhibitors (HDACi) – a known epigenetic drug that modulate gene expression to promote regeneration and inhibit fibrosis in DMD, and is currently in clinical trial with DMD boys. We observed that FAPs-derived EVs exhibit standard features of exosomes, and their accumulation in muscle interstitial space of regenerating muscles suggests a role in promoting muscle regeneration. Exosomes are nanovesicles involved in intracellular communication, which can transfer a cargo of genetic (mRNA, microRNA) or proteic information to recipient target cells. In order to decipher exosomal content transferred from dystrophic FAPs to MuSCs to support MuSCs myogenic activity, we analyzed microRNAs (miRs) cargo of FAPs derived exosomes. We found that exosomes from FAPs of mdx mice (the mouse model of DMD) that have been exposed to histone deacetylases inhibitor (HDACi) can replicate most of the beneficial effects of HDACi, including the increase in muscle regeneration and the inhibition of inflammation and fibrotic deposition. Transwell co-culture experiments show that exosomes from FAPs of mdx mice exposed to HDACi enhance muscle satellite cell (MuSCs) expansion and differentiation into myotubes. Of note, these exosomes showed enrichment in miRs involved into muscle regeneration, with the myomiR miR-206 being the most up- regulated. Functional evidence demonstrates that antagomiR- mediated targeting of miR-206 abrogated exosome ability to support MuSC expansion and formation of myotubes. We also performed a functional analysis of FAPs-derived exosomes by intramuscular injections of exosomes isolated from treated or untreated dystrophic FAPs, previously transfected or not with antagomiR-206. We show that increased amounts of exosomal miR-206 are required for exosomes of HDACi-treated FAP- derived to promote compensatory regeneration and inhibit fibrosis in mdx muscles. Our findings reported the first evidence of pharmacological manipulation of the miR content of cell type specific-derived exosomes to promote compensatory regeneration of dystrophic muscles. This evidence points to the potential for pharmacological modulation of FAP-derived exosome’s content as tool for selective therapeutic interventions in muscular diseases.