Epigenetic reprogramming of muscle progenitors: inspiration for clinical therapies

In the context of regenerative medicine, based on the potential of stem cells to restore diseased tissues, epigenetics is becoming a pivotal area of interest. Therapeutic interventions that promote tissue and organ regeneration have as primary objective the selective control of gene expression in adult stem cells. This requires a deep understanding of the epigenetic mechanisms controlling transcriptional programs in tissue progenitors. This review attempts to elucidate the principle epigenetic regulations responsible of stem cells differentiation. In particular we focus on the current understanding of the epigenetic networks that regulate differentiation of muscle progenitors by the concerted action of chromatin-modifying enzymes and noncoding RNAs. The novel exciting role of exosome-bound microRNA in mediating epigenetic information transfer is also discussed. Finally we show an overview of the epigenetic strategies and therapies that aim to potentiate muscle regeneration and counteract the progression of Duchenne Muscular Dystrophy (DMD)

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

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

Advanced methods to study the cross talk between fibro-adipogenic progenitors and muscle stem cells

Functional interactions between muscle (satellite) stem cells—MuSCs—and other cellular components of their niche (the fibro-adipogenic progenitors—FAPs) coordinate regeneration of injured as well as diseased skeletal muscles. These interactions are largely mediated by secretory networks, whose integrity is critical to determine whether repair occurs by compensatory regeneration leading to formation of new contractile fibers, or by maladaptive formation of fibrotic scars and fat infiltration. Here we provide the description of methods for isolation of FAPs and MuSCs from muscles of wild type and dystrophic mice, and protocols of cocultures as well as MuSC’s exposure to FAP- derived exosomes. These methods and protocols can be exploited in murine models of acute muscle injury to investigate salient features of physiological repair, and in models of muscular diseases to identify dysregulated networks that compromise functional interactions between cellular components of the regeneration environment during disease progression. We predict that exporting these procedures to patient-derived muscle samples will contribute to advance our understanding of human skeletal myogenesis and related disorders

CFTR corrector C17 is effective in muscular dystrophy, in vivo proof of concept in LGMDR3

Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with β- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer

HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles

Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC–myomiR–BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a “latent” myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles