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)

HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes

Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7(+) cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion

HDAC4 is necessary for satellite cell differentiation and muscle regeneration

In response to injury, skeletal muscle exhibits high capacity to regenerate and epigenetics controls multiple steps of this process (Giordani et al., 2013). It has been demonstrated in vitro that completion of muscle differentiation requires shuttling of histone deacetylase 4 (HDAC4), a member of class IIa HDACs, from the nucleus to the cytoplasm and consequent activation of MEF2-dependent differentiation genes (McKinsey et al., 2000). In vivo, HDAC4 expression is up-regulated in skeletal muscle upon injury, suggesting a role for this protein in muscle regeneration. With the aim to elucidate the role of HDAC4 in skeletal muscle regeneration, we generate mice lacking HDAC4 in the satellite cells (HDAC4fl/fl;Pax7CE Cre). Lack of HDAC4 inhibits satellite cell differentiation. Despite having similar amount of sorted cells, HDAC4 KO satellite cells proliferate less and have less pax7 than controls. Importantly, muscle regeneration in vivo is impaired in HDAC4fl/fl;Pax7CE Cre mice. These results are confirmed by molecular analyses of the expression of myogenic markers. All together, these data delineate the importance of HDAC4 in muscle regeneration and suggest a protective role in response to muscle damage

PKC Theta Ablation Improves Healing in a Mouse Model of Muscular Dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ−/−, where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ−/− mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells

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

Computational and Experimental Studies on beta-Sheet Breakers Targeting Abeta(1-40) Fibrils

In this work we present and compare with experiments the results of extensive Molecular Dynamics simulations of model systems comprising an Aβ1-40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17-21 region of the Aβ1-40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, Circular Dichroism and Mass Spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ1-40 peptide. We show that Molecular Dynamics simulations and far UV Circular Dichroism provide consistent evidence that the new Ac-LPFFN-NH2 β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ1-40. In agreement with these results Thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ1-40 aggregation. Furthermore, Mass Spectrometry data and Molecular Dynamics simulations consistently identified the 17-21 Aβ1-40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2β-sheet breaker