RESUMEN
The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.
Asunto(s)
Tipificación del Cuerpo , Corazón , Miocardio , Animales , Humanos , Ratones , Corazón/anatomía & histología , Corazón/embriología , Cardiopatías/metabolismo , Cardiopatías/patología , Ventrículos Cardíacos/anatomía & histología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/embriología , Hibridación Fluorescente in Situ , Modelos Animales , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Motivation: Unsupervised clustering of single-cell transcriptomics is a powerful method for identifying cell populations. Static visualization techniques for single-cell clustering only display results for a single resolution parameter. Analysts will often evaluate more than one resolution parameter but then only report one. Results: We developed Cell Layers, an interactive Sankey tool for the quantitative investigation of gene expression, co-expression, biological processes and cluster integrity across clustering resolutions. Cell Layers enhances the interpretability of single-cell clustering by linking molecular data and cluster evaluation metrics, providing novel insight into cell populations. Availability and implementation: https://github.com/apblair/CellLayers.
RESUMEN
The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.
RESUMEN
Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease-associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type-specific gene regulation in human hearts during health and disease.
Asunto(s)
Corazón , Secuencias Reguladoras de Ácidos Nucleicos , Humanos , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismoRESUMEN
Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.
Asunto(s)
Proteína Homeótica Nkx-2.5/metabolismo , Mesodermo/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Secuencia de Bases , Bioimpresión , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Mutación con Pérdida de Función/genética , Modelos Cardiovasculares , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre/metabolismo , Pez CebraRESUMEN
Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture.
Asunto(s)
Cromatina/genética , Retrovirus Endógenos/genética , Regulación de la Expresión Génica , Células Madre Pluripotentes/citología , Elementos de Respuesta , Retroelementos/genética , Transcripción Genética , Animales , Diferenciación Celular , Humanos , Células Madre Pluripotentes/fisiología , Primates , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide; yet how genetic variation and environmental factors impact DCM heritability remains unclear. Here, we report that compound genetic interactions between DNA sequence variants contribute to the complex heritability of DCM. By using genetic data from a large family with a history of DCM, we discovered that heterozygous sequence variants in the TROPOMYOSIN 1 (TPM1) and VINCULIN (VCL) genes cose-gregate in individuals affected by DCM. In vitro studies of patient-derived and isogenic human-pluripotent-stem-cell-derived cardio-myocytes that were genome-edited via CRISPR to create an allelic series of TPM1 and VCL variants revealed that cardiomyocytes with both TPM1 and VCL variants display reduced contractility and sarcomeres that are less organized. Analyses of mice genetically engineered to harbour these human TPM1 and VCL variants show that stress on the heart may also influence the variable penetrance and expressivity of DCM-associated genetic variants in vivo. We conclude that compound genetic variants can interact combinatorially to induce DCM, particularly when influenced by other disease-provoking stressors.
Asunto(s)
Cardiomiopatía Dilatada/genética , Predisposición Genética a la Enfermedad , Variación Genética , Animales , Cardiomiopatía Dilatada/fisiopatología , Matriz Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Patrón de Herencia/genética , Masculino , Ratones , Modelos Biológicos , Contracción Muscular/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Linaje , Células Madre Pluripotentes/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200- cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications.