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Neurogenic muscle atrophy is a hallmark of several degenerative processes involved in different pathologies, such as Amyotrophic Lateral Sclerosis (ALS). This condition is characterized by progressive motor neurons (MNs) degeneration, which leads to a deterioration of neuromuscular functions, causing weakness, paralysis and atrophy of skeletal muscles. During neurogenic muscle atrophy, the interruption of transmission of neurogenic signals to muscles, caused by loss of neuromuscular junction (NMJ) integrity, activates protein breakdown and reduces protein synthesis, leading to the loss of muscle mass and contractile activity. To dissect the role of muscle resident cells in the maintenance of the cross talk between muscle and nerve, we use next generation sequencing, in-vitro and in-vivo studies. Thanks to these complementary approaches, we identified new cellular and molecular players that underlie the neurogenic muscle atrophy. Perturbations of cellular response, that includes the activation of muscle resident cells (FAPs and Glial cells) that partecipate to neurogenic muscle atrophy has been recently observed (Madaro et al. 2018; Proietti et al. 2021). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. The incidence of obesity has reached alarming proportions worldwide, and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. However, to date, mechanisms underlying intergenerational heart disease risks have yet to be elucidated. Here we report that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. We provide evidence that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, we find that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations. Animal studies have shown that the nutritional status of parents can predispose the offspring to obesity and obesity-related diseases. Here the authors show that cardiac dysfunction induced by a high-fat diet persists for two generations in Drosophila, and that targeted expression of ATGL/bmm in the offspring, as well as inhibition of H3K27 trimethylation, is cardioprotective. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate muscle gene expression. HDAC inhibitors (HDACis) enhance the process of skeletal myogenesis. HDACis inhibit fibro-adipogenic degeneration, while promoting muscle regeneration in muscles of animal models and patients with Duchenne muscular dystrophy (DMD). Fibro-adipogenic progenitors (FAPs) are central cellular mediators of HDACi effects in DMD muscles. Extracellular vesicles isolated from FAPs exposed to HDACis might exert a therapeutic effect equivalent to that of HDACis. HDACi-based treatment for DMD can be improved by developing new compounds with optimized therapeutic profiles. Earlier evidence that targeting the balance between histone acetyltransferases (HATs) and deacetylases (HDACs), through exposure to HDAC inhibitors (HDACis), could enhance skeletal myogenesis, prompted interest in using HDACis to promote muscle regeneration. Further identification of constitutive HDAC activation in dystrophin-deficient muscles, caused by dysregulated nitric oxide (NO) signaling, provided the rationale for HDACi-based therapeutic interventions for Duchenne muscular dystrophy (DMD). In this review, we describe the molecular, preclinical, and clinical evidence supporting the efficacy of HDACis in countering disease progression by targeting pathogenic networks of gene expression in multiple muscle-resident cell types of patients with DMD. Given that givinostat is paving the way for HDACi-based interventions in DMD, next-generation HDACis with optimized therapeutic profiles and efficacy could be also explored for synergistic combinations with other therapeutic strategies. [ABSTRACT FROM AUTHOR]

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 &y& Elsevier]

The recent article from Gómez-del Arco et al () in Cell Metabolism reveals the essential role of chromodomain-helicase-DNA-binding protein 4 ( CHD4) in the control of alternative gene expression in cell types that share seemingly redundant, yet distinct, biological properties, such as the contractile activities of striated cardiac or skeletal muscles. An altered expression of genes for alternative sarcomeric and metabolic programs occurred upon genetic inactivation of Chd4 in skeletal or cardiac cells, leading to formation of 'hybrid' striated muscles and pathological outcomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair. [Display omitted] • Activated satellite cells (ASCs) show micro-heterogeneity of surface CRIPTO protein • CRIPTO micro-heterogeneity identifies phenotypic and molecular variants within ASCs • Fluctuation of surface CRIPTO preserves ASC self-renewal/differentiation equilibrium • CRIPTO micro-heterogeneity secures ASC adaptive response to microenvironmental cues Guardiola et al. investigate intrinsic cell-to-cell variability in mouse-activated satellite cell (ASC) population in skeletal muscle regeneration. ASCs display a continuum of metastable cell states along with CRIPTO expression gradient, whose dynamic fluctuation enables rapid adaptation to changing microenvironment and preserves muscle stem cell population diversity. [ABSTRACT FROM AUTHOR]

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene leading to dystrophin deficiency, muscle fiber degeneration and progressive fibrotic replacement of muscles. Givinostat, a histone deacetylase (HDAC) inhibitor, significantly reduced fibrosis and promoted compensatory muscle regeneration in mdx mice. This study was conducted to evaluate whether the beneficial histological effects of Givinostat could be extended to DMD boys. Twenty ambulant DMD boys aged 7 to <11 years on stable corticosteroid treatment were enrolled in the study and treated for ≥12 months with Givinostat. A muscle biopsy was collected at the beginning and at the end of treatment to evaluate the amount of muscle and fibrotic tissue. Histological effects were the primary objectives of the study. Treatment with Givinostat significantly increased the fraction of muscle tissue in the biopsies and reduced the amount of fibrotic tissue. It also substantially reduced tissue necrosis and fatty replacement. Overall the drug was safe and tolerated. Improvement in functional tests was not observed in this study, but the sample size of the study was not sufficient to draw definitive conclusions. This study showed that treatment with Givinostat for more than 1 year significantly counteracted histological disease progression in ambulant DMD boys aged 7 to 10 years. [ABSTRACT FROM AUTHOR]

The progressive loss of muscle regenerative capacity with age or disease results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair. However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions. The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting, and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues. Here we show that IL-6-activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myod1) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition on cell fate and proliferation were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells in pathological conditions, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases. [ABSTRACT FROM AUTHOR]

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26?years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased ?-catenin activity in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-?) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-? pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2?months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies. [ABSTRACT FROM AUTHOR]

Previous work has established the existence of dystrophin–nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice—the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients. [ABSTRACT FROM AUTHOR]

Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38? kinase is the signal that promotes incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues. [ABSTRACT FROM AUTHOR]

Histone deacetylases inhibitors (HDACi) include a growing number of drugs that share the ability to inhibit the enzymatic activity of some or all the HDACs. Experimental and preclinical evidence indicates that these epigenetic drugs not only can be effective in the treatment of malignancies, inflammatory diseases and degenerative disorders, but also in the treatment of genetic diseases, such as muscular dystrophies. The ability of HDACi to counter the progression of muscular dystrophies points to HDACs as a crucial link between specific genetic mutations and downstream determinants of disease progression. It also suggests the contribution of epigenetic events to the pathogenesis of muscular dystrophies. Here we describe the experimental evidence supporting the key role of HDACs in the control of the transcriptional networks underlying the potential of dystrophic muscles either to activate compensatory regeneration or to undergo fibroadipogenic degeneration. Studies performed in mouse models of Duchenne muscular dystrophy (DMD) indicate that dystrophin deficiency leads to deregulated HDAC activity, which perturbs downstream networks and can be restored directly, by HDAC blockade, or indirectly, by reexpression of dystrophin. This evidence supports the current view that HDACi are emerging candidate drugs for pharmacological interventions in muscular dystrophies, and reveals unexpected common beneficial outcomes of pharmacological treatment or gene therapy. [ABSTRACT FROM AUTHOR]

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 led 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. • MYOD drives the re-wiring of chromatin interactions during trans -differentiation • MYOD alters chromatin interactions between cis -regulatory elements • MYOD re-wires insulated neighborhoods, hotspots of differential interactions • MYOD re-wiring of chromatin interactions temporally precedes transcriptional changes Dall'Agnese et al. find that the myogenic master transcription factor MYOD drives significant re-wiring of the 3D chromatin architecture during somatic reprogramming toward transdifferentiation, in order to erase the cell-of-origin transcriptional program and activate skeletal myogenesis. MYOD-directed reconfiguration of chromatin interactions involves cis -regulatory and structural genomic elements and temporally precedes transcriptional regulation of target genes. [ABSTRACT FROM AUTHOR]

An abstract of the article "Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment" by Wenqing Cai and colleagues is presented.

Computational enhancement is an important strategy for inferring high-resolution features from genome-wide chromosome conformation capture (Hi-C) data, which typically have limited resolution. Deep learning has been highly successful in this task but we show that it creates prevalent artificial structures in the enhanced data due to the need to divide the large contact matrix into small patches. In addition, previous deep learning methods largely focus on local patterns, which cannot fully capture the complexity of Hi-C data. Here we propose Smooth, High-resolution, and Accurate Reconstruction of Patterns (SHARP) for enhancing Hi-C data. It uses the novel approach of decomposing the data into three types of signals, due to one-dimensional proximity, contiguous domains, and other fine structures, and applies deep learning only to the third type of signals, such that enhancement of the first two is unaffected by the patches. For the deep learning part, SHARP uses both local and global attention mechanisms to capture multi-scale contextual information. We compare SHARP with state-of-the-art methods extensively, including application to data from new samples and another species, and show that SHARP has superior performance in terms of resolution enhancement accuracy, avoiding creation of artificial structures, identifying significant interactions, and enrichment in chromatin states.

Age-related reduction in muscle stem cell (MuSC) regenerative capacity is associated with cell-autonomous and non-cell-autonomous changes caused by alterations in systemic and skeletal muscle environments, ultimately leading to a decline in MuSC number and function. Previous studies demonstrated that STAT3 plays a key role in driving MuSC expansion and differentiation after injury-activated regeneration, by regulating autophagy in activated MuSCs. However, autophagy gradually declines in MuSCs during lifespan and contributes to the impairment of MuSC-mediated regeneration of aged muscles. Here, we show that STAT3 inhibition restores the autophagic process in aged MuSCs, thereby recovering MuSC ability to promote muscle regeneration in geriatric mice. We show that STAT3 inhibition could activate autophagy at the nuclear level, by promoting transcription of autophagy-related genes, and at the cytoplasmic level, by targeting STAT3/PKR phosphorylation of eIF2α. These results point to STAT3 inhibition as a potential intervention to reverse the age-related autophagic block that impairs MuSC ability to regenerate aged muscles. They also reveal that STAT3 regulates MuSC function by both transcription-dependent and transcription-independent regulation of autophagy., (© 2024 Catarinella et al.)

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast- and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (≤2 × 10 8 particles/mL) and were inhibitory at the highest dose tested (2 × 10 11 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insulin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research., Competing Interests: M.J.A.W. and S.ELA. are founders, shareholders, and consultants for Evox Therapeutics, a biotech company that aims to commercialize extracellular vesicles., (© 2023 The Authors.)

We performed scRNA-seq/snATAC-seq of skeletal muscles post sciatic nerve transection to delineate cell type-specific patterns of gene expression/chromatin accessibility at different time points post-denervation. Unlike myotrauma, denervation selectively activates glial cells and Thy1 /CD90-expressing mesenchymal cells. Glial cells expressed Ngf receptor (Ngfr) and were located near neuromuscular junctions (NMJs), close to Thy1 /CD90-expressing cells, which provided the main cellular source of NGF post-denervation. Functional communication between these cells was mediated by NGF/NGFR, as either recombinant NGF or co-culture with Thy1 /CD90-expressing cells could increase glial cell number ex vivo . Pseudo-time analysis in glial cells revealed an initial bifurcation into processes related to either cellular de-differentiation/commitment to specialized cell types (e.g., Schwann cells), or failure to promote nerve regeneration, leading to extracellular matrix remodeling toward fibrosis. Thus, interactions between denervation-activated Thy1 /CD90-expressing and glial cells represent an early abortive process toward NMJs repair, ensued by the conversion of denervated muscles into an environment hostile for NMJ repair., Competing Interests: The authors declare no competing interests.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Defining the mechanisms safeguarding cell fate identity in differentiated cells is crucial to improve 1) - our understanding of how differentiation is maintained in healthy tissues or altered in a disease state, and 2) - our ability to use cell fate reprogramming for regenerative purposes. Here, using a genome-wide transcription factor screen followed by validation steps in a variety of reprogramming assays (cardiac, neural and iPSC in fibroblasts and endothelial cells), we identified a set of four transcription factors (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming in both lineage and cell type independent manners. Mechanistically, our integrated multi-omics approach (ChIP, ATAC and RNA-seq) revealed that AJSZ oppose cell fate reprogramming by 1) - maintaining chromatin enriched for reprogramming TF motifs in a closed state and 2) - downregulating genes required for reprogramming. Finally, KD of AJSZ in combination with MGT overexpression, significantly reduced scar size and improved heart function by 50%, as compared to MGT alone post-myocardial infarction. Collectively, our study suggests that inhibition of barrier to reprogramming mechanisms represents a promising therapeutic avenue to improve adult organ function post-injury., (© 2023. The Author(s).)

Pancreatic cancer is characterized by extensive resistance to conventional therapies, making clinical management a challenge. Here we map the epigenetic dependencies of cancer stem cells, cells that preferentially evade therapy and drive progression, and identify SWI/SNF complex member SMARCD3 as a regulator of pancreatic cancer cells. Although SWI/SNF subunits often act as tumor suppressors, we show that SMARCD3 is amplified in cancer, enriched in pancreatic cancer stem cells and upregulated in the human disease. Diverse genetic mouse models of pancreatic cancer and stage-specific Smarcd3 deletion reveal that Smarcd3 loss preferentially impacts established tumors, improving survival especially in context of chemotherapy. Mechanistically, SMARCD3 acts with FOXA1 to control lipid and fatty acid metabolism, programs associated with therapy resistance and poor prognosis in cancer. These data identify SMARCD3 as an epigenetic modulator responsible for establishing the metabolic landscape in aggressive pancreatic cancer cells and a potential target for new therapies., (© 2023. The Author(s).)

In this issue of Cell Stem Cell, Porpiglia et al. 1 report on alterations in CD47 and THBS1 expression and function in aged muscle stem cells that disrupt their regeneration capacity. Targeting THBS1-CD47 cross-signaling is sufficient to reverse sarcopenia and restore muscle mass and function in aged mice., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-β signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1-a transcriptional target of EPR-modulates translation of select transcripts., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre-clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late-stage mdx mice exhibit aberrant HDAC activity and genome-wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo-acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late-stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi-induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper-acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late-stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease-associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti-fibrotic effects at late stages of DMD., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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 FAP CD90+ . We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPs CD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAP CD90+ 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 H 2 O 2 -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., Competing Interests: The authors declare no financial interest., (© 2021 The Author(s).)

Here, we report on the identification of Itga7-expressing muscle-resident glial cells activated by loss of neuromuscular junction (NMJ) integrity. Gene expression analysis at the bulk and single-cell level revealed that these cells are distinct from Itga7-expressing muscle satellite cells. We show that a selective activation and expansion of Itga7+ glial cells occur in response to muscle nerve lesion. Upon activation, muscle glial-derived progenies expressed neurotrophic genes, including nerve growth factor receptor, which enables their isolation by FACS. We show that activated muscle glial cells also expressed genes potentially implicated in extracellular matrix remodeling at NMJs. We found that tenascin C, which was highly expressed by muscle glial cells, activated upon nerve injury and preferentially localized to NMJ. Interestingly, we observed that the activation of muscle glial cells by acute nerve injury was reversible upon NMJ repair. By contrast, in a mouse model of ALS, in which NMJ degeneration is progressive, muscle glial cells steadily increased over the course of the disease. However, they exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.

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., (© 2020 The Authors.)

Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (SOD G93A 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.