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In this issue of Cell Stem Cell, Porpiglia et al.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)—the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle. Sin financiación 31.373 JCR (2021) Q1, 8/195 Cell Biology 9.297 SJR (2021) Q1, 9/293 Cell Biology No data IDR 2021 UEM

While Fibro-Adipogenic Progenitors (FAPs) have been originally identified as muscle-interstitial mesenchymal cells activated in response to muscle injury and endowed with inducible fibrogenic and adipogenic potential, subsequent studies have expanded their phenotypic and functional repertoire and revealed their contribution to skeletal muscle response to a vast range of perturbations. Here we review the emerging contribution of FAPs to skeletal muscle responses to motor neuron injuries and to systemic physiological (e.g., exercise) or pathological metabolic (e.g., diabetes) perturbations. We also provide an initial blueprint of discrete sub-clusters of FAPs that are activated by specific perturbations and discuss their role in muscle adaptation to these conditions.

The comprehensive characterization of mechanisms safeguarding cell fate identity in differentiated cells is crucial for 1) our understanding of how differentiation is maintained in healthy tissues or misregulated in disease states and 2) to improve our ability to use direct reprogramming for regenerative purposes. To uncover novel fate-stabilizing regulators, we employed a genome-wide TF siRNA screen followed by a high-complexity combinatorial evaluation of top performing hits, in a cardiac reprogramming assay in mouse embryonic fibroblasts, and subsequently validated our findings in cardiac, neuronal and iPSCs reprogramming assays in primary human fibroblasts and adult endothelial cells. This approach identified a conserved set of 4 TFs (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming, as demonstrated by up to 6-fold increases in efficiency upon AJSZ knockdown in both lineage- and cell type-independent manners. Mechanistically, ChIP-seq and single-cell ATAC-seq analyses, revealed that AJSZ bind to both open and closed chromatin in a genome-wide and regionalized fashion, thereby limiting reprogramming TFs access to target DNA and ability to remodel the chromatin. In parallel, integration of ChIP-seq and RNA-seq data followed by systematic functional gene testing, identified that AJSZ also promote cell fate stability by proximally down-regulating a conserved set of genes involved in the regulation of cell fate specification (MEF2C), proteome remodeling (TPP1, PPIC), ATP homeostasis (EFHD1), and inflammation signaling (IL7R), thereby limiting cells ability to undergo large-scale phenotypic changes. Finally, simultaneous knock-down of AJSZ in combination with cardiac reprogramming TFs overexpression improved heart function by 250% as compared to no treatment and 50% as compared to MGT, 1 month after myocardial infarction. In sum, this study uncovers a novel evolutionarily conserved mechanism mediating cell fate stability in differentiated cells and also identifies AJSZ as promising therapeutic targets for regenerative purposes in adult organs.Significance StatementDifferentiated cells can be converted from one cell type into another by overexpressing lineage-determining transcription factors. Direct lineage reprogramming represents a promising strategy for regenerative medicine, but current clinical applications remain limited by the low yield of the reprogramming process. Here, we present the identification and detailed mechanistic study of a novel mechanisms opposing reprogramming process and promoting cell fate stability. Using a highthroughput screening technique, we identified four transcription factors that act as blockades to cell type change. By tracking chromatin and gene expression changes, we reconstructed a conserved pathway mediating cell fate stability in differentiated cells.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices., Induction of mitochondrial oxidative respiration is required for stem cell differentiation, but the mechanisms underlying this process are poorly understood. Here, the authors report that Stat3 promotes muscle stem cell differentiation by stimulating mitochondrial respiration via Fam3a.

International audience; During denervation induced muscle atrophy, the loss of neuro-muscular junction (NMJ) integrity and the consequent cessation of nerve signal transmission to muscle, lead to a decline in myofiber size mass and contractile activity. However, the identity of the cell types implicated in the muscle response to nerve injury has not been clearly defined. Here, we describe a subpopulation of muscle resident glial cells activated by loss of NMJ integrity. Gene expression analysis at bulk and single cell level revealed the existence of a population of Itga7-expressing cells, which are distinct from muscle satellite cells and are selectively activated upon nerve injury. Upon nerve lesion, these cells expanded and activated a neurotrophic gene program, including the expression of a prospective selection marker - Ngfr - and a number of neurotrophic genes as well as ECM components. Among them, we observed that Tenascin C (Tnc) was specifically produced by muscle glial cells activated by nerve injury and preferentially localized to NMJ. Activation of muscle-resident glial cells by nerve injury induced a neurotrophic phenotype, which was reversible upon recovery of NMJ integrity; by contrast, muscle-resident glial cells in skeletal muscles of a mouse model of Amyotrophic Lateral Sclerosis (ALS) steadily increased over the course of the disease and exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.

SummaryDevelopmental synaptogenesis toward formation of neuromuscular junctions (NMJs) is regulated by the reciprocal exchange of signals derived from nerve or muscle ends, respectively. These signals are re-deployed in adult life to repair NMJ lesions. The emerging heterogeneity of skeletal muscle cellular composition and the functional interplay between different muscle-resident cell types activated in response to homeostatic perturbations challenge the traditional notion that muscle-derived signals uniquely derive from myofibers. We have used single cell RNA sequencing (scRNA-seq) for a longitudinal analysis of gene expression profiles in cells isolated from skeletal muscles subjected to denervation by complete sciatic nerve transection. Our data show that, unlike muscle injury, which massively activates multiple muscle-resident cell types, denervation selectively induced the expansion of two cell types - muscle glial cells and activated fibroblasts. These cells were also identified as putative sources of muscle-derived signals implicated in NMJ repair and extracellular matrix (ECM) remodelling. Pseudo-time analysis of gene expression in muscle glial-derived cells at sequential timepoints post-denervation revealed an initial bifurcation into distinct processes related to either cellular de-differentiation and commitment to specialized cell types, such as Schwann cells, or ECM remodeling. However, at later time points muscle glial-derived cells appear to adopt a more uniform pattern of gene expression, dominated by a reduction of neurogenic signals. Consensual activation of pro-fibrotic and pro-atrophic genes from fibroblasts and other muscle-resident cell types suggests a global conversion of denervated muscles into an environment hostile for NMJ repair, while conductive for progressive development of fibrosis and myofiber atrophy.

Background In Duchenne muscular dystrophy (DMD), DYSTROPHIN deficiency exposes myofibers to repeated cycles of contraction/degeneration, ultimately leading to muscle loss and replacement by fibrotic tissue. DMD pathology is typically exacerbated by excessive secretion of TGFβ and consequent accumulation of pro-fibrotic components of the extra-cellular matrix (ECM), which in turn impairs compensatory regeneration and complicates the efficacy of therapeutic strategies. It is currently unclear whether DMD skeletal muscle fibers directly contribute to excessive activation of TGFβ. Development of skeletal myofibers from DMD patient-derived induced pluripotent stem cells (iPSC), as an “in dish” model of disease, can be exploited to determine the myofiber contribution to pathogenic TGFβ signaling in DMD and might provide a screening platform for the identification of anti-fibrotic interventions in DMD. Methods We describe a rapid and efficient method for the generation of contractile human skeletal muscle cells from DMD patient-derived hiPSC, based on the inducible expression of MyoD and BAF60C (encoded by SMARCD3 gene), using an enhanced version of piggyBac (epB) transposone vectors. DMD iPSC-derived myotubes were tested as an “in dish” disease model and exposed to environmental and mechanical cues that recapitulate salient pathological features of DMD. Results We show that DMD iPSC-derived myotubes exhibit a constitutive activation of TGFβ-SMAD2/3 signaling. High-content screening (HCS)-based quantification of nuclear phosphorylated SMAD2/3 signal revealed that DMD iPSC-derived myotubes also exhibit increased activation of the TGFβ-SMAD2/3 signaling following exposure to either recombinant TGFβ or electrical pacing-induced contraction. Conclusions Acute conversion of DMD patient-derived iPSC into skeletal muscles, by the ectopic expression of MyoD and BAF60C, provides a rapid and reliable protocol for an “in dish” DMD model that recapitulates key pathogenic features of disease pathology, such as the constitutive activation of the TGFβ/SMAD signaling as well as the deregulated response to pathogenic stimuli, e.g., ECM-derived signals or mechanical cues. Thus, this model is suitable for the identification of new therapeutic targets in DMD patient-specific muscles.

Aging and type 2 diabetes mellitus (T2DM) are associated with impaired skeletal muscle function and degeneration of the skeletal muscle microenvironment. However, the origin and mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscles of T2DM patients exhibit pathological degenerative remodeling of the extracellular matrix that was associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90) - the FAPCD90+. We identified Platelet-derived growth factor (PDGF) signaling as key regulator of human FAP biology, as it promotes proliferation and collagen production at the expense of adipogenesis, an effect accompanied with a metabolic shift towards glycolytic lactate fermentation. FAPsCD90+ showed a PDGF-mimetic phenotype, with high proliferative activity and clonogenicity, increased production of extracellular matrix production and enhanced glycolysis. Importantly, the pathogenic phenotype of T2DM FAPCD90+ was reduced by treatment with the anti-diabetic drug Metformin. These data identify PDGF-driven conversion of a sub-population of FAPs as a key event in the pathogenic accumulation of extracellular matrix in T2DM muscles.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation. Research in Beato's lab is supported by funds from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC Synergy grant agreement 609989 (4DGenome), from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Program/Generalitat de Catalunya. L.B was supported by the Forschungskredit 2019 of the University of Zurich and K.G by a grant of the Oncosuisse (Nr. KFS-3740-08-2015-R). ADP-ribosylation research in the laboratory of MOH is funded by the Kanton of Zurich and the Swiss National Science Foundation, Switzerland (grant 31003A_176177).

Functional interactions between cellular components of the muscle stem cell (MuSC) niche regulate the regenerative ability of skeletal muscles in physiological and pathological conditions; however, the identity of the mediators of these interactions remains largely unknown. We show here that fibro-adipogenic progenitor (FAP)-derived Extracellular Vesicles (EVs) mediate microRNA transfer to MuSCs, and that exposure of dystrophic FAPs to HDAC inhibitors (HDACi) increases the intra-EV levels of a subset of microRNAs (miRs), which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis and inflammation. In particular, we found that increased levels of miR206 in EVs released from FAPs of muscles from Duchenne dystrophic patients or mice (mdx) exposed to HDACi were associated with enhanced regeneration and inhibition of fibrosis of dystrophic muscles. Consistently, EVs from HDACi-treated dystrophic FAPs could stimulated MuSC activation and expansion ex vivo, and promoted regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation, in vivo. These data reveal a potential for pharmacological modulation of FAP-derived EV’s content as novel strategy for focal therapeutic interventions in Duchenne Muscular Dystrophy (DMD) and possibly other muscular diseases.Brief SummaryExtracellular Vesicles from HDACi-treated dystrophic FAPs promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles

We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors--FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.

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.

Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD–Baf60c–Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression., Long non-coding RNAs are important regulators of many diverse biological processes. Here the authors describe Linc-RAM, which regulates myogenesis by binding MyoD and promoting the assembly of the MyoD–Baf60c–Brg1 complex at target genes.

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles., Author summary Muscular dystrophies are a heterogenous group of genetic disorders characterized by muscle wasting, leading to loss of mobility and eventually to death due to respiratory or cardiac failure. Duchenne Muscular Dystrophy (DMD) is one of the most severe dystrophies and is caused by the loss of functional dystrophin protein owing to genetic mutations, consequently, the sarcolemma becomes fragile and susceptible to muscle damage induced by contraction. Satellite cells (SCs) are skeletal muscle stem cells that mediate the repair process leading to muscle regeneration. Dystrophic muscles undergo continuous cycles of degeneration and regeneration eventually culminating in myofiber loss and deposition of fibrous and fatty connective tissue. Inflammation is always associated with the muscle regeneration process. Among different types of inflammatory cells, mainly macrophages (MΦs) are present in regenerating skeletal muscles and are involved in the regenerative process both after an acute injury and during pathological conditions such as DMD. We focused on the cross-talk between MΦs and SCs in a mouse model of DMD and highlighted a role of MΦs in preserving the SC identity.

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.

The endocannabinoid system refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. However, the potential role of endocannabinoids in skeletal muscle disorders remains unknown. Here we report the role of the endocannabinoid CB1 receptors in Duchenne’s muscular dystrophy. In murine and human models, CB1 transcripts show the highest degree of expression at disease onset, and then decline overtime. Similar changes are observed for PAX7, a key regulator of muscle stem cells. Bioinformatics and biochemical analysis reveal that PAX7 binds and upregulates the CB1 gene in dystrophic more than in healthy muscles. Rimonabant, an antagonist of CB1, promotes human satellite cell differentiation in vitro, increases the number of regenerated myofibers, and prevents locomotor impairment in dystrophic mice. In conclusion, our study uncovers a PAX7–CB1 cross talk potentially exacerbating DMD and highlights the role of CB1 receptors as target for potential therapies., The regenerative capacity of muscle stem cells is impaired in Duchenne muscular dystrophy (DMD). Here, the authors show that the endocannabinoid receptor CB1 is activated by PAX7 in muscle stem cells, and that pharmacological inhibition of CB1 promotes stem cell activation and ameliorates symptoms in DMD mouse models.

Since its discovery as a skeletal muscle-specific transcription factor able to reprogram somatic cells into differentiated myofibers, MyoD has provided an instructive model to understand how transcription factors regulate gene expression. Reciprocally, studies of other transcriptional regulators have provided testable hypotheses to further understand how MyoD activates transcription. Using MyoD as a reference, in this review, we discuss the similarities and differences in the regulatory mechanisms employed by tissue-specific transcription factors to access DNA and regulate gene expression by cooperatively shaping the chromatin landscape within the context of cellular differentiation.

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3-IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3-IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2high or Tie2low and Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome., Fibro-adipogenic progenitors (FAPs) resident in skeletal muscle are involved in both regeneration and maladaptive processes. Here, the authors identify subpopulations of FAPs with biological activities implicated in physiological muscle repair that are altered in pathological conditions such as muscular dystrophies.

International audience; Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix–loop–helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation—Tcf3 and Foxa2—and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1–4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.

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.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs leads to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.

Age-related muscle decline is associated with functional impairment of satellite cells (SCs). Conflicting data suggest dysregulation of cell-extrinsic or -intrinsic factors can independently contribute to such impairment. Here, we emphasize the importance of identifying nodes that integrate these factors into feed-forward circuits, which could provide targets for therapeutic intervention.

SummaryDirect generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for “disease in a dish” models of muscular physiology and dysfunction.

Rationale: Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level. Objective: We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity. Methods and Results: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1β and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 β/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process. Conclusions: We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.

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.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.

Autophagy is emerging as a key regulatory process during skeletal muscle development, regeneration and homeostasis, and deregulated autophagy has been implicated in muscular disorders and age-related muscle decline. We have monitored autophagy in muscles of mdx mice and human Duchenne muscular dystrophy (DMD) patients at different stages of disease. Our data show that autophagy is activated during the early, compensatory regenerative stages of DMD. A progressive reduction was observed during mdx disease progression, in coincidence with the functional exhaustion of satellite cell-mediated regeneration and accumulation of fibrosis. Moreover, pharmacological manipulation of autophagy can influence disease progression in mdx mice. Of note, studies performed in regenerating muscles of wild-type mice revealed an essential role of autophagy in the activation of satellite cells upon muscle injury. These results support the notion that regeneration-associated autophagy contributes to the early compensatory stage of DMD progression, and interventions that extend activation of autophagy might be beneficial in the treatment of DMD. Thus, autophagy could be a ‘disease modifier’ targeted by interventions aimed to promote regeneration and delay disease progression in DMD.

Skeletal muscle differentiation relies on the coordinated activation and repression of specific subsets of genes. This reflects extensive changes in chromatin architecture, composition of chromatin-associated complexes and histone modifications at the promoter/enhancer elements of skeletal muscle genes. An early, key event in the activation of muscle-specific gene transcription is the disruption of the repressive conformation imposed by nucleosomes, which impede the access of pioneer transcription factors, such as the muscle-specific basic helix-loop-helix (bHLH) factors MyoD and Myf5, to their DNA-binding sites. This review focuses on our current understanding of the role of the SWI/SNF ATP-dependent chromatin-remodeling complex in the activation of the myogenic program, by inducing conformational changes permissive for muscle-gene expression. Recent findings suggest that specific combinations of individual SWI/SNF components can generate sub-complexes with specialized functions that are engaged at sequential stages of muscle-gene activation--e.g., initial displacement of the nucleosome followed by the loading of the complete myogenic transcriptosome that promotes gene transcription. SWI/SNF composition and function is regulated by the exchange of specific variants of structural sub-units. In turn, an exchange of histone variants and related epigenetic modifications might reflect the impact of distinct SWI/SNF complexes on the architecture and activity of target promoter/enhancer elements. Thus, the SWI/SNF complexes should be regarded not just as simple executors of the program imposed by transcription factors, but as multifaceted "readers" and "shapers" of the chromatin/DNA landscape within target muscle genes along the transition from myoblasts to myotubes.

Summary How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified an inflammation-activated signaling in muscle stem (satellite) cells, by which the polycomb repressive complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38α kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EZH2, the enzymatic subunit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. TNF-α antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signaling—p38α and EZH2—invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signaling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signaling to Pax7 and satellite cell decision to proliferate or differentiate.

Muscle regeneration provides a paradigm by which to study how extrinsic signals coordinate gene expression in somatic stem cells (satellite cells) by directing the genome distribution of chromatin-modifying complexes. Understanding the signal-dependent control of the epigenetic events underlying the transition of muscle stem cells through sequential regeneration stages holds the promise to reveal new targets for selective interventions toward repairing diseased muscles. This review describes the latest findings on how regeneration cues are integrated at the chromatin level to build the transcription network that regulates progression of endogenous muscle progenitors throughout the myogenic program. In particular, we describe how specific epigenetic signatures can confer responsiveness to extrinsic cues on discrete regions of the muscle stem cell genome.

The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1α promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1α and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway.

Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.

This review focuses on our current knowledge of the epigenetic changes regulating gene expression at the chromatin and DNA level, independently on the primary DNA sequence, to reprogram the nuclei of muscle precursors during developmental myogenesis and muscle regeneration. These epigenetic marks provide the blueprint by which the extra-cellular cues are interpreted at the nuclear level by the transcription machinery to select the repertoire of tissue-specific genes to be expressed. The reversibility of some of these changes necessarily reflects the dynamic nature of skeletal myogenesis, which entails the progression through two antagonistic processes--proliferation and differentiation. Other epigenetic modifications are instead associated to events conventionally considered as irreversible--e.g. maintenance of lineage commitment and terminal differentiation. However, recent results support the possibility that these events can be reversed, at least upon certain experimental conditions, thereby revealing a dynamic nature of many of the epigenetic modifications underlying skeletal myogenesis. The elucidation of the epigenetic network that regulates transcription during developmental myogenesis and muscle regeneration might provide the information instrumental to devise pharmacological interventions toward selective manipulation of gene expression to promote regeneration of skeletal muscles and possibly other tissue.

Transcriptional and posttranscriptional processes regulate expression of genetic networks in response to environmental cues. The extracellular signal-activated p38 MAP kinase (p38) pathway plays a fundamental role in conversion of myoblasts to differentiated myocytes. p38 phosphorylates specific transcription factors and chromatin-associated proteins promoting assembly of the myogenic transcriptome. Here, we demonstrate that p38 alpha and beta isoforms also control muscle-gene expression posttranscriptionally, by stabilizing critical myogenic transcripts. KSRP, an important factor for AU-rich element (ARE)-directed mRNA decay, undergoes p38-dependent phosphorylation during muscle differentiation. KSRP phosphorylated by p38 displays compromised binding to ARE-containing transcripts and fails to promote their rapid decay, although it retains the ability to interact with the mRNA degradation machinery. Overexpression of KSRP selectively impairs induction of ARE-containing early myogenic transcripts, without affecting p38-mediated transcriptional responses. Our results uncover an unanticipated role for KSRP in establishing a biochemical link between differentiation-activated p38 signaling and turnover of myogenic mRNAs.

During skeletal myogenesis, muscle-regulatory factors bHLH and MEF2 promote the expression of muscle-specific genes by recruiting several chromatin-modifying complexes on specific DNA regulatory sequences. A number of MyoD-interacting proteins have been reported, but whether they are recruited to the chromatin of myogenic loci, and the relationship with other chromatin bound proteins is unknown. We show that MyoD recruits cdk9/cyclin T2, together with the histone acetyltransferases p300 and PCAF, and the chromatin remodeling complex SWI/SNF, on promoters and enhancers of muscle-specific genes, and that this event correlates with the acetylation of histone tails, remodeling of chromatin, and phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II at these elements.

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

Angiotensin-II (AII), the dominant effector of the renin-angiotensin system, is involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin in endothelial cells by inflammatory cytokines through nuclear factor kappa B (NFkappaB) activation is implicated in formation and progression of atherosclerotic plaque. Here we show that AII induces NFkappaB-dependent transcription in primary endothelial cell lines, leading to the upregulation of ICAM-1 and VCAM-1 expression. NFkappaB activation by AII is mediated by the NFkappaB-inducing kinase (NIK), a common mediator of NFkappaB activation by inflammatory cytokines, such as TNF-alpha. However, NFkappaB stimulation by AII differs from that of TNF-alpha since a TNF-receptor associated factor 2 (TRAF-2) dominant negative mutant does not prevent AII-mediated NFkappaB activation. In analogy with TNF-alpha-dependent activation of NFkappaB, treatment with either the anti-oxidant N-acetyl cysteine (NAC) or the cyclooxygenase (COX) inhibitor acetyl salicylic acid (aspirin), but not indometacin, prevents the induction of NFkappaB-dependent transcription by AII. Thus, production of reactive oxygen species, aspirin (asp)-sensitive enzymes of the arachidonate metabolism, and NIK are common transducers of AII- and TNF-dependent pathways to NFkappaB. AII also activates the inflammatory p38 kinase in endothelial cells, an effect inhibited by exposure to either NAC or asp. Pharmacological interference of the p38 pathway, with the inhibitor SB 202190, prevented AII-mediated activation of the NFkappaB target V-CAM, without affecting degradation of IkappaBalpha. These results support a pro-inflammatory effect of the vasoactive peptide AII in endothelial cells, through at least two pathways-NFkappaB and p38-both of which are sensitive to asp and antioxidants.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.