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Cardiovascular diseases (CVDs) are a major burden on the healthcare system: indeed, over two million new cases are diagnosed every year worldwide. Unfortunately, important drawbacks for the treatment of these patients derive from our current inability to stop the structural alterations that lead to heart failure, the common endpoint of many CVDs. In this scenario, a better understanding of the role of epigenetics - hereditable changes of chromatin that do not alter the DNA sequence itself - is warranted. To date, hyperacetylation of histones has been reported in hypertension and myocardial infarction, but the use of inhibitors for treating CVDs remains limited. Here, we studied the effect of the histone deacetylase inhibitor Givinostat on a mouse model of acute myocardial infarction. We found that it contributes to decrease endothelial-to-mesenchymal transition and inflammation, reducing cardiac fibrosis and improving heart performance and protecting the blood vessels from apoptosis through the modulatory effect of cardiac fibroblasts on endothelial cells. Therefore, Givinostat may have potential for the treatment of CVDs.

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.

Single cell gene expression profiling is a fundamental tool for studying the heterogeneity of a cell population by addressing the phenotypic and functional characteristics of each cell. Technological advances that have coupled microfluidic technologies with high-throughput quantitative RT-PCR analyses have enabled detailed analyses of single cells in various biological contexts. In this chapter, we describe the procedure for isolating the skeletal muscle interstitial cells termed Fibro-Adipogenic Progenitors (FAPs ) and their gene expression profiling at the single cell level. Moreover, we accompany our bench protocol with bioinformatics analysis designed to process raw data as well as to visualize single cell gene expression data. Single cell gene expression profiling is therefore a useful tool in the investigation of FAPs heterogeneity and their contribution to muscle homeostasis.

SWI/SNF chromatin-remodeling complexes are key regulators of the epigenetic modifications that determine whether stem cells maintain pluripotency or commit toward specific lineages through development and during postnatal life. Dynamic combinatorial assembly of multiple variants of SWI/SNF subunits is emerging as the major determinant of the functional versatility of SWI/SNF. Here, we summarize the current knowledge on the structural and functional properties of the alternative SWI/SNF complexes that direct stem cell fate toward skeletal muscle lineage and control distinct stages of skeletal myogenesis. In particular, we will refer to recent evidence pointing to the essential role of two SWI/SNF components not expressed in embryonic stem cells-the catalytic subunit BRM and the structural component BAF60C-whose induction in muscle progenitors coincides with the expansion of their transcriptional repertoire.

Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex--Brahma (Brm) and Brg1--are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes., (© 2015 The Authors.)

Previous work linked nitric oxide (NO) signaling to histone deacetelyases (HDACs) in the control of tissue homeostasis and suggested that deregulation of this signaling contributes to human diseases. In the previous issue of Chemistry & Biology, Kong and colleagues showed that coordinated NO signaling and histone acetylation are required for proper cranial neural crest development and craniofacial morphogenesis and suggested that alterations of NO/acetylation network can contribute to the pathogenesis of craniofacial malformations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

During embryonic development, pluripotent cells are genetically committed to specific lineages by the expression of cell-type-specific transcriptional activators that direct the formation of specialized tissues and organs in response to developmental cues. Chromatin-modifying proteins are emerging as essential components of the epigenetic machinery, which establishes the nuclear landscape that ultimately determines the final identity and functional specialization of adult cells. Recent evidence has revealed that discrete populations of adult cells can retain the ability to adopt alternative cell fates in response to environmental cues. These cells include conventional adult stem cells and a still poorly defined collection of cell types endowed with facultative phenotype and functional plasticity. Under physiological conditions or adaptive states, these cells cooperate to support tissue and organ homeostasis, and to promote growth or compensatory regeneration. However, during chronic diseases and aging these cells can adopt a pathological phenotype and mediate maladaptive responses, such as the formation of fibrotic scars and fat deposition that progressively replaces structural and functional units of tissues and organs. The molecular determinants of these phenotypic transitions are only emerging from recent studies that reveal how dynamic chromatin states can generate flexible epigenetic landscapes, which confer on cells the ability to retain partial pluripotency and adapt to environmental changes. This review summarizes our current knowledge on the role of the epigenetic machinery as a 'filter' between genetic commitment and environmental signals in cell types that can alternatively promote skeletal muscle regeneration or fibro-adipogenic degeneration., (© 2013 FEBS.)

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

The capacity of adult skeletal muscle for regeneration appears to be limited, with progressive impairment in repair efficiency of injured muscles observed in chronic muscular disorders and during aging. While satellite cells, the committed adult muscle stem cells, are the main direct cell source supporting the regenerative potential of adult skeletal muscles, the characterization of the cell types and signals that constitute the functional "niche" of satellite cells is currently the object of intense investigation. Recent studies have identified a functional relationship between satellite cells and various cell types located in key anatomical position, such as the interstitium of skeletal muscles. This heterogeneous population of muscle interstitial cells (MICs) appears to retain an intrinsic multipotency within the mesodermal lineage, and their direct or indirect contribution to myofiber turnover, repair and degeneration has been suggested by many studies that will be reviewed here. Given the existing gap of knowledge on lineage identity and functional properties of MICs, their detailed characterization at the single cell level holds the promise to provide key insight into the composition of this heterogeneous population and the dynamic transition through distinct sub-populations in healthy, diseased and aging muscles. This review provides an overview of the results of various studies describing the phenotype and the function of cells isolated from skeletal muscle interstitium, and discusses the importance of single cell transcription profiling in order to decipher the functional and phenotypical heterogeneity of muscle interstitial cells (MICs).

Skeletal muscle diseases heavily impair the strength and the movement of patients. Muscles loose their adaptive capacity, undergoing atrophy or wasting, due to a number of pathological insults. Metabolic changes, such as those occurring during aging, contribute to the progressive reduction of myofiber size and decline of skeletal muscle performance that is typically observed in the elderly. The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 has been involved in the protection against metabolic disorders, against cancers and in the enhancement of life span. Here we discuss the current evidence placing SIRT1 at the crossroad between energy homeostasis, fiber strength, and regeneration from damage in the skeletal muscle. Furthermore, we underline how cell type specific targeting of SIRT1 could be beneficial in the treatment of skeletal muscle diseases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

Nuclear acetyltransferases promote and deacetylases inhibit skeletal muscle-gene expression, suggesting the potential effectiveness of deacetylase inhibitors (DIs) in modulating skeletal myogenesis. Surprisingly, previous studies have indicated that DIs suppress myogenesis. The recent observations that histone deacetylases associate with the muscle-regulatory proteins MyoD and MEF2C only in undifferentiated myoblasts prompted us to evaluate the effect of DIs at distinct stages of the myogenic program. We found that exposure of established rodent and human muscle cells to distinct DIs has stage-specific effects. Exposure of undifferentiated skeletal myoblasts to DIs, followed by incubation in differentiation medium, enhanced the expression of muscle-specific reporters and increased the levels of endogenous muscle proteins, leading to a dramatic increase in the formation of multinucleated myotubes. By contrast, simultaneous exposure of muscle cells to differentiation medium and DIs inhibited the myogenic program. Likewise, embryos exposed in utero to nonteratogenic doses of DI at the early stages of somitic myogenesis (embryonic day 8.5) exhibited an increased number of somites and augmented expression of a muscle-specific transgene as well as endogenous muscle genes. The functional effects induced by DIs were mirrored by changes in the state of acetylation of histones present at a muscle-gene enhancer and of MyoD itself. These results represent the first evidence that DIs can enhance muscle differentiation and suggest the rationale for their use in manipulating adult and embryonic skeletal myogenesis.