RESUMEN
Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI-67. The characterization of different senescence-associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single-cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry-based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well-established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy-related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence-related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL-6. Overall, the analysis framework prioritizes multiple senescence-associated proteins to characterize putative senescent cells sourced from tissue made of different cell types.
Asunto(s)
Biomarcadores , Senescencia Celular , Citometría de Flujo , Músculo Esquelético , Animales , Senescencia Celular/fisiología , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Citometría de Flujo/métodos , Biomarcadores/metabolismo , Femenino , Masculino , Ratones Endogámicos C57BL , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that CARDEL (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. CARDEL knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of CARDEL during differentiation. Altogether, we provide physiological and molecular evidence that CARDEL expression contributes to sculpting the cardiac program during cell-fate commitment.
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Diferenciación Celular , Corazón , Homeostasis , Miocitos Cardíacos , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Diferenciación Celular/genética , Corazón/embriología , Corazón/fisiología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Linaje de la Célula/genética , Organogénesis/genéticaRESUMEN
In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.
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Genes Homeobox , Proteínas de Homeodominio , Células Madre Embrionarias de Ratones , Factores de Transcripción , Animales , Ratones , Diferenciación Celular , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.
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Arbovirus , Infecciones por Bunyaviridae , Encefalitis de California , Virus La Crosse , Orthobunyavirus , Humanos , Niño , Animales , Ratones , Replicación Viral , MúsculosRESUMEN
Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.
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Células Madre Pluripotentes Inducidas , Humanos , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo , Miosinas Ventriculares/farmacología , Edición Génica , Miocardio , Miocitos Cardíacos/metabolismo , Diferenciación Celular , Miosinas/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismoRESUMEN
This scientific commentary refers to 'The FSHD muscle-blood biomarker: a circulating transcriptomic biomarker for clinical severity in facioscapulohumeral muscular dystrophy', by Banerji et al. (https://doi.org/10.1093/braincomms/fcad221).
RESUMEN
Double homeobox (DUX) genes are unique to eutherian mammals, expressed transiently during zygotic genome activation (ZGA) and involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer when misexpressed. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 cytotoxicity is not shared with DUXA or DUXB, but surprisingly is shared with platypus sDUX, which binds DNA as a homodimer and activates numerous ZGA genes and long terminal repeat (LTR) elements. DUXA, although transcriptionally inactive, has DNA binding overlap with DUX4, and DUXA-VP64 activates DUX4 targets and is cytotoxic. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is a DUX4 target gene, this competition potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises antagonistic members of opposing function, with implications for their roles in ZGA, FSHD, and cancer.
RESUMEN
A potential therapeutic target to curb obesity and diabetes is thermogenic beige adipocytes. However, beige adipocytes quickly transition into white adipocytes upon removing stimuli. Here, we define the critical role of cyclin dependent kinase inhibitor 2A (Cdkn2a) as a molecular pedal for the beige-to-white transition. Beige adipocytes lacking Cdkn2a exhibit prolonged lifespan, and male mice confer long-term metabolic protection from diet-induced obesity, along with enhanced energy expenditure and improved glucose tolerance. Mechanistically, Cdkn2a promotes the expression and activity of beclin 1 (BECN1) by directly binding to its mRNA and its negative regulator BCL2 like 1 (BCL2L1), activating autophagy and accelerating the beige-to-white transition. Reactivating autophagy by pharmacological or genetic methods abolishes beige adipocyte maintenance induced by Cdkn2a ablation. Furthermore, hyperactive BECN1 alone accelerates the beige-to-white transition in mice and human. Notably, both Cdkn2a and Becn1 exhibit striking positive correlations with adiposity. Hence, blocking Cdkn2a-mediated BECN1 activity holds therapeutic potential to sustain beige adipocytes in treating obesity and related metabolic diseases.
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Adipocitos Beige , Tejido Adiposo Beige , Obesidad , Animales , Humanos , Masculino , Ratones , Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Tejido Adiposo Beige/metabolismo , Tejido Adiposo Blanco/metabolismo , Adiposidad/genética , Adiposidad/fisiología , Obesidad/genética , Obesidad/metabolismo , TermogénesisRESUMEN
Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.
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ADN (Citosina-5-)-Metiltransferasas , ADN Metiltransferasa 3A , Animales , Humanos , Ratones , Diferenciación Celular/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Células Madre Hematopoyéticas/metabolismoRESUMEN
The muscle satellite cell population is responsible for homeostatic maintenance of muscle fibers in response to muscle injury and normal wear and tear. This population is heterogeneous, and its capacity for self-renewal and differentiation can be altered either by mutation of genes that regulate these processes or with natural processes such as aging. The satellite cell colony assay is a facile way to extract information about the proliferation and differentiation potential of individual cells. Here, we provide a detailed protocol for the isolation, single cell plating, culture, and evaluation of colonies derived from single satellite cells. The variables of cell survival (cloning efficiency), proliferative potential (nuclei per colony), and differentiation propensity (ratio of nuclei within myosin heavy chain-positive cytoplasm to total nuclei) can thus be obtained.
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Enfermedades Musculares , Células Satélite del Músculo Esquelético , Humanos , Fibras Musculares Esqueléticas , Diferenciación Celular/fisiología , Músculo Esquelético , Células CultivadasRESUMEN
During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.
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Células Madre Pluripotentes Inducidas , Ingeniería de Tejidos , Humanos , Ingeniería de Tejidos/métodos , Miocitos Cardíacos/fisiología , Miocardio , Diferenciación Celular/fisiología , Proliferación CelularRESUMEN
Double homeobox (DUX) genes are unique to eutherian mammals and normally expressed transiently during zygotic genome activation. The canonical member, DUX4, is involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer, when misexpressed in other contexts. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 activities are not shared with DUXA or DUXB, which lack transcriptional activation potential, but surprisingly are shared with platypus sDUX. In human myoblasts, platypus sDUX drives cytotoxicity, inhibits myogenesis, and induces DUX4 target genes, particularly those associated with zygotic genome activation (ZGA), by binding DNA as a homodimer in a way that overlaps the DUX4 homeodomain crystal structure. DUXA lacks transcriptional activity but has DNA-binding and chromatin accessibility overlap with DUX4 and sDUX, including on ZGA genes and LTR elements, and can actually be converted into a DUX4-like cytotoxic factor by fusion to a synthetic transactivation domain. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is an early DUX4 target gene, this activity potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. HIGHLIGHTS: Platypus sDUX is toxic and inhibits myogenic differentiation.DUXA targets overlap substantially with those of DUX4.DUXA fused to a synthetic transactivation domain acquires DUX4-like toxicity.DUXA behaves as a competitive inhibitor of DUX4.
RESUMEN
AIM: It has been suggested that the proliferation and early differentiation of myoblasts are impaired in Marfan syndrome (MFS) mice during muscle regeneration. However, the underlying cellular and molecular mechanisms remain poorly understood. Here, we investigated muscle regeneration in MFS mouse models by analyzing the influence of the fibrotic niche on satellite cell function. METHODS: In vivo, ex vivo, and in vitro experiments were performed. In addition, we evaluated the effect of the pharmacological inhibition of fibrosis using Ang-(1-7) on regenerating skeletal muscles of MFS mice. RESULTS: The skeletal muscle of MFS mice shows an increased accumulation of collagen fibers (81.2%), number of fibroblasts (157.1%), and Smad2/3 signaling (110.5%), as well as an aberrant number of fibro-adipogenic progenitor cells in response to injury compared with wild-type mice. There was an increased number of proinflammatory and anti-inflammatory macrophages (3.6- and 3.1-fold, respectively) in regenerating muscles of wild-type mice, but not in the regenerating muscles of MFS mice. Our data show that proliferation and differentiation of satellite cells are altered (p ≤ 0.05) in MFS mice. Myoblast transplantation assay revealed that the regenerating muscles from MFS mice have reduced satellite cell self-renewal capacity (74.7%). In addition, we found that treatment with Ang-(1-7) reduces fibrosis (71.6%) and ameliorates satellite cell dysfunction (p ≤ 0.05) and muscle contractile function (p ≤ 0.05) in MFS mice. CONCLUSION: The fibrotic niche, caused by Fbn1 mutations, reduces the myogenic potential of satellite cells, affecting structural and functional muscle regeneration. In addition, the fibrosis inhibitor Ang-(1-7) partially counteracts satellite cell abnormalities and restores myofiber size and contractile force in regenerating muscles.
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Síndrome de Marfan , Células Satélite del Músculo Esquelético , Ratones , Animales , Síndrome de Marfan/patología , Músculo Esquelético/fisiología , Células Satélite del Músculo Esquelético/fisiología , Diferenciación Celular , Modelos Animales de Enfermedad , Regeneración/fisiología , FibrosisRESUMEN
Muscle fibers express particular isoforms of contractile proteins, depending on the fiber's function and the organism's developmental stage. In the adult, after a muscle injury, newly generated fibers transition through embryonic and neonatal myosins, prior to selecting their distinctive adult myosin isoform. In this issue of the JCI, Wang et al. discover a checkpoint that regulates the neonatal-to-adult myosin isoform transition. They found that HIF-1α regulated this checkpoint, with elevated HIF-1α levels blocking progression, while HIF-1α knockout accelerated the transition. They further related these findings to centronuclear myopathy, a disease in which HIF-1α is similarly elevated and neonatal myosin expression is maintained. These findings highlight a maturation checkpoint that impacts the skeletal muscle regeneration following ischemic injury, providing a pharmacologically accessible pathway in injury and diseases such as centronuclear myopathy.
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Fibras Musculares Esqueléticas , Miopatías Estructurales Congénitas , Adulto , Recién Nacido , Humanos , RegeneraciónRESUMEN
Acute skeletal muscle injury is followed by satellite cell activation, proliferation, and differentiation to replace damaged fibers with newly regenerated muscle fibers, processes that involve satellite cell interactions with various niche signals. Here we show that satellite cell specific deletion of the chemokine receptor CXCR4, followed by suppression of recombination escapers, leads to defects in regeneration and satellite cell pool repopulation in both the transplantation and in situ injury contexts. Mechanistically, we show that endothelial cells and FAPs express the gene for the ligand, SDF1α, and that CXCR4 is principally required for proper activation and for transit through the first cell division, and to a lesser extent the later cell divisions. In the absence of CXCR4, gene expression in quiescent satellite cells is not severely disrupted, but in activated satellite cells a subset of genes normally induced by activation fail to upregulate normally. These data demonstrate that CXCR4 signaling is essential to normal early activation, proliferation, and self-renewal of satellite cells.
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Many developmental signaling pathways have been implicated in lineage-specific differentiation; however, mechanisms that explicitly control differentiation timing remain poorly defined in mammals. We report that murine Hedgehog signaling is a heterochronic pathway that determines the timing of progenitor differentiation. Hedgehog activity was necessary to prevent premature differentiation of second heart field (SHF) cardiac progenitors in mouse embryos, and the Hedgehog transcription factor GLI1 was sufficient to delay differentiation of cardiac progenitors in vitro. GLI1 directly activated a de novo progenitor-specific network in vitro, akin to that of SHF progenitors in vivo, which prevented the onset of the cardiac differentiation program. A Hedgehog signaling-dependent active-to-repressive GLI transition functioned as a differentiation timer, restricting the progenitor network to the SHF. GLI1 expression was associated with progenitor status across germ layers, and it delayed the differentiation of neural progenitors in vitro, suggesting a broad role for Hedgehog signaling as a heterochronic pathway.
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Redes Reguladoras de Genes , Proteínas Hedgehog , Animales , Diferenciación Celular/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ratones , Transducción de Señal/fisiología , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a genetically dominant progressive myopathy caused by improper silencing of the DUX4 gene, leading to fibrosis, muscle atrophy, and fatty replacement. Approaches focused on muscle regeneration through the delivery of stem cells represent an attractive therapeutic option for muscular dystrophies. To investigate the potential for cell transplantation in FSHD, we have used the doxycycline-regulated iDUX4pA-HSA mouse model in which low-level DUX4 can be induced in skeletal muscle. We find that mouse pluripotent stem cell (PSC)-derived myogenic progenitors engraft in muscle actively undergoing DUX4-mediated degeneration. Donor-derived muscle tissue displayed reduced fibrosis and importantly, engrafted muscles showed improved contractile specific force compared to non-transplanted controls. These data demonstrate the feasibility of replacement of diseased muscle with PSC-derived myogenic progenitors in a mouse model for FSHD, and highlight the potential for the clinical benefit of such a cell therapy approach.
RESUMEN
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.
Asunto(s)
Células Madre Pluripotentes Inducidas , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Recién Nacido , Miocitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
[This corrects the article DOI: 10.3389/fphys.2020.01008.].