RESUMO
Single-cell technology has allowed researchers to probe tissue complexity and dynamics at unprecedented depth in health and disease. However, the generation of high-dimensionality single-cell atlases and virtual three-dimensional tissues requires integrated reference maps that harmonize disparate experimental designs, analytical pipelines, and taxonomies. Here, we present a comprehensive single-cell transcriptome integration map of cardiac fibrosis, which underpins pathophysiology in most cardiovascular diseases. Our findings reveal similarity between cardiac fibroblast (CF) identities and dynamics in ischemic versus pressure overload models of cardiomyopathy. We also describe timelines for commitment of activated CFs to proliferation and myofibrogenesis, profibrotic and antifibrotic polarization of myofibroblasts and matrifibrocytes, and CF conservation across mouse and human healthy and diseased hearts. These insights have the potential to inform knowledge-based therapies.
Assuntos
Fibroblastos , Fibrose , Análise de Célula Única , Transcriptoma , Animais , Análise de Célula Única/métodos , Humanos , Fibroblastos/metabolismo , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Perfilação da Expressão GênicaRESUMO
Migfilin is a LIM-domain-containing protein of the zyxin family of adaptor proteins and is found at cell-matrix and cell-cell adhesion sites and in the nucleus. In vitro studies have suggested that migfilin promotes ß1 integrin activity, regulates cell spreading and migration and induces cardiomyocyte differentiation. To test directly the function of migfilin in vivo, we generated a migfilin-null mouse strain. Here, we report that loss of migfilin expression permits normal development and normal postnatal aging. Fibroblasts and keratinocytes from migfilin-null mice display normal spreading and adhesion, and normal integrin expression and activation. The migration velocity and directionality of migfilin-null embryonic fibroblasts were normal, whereas the velocity of migfilin-null keratinocytes in wound scratch assays was slightly but significantly reduced. Our findings indicate that the roles of migfilin are functionally redundant during mouse development and tissue homeostasis.
Assuntos
Moléculas de Adesão Celular/genética , Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/metabolismo , Animais , Moléculas de Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas com Domínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , GravidezRESUMO
Cardiovascular disease is the leading cause of death globally. An advanced understanding of cardiovascular disease mechanisms is required to improve therapeutic strategies and patient risk stratification. State-of-the-art, large-scale, single-cell and single-nucleus transcriptomics facilitate the exploration of the cardiac cellular landscape at an unprecedented level, beyond its descriptive features, and can further our understanding of the mechanisms of disease and guide functional studies. In this Review, we provide an overview of the technical challenges in the experimental design of single-cell and single-nucleus transcriptomics studies, as well as a discussion of the type of inferences that can be made from the data derived from these studies. Furthermore, we describe novel findings derived from transcriptomics studies for each major cardiac cell type in both health and disease, and from development to adulthood. This Review also provides a guide to interpreting the exhaustive list of newly identified cardiac cell types and states, and highlights the consensus and discordances in annotation, indicating an urgent need for standardization. We describe advanced applications such as integration of single-cell data with spatial transcriptomics to map genes and cells on tissue and define cellular microenvironments that regulate homeostasis and disease progression. Finally, we discuss current and future translational and clinical implications of novel transcriptomics approaches, and provide an outlook of how these technologies will change the way we diagnose and treat heart disease.
Assuntos
Doenças Cardiovasculares , Cardiopatias , Humanos , Transcriptoma , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/genética , Perfilação da Expressão Gênica , Coração , Cardiopatias/diagnóstico , Cardiopatias/genética , Cardiopatias/terapiaRESUMO
Piezo channels are critical cellular sensors of mechanical forces. Despite their large size, ubiquitous expression, and irreplaceable roles in an ever-growing list of physiological processes, few Piezo channel-binding proteins have emerged. In this work, we found that MyoD (myoblast determination)-family inhibitor proteins (MDFIC and MDFI) are PIEZO1/2 interacting partners. These transcriptional regulators bind to PIEZO1/2 channels, regulating channel inactivation. Using single-particle cryogenic electron microscopy, we mapped the interaction site in MDFIC to a lipidated, C-terminal helix that inserts laterally into the PIEZO1 pore module. These Piezo-interacting proteins fit all the criteria for auxiliary subunits, contribute to explaining the vastly different gating kinetics of endogenous Piezo channels observed in many cell types, and elucidate mechanisms potentially involved in human lymphatic vascular disease.
Assuntos
Canais Iônicos , Fatores de Regulação Miogênica , Humanos , Microscopia Crioeletrônica , Células HEK293 , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Cinética , Doenças Linfáticas/genética , Mutação , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Domínios Proteicos , Mioblastos/metabolismo , Animais , CamundongosRESUMO
Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.
Assuntos
Isquemia Encefálica , Forame Oval Patente , Cardiopatias Congênitas , Acidente Vascular Cerebral , Humanos , Camundongos , Animais , Forame Oval Patente/genética , Fenótipo , Perfilação da Expressão GênicaRESUMO
Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor ß (PDGFRß) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.
Assuntos
Células Endoteliais , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Pericitos/metabolismo , Transdução de Sinais , Organoides/metabolismoRESUMO
Following myocardial infarction, damaged myocardium is replaced with a fibrotic scar that preserves cardiac structural integrity. Scar area measured from sample 2D images of serial heart sections does not faithfully measure the extent of fibrosis due to structural heterogeneity caused by tissue dynamics. Here, we present an X-ray microcomputed tomography (micro-CT) workflow that generates accurate volumetric quantification of scar and surviving myocardium in infarcted mouse hearts. This workflow could be applied to other fibrotic organs or hearts from other species. For complete details on the use and execution of this protocol, please refer to Janbandhu et al. (2021).
Assuntos
Cicatriz , Infarto do Miocárdio , Animais , Cicatriz/patologia , Fibrose , Camundongos , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/patologia , Microtomografia por Raio-X/métodosRESUMO
Quantitative assessment of post-ischemic cardiac remodeling is often hampered by tissue complexity and structural heterogeneity of the scar. Automated quantification of microscopy images offers an unbiased approach to reduce inter-observer variability. Here, we present a CellProfiler-based analytical pipeline for the high-throughput analysis of confocal images to quantify post-ischemic cardiac parameters. We describe image preprocessing and the quantification of capillary rarefaction, immune cell infiltration, cell death, and proliferating fibroblasts. This protocol can be adapted to other tissue types. For complete details on the use and execution of this profile, please refer to Janbandhu et al. (2021).
Assuntos
Coração , Processamento de Imagem Assistida por Computador , Cicatriz , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was â¼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.
Assuntos
Infarto do Miocárdio , Animais , Antioxidantes/metabolismo , Proliferação de Células , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Espécies Reativas de Oxigênio/metabolismoRESUMO
NF-kappaB family members play a pivotal role in many cellular and organismal functions, including the cell cycle. As an activator of cyclin D1 and p21(Waf1) genes, NF-kappaB has been regarded as a critical modulator of cell cycle. To study the involvement of NF-kappaB in G(1)/S phase regulation, the levels of selected transcriptional regulators were monitored following overexpression of NF-kappaB or its physiological induction by tumor necrosis factor-alpha. Cyclin E gene was identified as a major transcriptional target of NF-kappaB. Recruitment of NF-kappaB to the cyclin E promoter was correlated with the transrepression of cyclin E gene. Ligation-mediated PCR and micrococcal nuclease-Southern assays suggested the nucleosomal nature of this region while chromatin immunoprecipitation analysis confirmed the exchange of cofactors following tumor necrosis factor-alpha treatment or release from serum starvation. There was a progressive reduction in cyclin E transcription along with the accumulation of catalytically inactive cyclin E-cdk2 complexes and arrest of cells in G(1)/S-phase. Thus, our study clearly establishes NF-kappaB as a negative regulator of cell cycle through transcriptional repression of cyclin E.
Assuntos
Ciclina E/biossíntese , Regulação da Expressão Gênica , Sinaptotagmina I/biossíntese , Catálise , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/química , Vetores Genéticos , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Besides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac stromal cell heterogeneity and flux in healthy and infarcted hearts using single-cell RNA sequencing (scRNA-seq) ( Farbehi et al., 2019 ). Here, we describe the FACS (Fluorescent Activated Cell Sorting)-based method used in that study for isolation of two cardiac cell fractions from adult mouse ventricles: the total interstitial cell population (TIP; non-cardiomyocytes) and enriched (Pdgfra-GFP+) cardiac fibroblasts.
RESUMO
High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 'UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra .
Assuntos
Regiões 3' não Traduzidas , Regulação da Expressão Gênica , Análise de Sequência de RNA , Análise de Célula Única , Software , Animais , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Miocárdio/metabolismo , Poli ARESUMO
Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.
Assuntos
Linhagem da Célula , Infarto do Miocárdio/patologia , Regeneração , Cicatrização , Animais , Comunicação Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Masculino , Camundongos , Análise de Célula ÚnicaRESUMO
Chemotherapeutic agents are well known to induce growth arrest of cancerous cells by inducing DNA damage/replicational stress and engaging cellular apoptotic machinery. Our studies on hydroxyurea (HU) recognized cyclin D1 destabilization as the initiator of growth arrest at G(1)/S-phase independent of other cell cycle regulators. Cyclin D1 degradation was associated with its phosphorylation at Thr286 by glycogen synthase kinase-3beta and inactivation of Akt kinase. Overexpression of the cyclin D1(T286A) mutant, or constitutively active Akt, conferred stability to cyclin D1 and helped bypass cell cycle arrest. Thus, growth arrest by HU seems to involve destabilization of cyclin D1 in addition to its well-established role as ribonucleotide reductase inhibitor.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hidroxiureia/farmacologia , Animais , Linhagem Celular , Replicação do DNA , Fase G1 , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase SRESUMO
The HBx (X protein of hepatitis B virus) is a promiscuous transactivator implicated to play a key role in hepatocellular carcinoma. However, HBx-regulated molecular events leading to deregulation of cell cycle or establishment of a permissive environment for hepatocarcinogenesis are not fully understood. Our cell culture-based studies suggested that HBx had a profound effect on cell cycle progression even in the absence of serum. HBx presence led to an early and sustained level of cyclin-cdk2 complex during the cell cycle combined with increased protein kinase activity of cdk2 heralding an early proliferative signal. The increased cdk2 activity also led to an early proteasomal degradation of p27(Kip1) that could be reversed by HBx-specific RNA interference and blocked by a chemical inhibitor of cdk2 or the T187A mutant of p27. Further, our co-immunoprecipitation and in vitro binding studies with recombinant proteins suggested a direct interaction between HBx and the cyclin E/A-cdk2 complex. Interference with different signalling cascades known to be activated by HBx suggested a constitutive requirement of Src kinases for the association of HBx with these complexes. Notably, the HBx mutant that did not interact with cyclin E/A failed to destabilize p27(Kip1) or deregulate the cell cycle. Thus HBx appears to deregulate the cell cycle by interacting with the key cell cycle regulators independent of its well-established role in transactivation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Bases , Carcinoma Hepatocelular , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27 , Primers do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Neoplasias Hepáticas , Fosforilação , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases Associadas a Fase S , Transativadores , Fatores de Transcrição/genéticaRESUMO
The spatiotemporal manipulations of gene expression by the Cre recombinase (Cre) of bacteriophage P1 has become an essential asset to understanding mammalian genetics. Accumulating evidence suggests that Cre activity can, in addition to excising targeted loxP sites, induce cytotoxic effects, including abnormal cell cycle progression, genomic instability, and apoptosis, which can accelerate cancer progression. It is speculated that these defects are caused by Cre-induced DNA damage at off-target sites. Here we report the formation of tetraploid keratinocytes in the epidermis of keratin 5 and/or keratin 14 promoter-driven Cre (KRT5- and KRT14-Cre) expressing mouse skin. Biochemical analyses and flow cytometry demonstrated that Cre expression also induces DNA damage, genomic instability, and tetraploidy in HCT116 cells, and live-cell imaging revealed an extension of the G 2 cell cycle phase followed by defective or skipping of mitosis as cause for the tetraploidy. Since tetraploidy eventually leads to aneuploidy, a hallmark of cancer, our findings highlight the importance of distinguishing non-specific cytopathic effects from specific Cre/loxP-driven genetic manipulations when using Cre-mediated gene deletions.