Heart Network

Heart Network - Overview

Single-cell RNA sequencing was applied to study the role of Pitx2 expression in heart development in mice.  Over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2-null embryos were amplified and sequenced.

A Cellular Atlas of Pitx2-Dependent Cardiac Development.

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

Pathological cardiac hypertrophy is a leading cause of heart failure, but knowledge of the full repertoire of cardiac cells and their gene expression profiles in the human hypertrophic heart is missing. Here, by using large-scale single-nucleus transcriptomics, we present the transcriptional response of human cardiomyocytes to pressure overload caused by aortic valve stenosis and describe major alterations in cardiac cellular crosstalk. Hypertrophied cardiomyocytes had reduced input from endothelial cells and fibroblasts. Genes encoding Eph receptor tyrosine kinases, particularly EPHB1, were significantly downregulated in cardiomyocytes of the hypertrophied heart. Consequently, EPHB1 activation by its ligand ephrin (EFN)B2, which is mainly expressed by endothelial cells, was reduced. EFNB2 inhibited cardiomyocyte hypertrophy in vitro, while silencing its expression in endothelial cells induced hypertrophy in co-cultured cardiomyocytes. Our human cell atlas of the hypertrophied heart highlights the importance of intercellular crosstalk in disease pathogenesis and provides a valuable resource.

A human cell atlas of the pressure-induced hypertrophic heart

We present a multiomic cell atlas of human lung development that combines single cell RNA and ATAC sequencing, high throughput spatial transcriptomics and single cell imaging. Coupling single cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial and erythrocyte/leukocyte compartments from 5-22 post conception weeks. We identify new cell states in all compartments. These include developmental-specific secretory progenitors that resemble cells in adult fibrotic lungs and a new subtype of neuroendocrine cell related to human small cell lung cancer; observations which strengthen the connections between development and disease/regeneration. Our datasets are available for the community to download and interact with through our web interface ( https://fetal-lung.cellgeni.sanger.ac.uk ). Finally, to illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signalling and transcription factor hierarchies which we test using organoid models. Highlights Spatiotemporal atlas of human lung development from 5-22 post conception weeks identifies 147 cell types/states. Tracking the developmental origins of multiple cell compartments, including new progenitor states. Functional diversity of fibroblasts in distinct anatomical signalling niches. Resource applied to interrogate and experimentally test the transcription factor code controlling neuroendocrine cell heterogeneity and the origins of small cell lung cancer.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for approximately 1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems. Overall design: Applying single cell chromatin accessibility assays to diverse human tissue types from four donors.

A single-cell atlas of chromatin accessibility in the human genome

The authors generated single-cell atlases of 23 lung, 16 kidney, 15 liver and 18 heart COVID-19 autopsy donor tissue samples, and spatial atlases of 14 lung donors.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

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.

Cardiac cell type-specific gene regulatory programs and disease risk association

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require deeper understanding of the healthy heart’s molecular processes. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavor. Here, using state-of-the-art analyses of large-scale single-cell and nuclei transcriptomes, we characterise six anatomical adult heart regions (left/right atria and ventricles, apex, interventricular septum). Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, revealing distinct atrial and ventricular subsets with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment we identify cardiac resident macrophages with inflammatory and protective transcriptional signatures. Further, inference of cell-cell interactions highlight different macrophage-fibroblast-cardiomyocyte networks between atria and ventricles that are distinct from skeletal muscle.  We expect this reference human cardiac cell atlas to advance mechanistic studies of heart homeostasis and disease.

Cells of the adult human heart

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

Recovery of cardiac function is the holy grail of heart failure therapy yet is infrequently observed and remains poorly understood. In this study, we performed single-nucleus RNA sequencing from patients with heart failure who recovered left ventricular systolic function after left ventricular assist device implantation, patients who did not recover and non-diseased donors. We identified cell-specific transcriptional signatures of recovery, most prominently in macrophages and fibroblasts. Within these cell types, inflammatory signatures were negative predictors of recovery, and downregulation of RUNX1 was associated with recovery. In silico perturbation of RUNX1 in macrophages and fibroblasts recapitulated the transcriptional state of recovery. Cardiac recovery mediated by BET inhibition in mice led to decreased macrophage and fibroblast Runx1 expression and diminished chromatin accessibility within a Runx1 intronic peak and acquisition of human recovery signatures. These findings suggest that cardiac recovery is a unique biological state and identify RUNX1 as a possible therapeutic target to facilitate cardiac recovery.

Defining cardiac functional recovery in end-stage heart failure at single-cell resolution.

The heart, the first organ to develop, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of CHD patients survive into adulthood but often suffer premature death from heart failure (HF) and non-cardiac causes. To gain insight into poorly understood disease progression, we performed single nuclear RNA sequencing (snRNA-seq) and analyzed 157,273 nuclei from donors and CHD patients, including hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF), two common forms of cyanotic CHD lesions, as well as, dilated (DCM) and hypertrophic (HCM) cardiomyopathies. We observed CHD specific cell states in cardiomyocytes (CMs) which had evidence of insulin resistance and increased FOXO and CRIM1 expression. Cardiac fibroblasts (CFs) in HLHS had enrichment for a low HIPPO and high YAP cell state characteristic of activated CFs. Imaging Mass Cytometry (IMC) uncovered the spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD in agreement with CHD predilection to infection and cancer. Our comprehensive CHD phenotyping provides a roadmap for future personalized medicine in CHD.

Integrated multiomic characterization of congenital heart disease

Atrial fibrillation disrupts contraction of the atria, leading to stroke and heart failure. We deciphered how immune and stromal cells contribute to atrial fibrillation. Single-cell transcriptomes from human atria documented inflammatory monocyte and SPP1+ macrophage expansion in atrial fibrillation. Combining hypertension, obesity, and mitral valve regurgitation (HOMER) in mice elicited enlarged, fibrosed, and fibrillation-prone atria. Single-cell transcriptomes from HOMER mouse atria recapitulated cell composition and transcriptome changes observed in patients. Inhibiting monocyte migration reduced arrhythmia in Ccr2-∕- HOMER mice. Cell-cell interaction analysis identified SPP1 as a pleiotropic signal that promotes atrial fibrillation through cross-talk with local immune and stromal cells. Deleting Spp1 reduced atrial fibrillation in HOMER mice. These results identify SPP1+ macrophages as targets for immunotherapy in atrial fibrillation.

Recruited macrophages elicit atrial fibrillation

we performed single cell RNA sequencing (scRNA-seq) in both normal and failed human heart. CD45- cells in the myocardium and immune cells (CD45+ cells) were isolated from myocardium and corresponding peripheral blood were sorted for scRNA-seq. Our findings were also validated with bulk RNAseq. Overall design: Establishing a comprehensive cell atlas of immune cells in human heart failure at single cell level.

Resolving the intertwining of inflammation and fibrosis in human heart failure at single-cell level

Background: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. Methods: Single nucleus RNA-sequencing (snRNA-seq) of 54,140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA-sequencing and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. Results: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. Conclusions: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.

Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor.

End stage, nonobstructive hypertrophic cardiomyopathy (HCM) is an intractable condition with no disease-specific therapies. To gain insights into the pathogenesis of nonobstructive HCM, we performed single nucleus RNA-sequencing (snRNA-seq) on human HCM hearts explanted at the time of cardiac transplantation and organ donor hearts serving as controls. Differential gene expression analysis revealed 64 differentially expressed genes linked to specific cell types and molecular functions. Analysis of ligand-receptor pair gene expression to delineate potential intercellular communication revealed significant reductions in expressed ligand-receptor pairs likely affecting the extracellular matrix, growth factor binding, peptidase regulator activity, platelet-derived growth factor binding and protease binding in the HCM tissue. Changes in Integrin-β1 receptor expression were responsible for many observed changes related to extracellular matrix interactions, by increasing in dendritic, smooth muscle and pericyte cells while decreasing in endothelial and fibroblast cells, suggesting potential mechanisms for fibrosis and microvascular disease in HCM and a potential role for dendritic cells. In contrast, there was an increase in ligand-receptor pair expression associated with adenylate cyclase binding, calcium channel molecular functions, channel inhibitor activity, ion channel inhibitor activity, phosphatase activator activity, protein kinase activator activity and titin binding, suggesting important shifts in various signaling cascades in nonobstructive, end stage HCM.

Single Nucleus RNA-sequencing Reveals Altered Intercellular Communication and Dendritic Cell Activation in Nonobstructive Hypertrophic Cardiomyopathy

Background: CD34+ cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34+ cell differentiation in vivo during heart failure and explore its intervention strategy.
Methods We performed single-cell RNA sequencing (scRNA-seq) of the total non-cardiomyocytes and enriched Cd34-tdTomato+ lineage cells in the murine (male Cd34-CreERT2; Rosa26-tdTomato mice) pressure overload model (transverse aortic constriction, TAC), and total non-cardiomyocytes from human adult hearts. Then, in order to determine the origin of CD34+ cell that plays a role in myocardial fibrosis, bone marrow transplantation model was performed. Furthermore, to further clarify the role of +cells in myocardial remodeling in response to TAC injury, we generated Cd34-CreERT2; Rosa26-eGFP-DTA (Cre/DTA) mice.
Results: By analyzing the transcriptomes of 59,505 single cells from the mouse heart and 22,537 single cells from the human heart, we illustrated the dynamics of cell landscape during the progression of heart hypertrophy, including CD34+ cells, fibroblasts, endothelial and immune cells. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone-marrow-derived CD34+ cells give rise to fibroblasts and endothelial cells, while bone-marrow-derived CD34+ cell turned into immune cells only in response to pressure overload. Interestingly, partial depletion of CD34+ cells alleviated the severity of myocardial fibrosis with a significant improvement of cardiac function in Cd34-CreERT2; Rosa26-eGFP-DTA model. Similar changes of non-cardiomyocyte composition and cellular heterogeneity of heart failure were also observed in human patient with heart failure. Furthermore, immunostaining showed a double labeling of CD34 and fibroblast markers in human heart tissue. Mechanistically, our single-cell pseudotime analysis of scRNA-seq data and in vitro cell culture study revealed that Wnt-β-catenin and TGFβ1/Smad pathways are critical in regulating CD34+ cell differentiation toward fibroblasts.
Conclusions: Our study provides a cellular landscape of CD34+ cell-derived cells in the hypertrophy heart of human and animal models, indicating that non-bone-marrow-derived CD34+ cells differentiating into fibroblasts largely account for cardiac fibrosis. These findings may provide novel insights for the pathogenesis of cardiac fibrosis and have further potential therapeutic implications for the heart failure.

Single cell and lineage tracing studies reveal the impact of CD34+ cells on myocardial fibrosis during heart failure

Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain.

Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury.

Genome-wide association studies (GWAS) have linked hundreds of loci to cardiac diseases. However, in most loci the causal variants and their target genes remain unknown. We developed a combined experimental and analytical approach that integrates single cell epigenomics with GWAS to prioritize risk variants and genes. We profiled accessible chromatin in single cells obtained from human hearts and leveraged the data to study genetics of Atrial Fibrillation (AF), the most common cardiac arrhythmia. Enrichment analysis of AF risk variants using cell-type-resolved open chromatin regions (OCRs) implicated cardiomyocytes as the main mediator of AF risk. We then performed statistical fine-mapping, leveraging the information in OCRs, and identified putative causal variants in 122 AF-associated loci. Taking advantage of the fine-mapping results, our novel statistical procedure for gene discovery prioritized 46 high-confidence risk genes, highlighting transcription factors and signal transduction pathways important for heart development. In summary, our analysis provides a comprehensive map of AF risk variants and genes, and a general framework to integrate single-cell genomics with genetic studies of complex traits.

Single-cell genomics improves the discovery of risk variants and genes of atrial fibrillation

Genome-wide association studies (GWAS) have linked hundreds of loci to cardiac diseases. However, in most loci the causal variants and their target genes remain unknown. We developed a combined experimental and analytical approach that integrates single cell epigenomics with GWAS to prioritize risk variants and genes. We profiled accessible chromatin in single cells obtained from human hearts and leveraged the data to study genetics of Atrial Fibrillation (AF), the most common cardiac arrhythmia. Enrichment analysis of AF risk variants using cell-type-resolved open chromatin regions (OCRs) implicated cardiomyocytes as the main mediator of AF risk. We then performed statistical fine-mapping, leveraging the information in OCRs, and identified putative causal variants in 122 AF-associated loci. Taking advantage of the fine-mapping results, our novel statistical procedure for gene discovery prioritized 45 high-confidence risk genes, highlighting transcription factors and signal transduction pathways important for heart development. We further leveraged our single-cell data to study genetics of gene expression. An unexpected finding from earlier studies is that expression QTLs (eQTLs) are often shared across tissues even though most regulatory elements are cell-type specific. We found that this sharing is largely driven by the limited power of eQTL studies using bulk tissues to detect cell-type-specific regulatory variants. This finding points to an important limitation of using eQTLs to interpret GWAS of complex traits. In summary, our analysis provides a comprehensive map of AF risk variants and genes, and a general framework to integrate single-cell genomics with genetic studies of complex traits.

Single-cell genomics improves the discovery of risk variants and genes of cardiac traits

Background: As core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing. Results: We perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novel COCH+ fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues. Conclusions: The adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs. Overall design: We performed single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generated an adult human cell atlas (AHCA). Keywords: Human cell atlas, Single-cell RNA sequencing, TCR, BCR, Transcriptome

Single-cell transcriptome profiling of an adult human cell atlas of 15 major organs

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy

Heart failure represents a major cause of morbidity and mortality worldwide. Single cell transcriptomics have revolutionized our understanding of cell composition and associated gene expression across human tissues. Through integrated analysis of single cell and single nucleus RNA sequencing data generated from 45 individuals, we define the cell composition of the healthy and failing human heart. We identify cell specific transcriptional signatures of heart failure and reveal the emergence of disease associated cell states. Intriguingly, cardiomyocytes converge towards a common disease associated cell state, while fibroblasts and myeloid cells undergo dramatic diversification. Endothelial cells and pericytes display global transcriptional shifts without changes in cell complexity. Collectively, our findings provide a comprehensive analysis of the cellular and transcriptomic landscape of human heart failure, identify cell type specific transcriptional programs and states associated with disease, and establish a valuable resource for the investigation of human heart failure. Overall design: Single cell n=7 (2 donor, 5 DCM), single nuclei n=38 (25 donor, 13 DCM).

Single-cell transcriptomics reveals cell-type-specific diversification in human heart failure

Ischemic cardiomyopathy (ICM) is the leading cause of heart failure worldwide, yet the cellular and molecular signature of this disease is largely unclear. Using single-nucleus RNA sequencing (snRNA-seq) and integrated computational analyses, we profile the transcriptomes of over 99,000 human cardiac nuclei from the non-infarct region of the left ventricle of 7 ICM transplant recipients and 8 non-failing (NF) controls. We find the cellular composition of the ischemic heart is significantly altered, with decreased cardiomyocytes and increased proportions of lymphatic, angiogenic, and arterial endothelial cells in patients with ICM. We show that there is increased LAMININ signaling from endothelial cells to other cell types in ICM compared with NF. Finally, we find that the transcriptional changes that occur in ICM are similar to those in hypertrophic and dilated cardiomyopathies and that the mining of these combined datasets can identify druggable genes that could be used to target end-stage heart failure.

Single-nucleus RNA sequencing in ischemic cardiomyopathy reveals common transcriptional profile underlying end-stage heart failure.

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFβ1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.

Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy

Myocardial infarction is a leading cause of mortality worldwide. While advances in the acute treatment have been made, the late-stage mortality is still elevated. We used single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of different physiological zones and timepoints of human myocardial infarction and human control myocardium to generate an integrative high-resolution map of cardiac tissue remodeling. This approach allowed us to increase spatial resolution of cell-type composition and provide spatially resolved insights into changes of the the cardiac transcriptome and epigenome with identification of distinct cellular zones of injury, repair and remodeling. This study provides an integrative molecular map of human myocardial infarction and represents a reference to advance mechanistic and therapeutic studies of cardiac disease.

Spatial multi-omic map of human myocardial infarction

A cell’s function is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here, we combine single-cell and spatial transcriptomic data to discover cellular niches within eight regions of the human heart. We map cells to micro-anatomic locations and integrate knowledge-based and unsupervised structural annotations. For the first time, we profile the cells of the human cardiac conduction system, revealing their distinctive repertoire of ion channels, G-protein coupled receptors, and cell interactions using a custom CellPhoneDB.org module. We show that the sinoatrial node is compartmentalised, with a core of pacemaker cells, fibroblasts and glial cells supporting paracrine glutamatergic signalling. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions, providing unexpected mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches which may contribute to infection defence. We define a ventricular myocardial-stress niche enriched for activated fibroblasts and stressed cardiomyocytes, cell states that are expanded in cardiomyopathies. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be deployed to other tissues and organs.

Spatially resolved multiomics of human cardiac niches

Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1, a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.

Stress-induced RNA–chromatin interactions promote endothelial dysfunction

We have created a compendium of single cell transcriptome data from the model
organism Mus musculus comprising more than 100,000 cells from 20 organs and
tissues. These data represent a new resource for cell biology, revealing gene
expression in poorly characterized cell populations and allowing for direct and
controlled comparison of gene expression in cell types shared between tissues, such
as T-lymphocytes and endothelial cells from distinct anatomical locations. Two
distinct technical approaches were used for most tissues: one approach, microfluidic
droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively
low coverage, while the other, FACS-based full length transcript analysis, enabled
characterization of cell types with high sensitivity and coverage. The cumulative
data provide the foundation for an atlas of transcriptomic cell biology.

Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution

Cardiac fibrosis is causally linked to heart failure pathogenesis and adverse clinical outcomes. However, the precise fibroblast populations that drive fibrosis in the human heart and the mechanisms that govern their emergence remain incompletely defined. Here, we performed Cellular Indexing of Transcriptomes and Epitomes by sequencing (CITE-seq) in 22 explanted human hearts from healthy donors, acute myocardial infarction (MI), and chronic ischemic and non-ischemic cardiomyopathy patients. We identified a fibroblast trajectory marked by fibroblast activator protein (FAP) and periostin (POSTN) expression that was independent of myofibroblasts, peaked early after MI, remained elevated in chronic heart failure, and displayed a transcriptional signature consistent with fibrotic activity. We assessed the applicability of cardiac fibrosis models and demonstrated that mouse MI, angiotensin II/phenylephrine infusion, and pressure overload models were superior compared to cultured human heart and dermal fibroblasts in recapitulating cardiac fibroblast diversity including pathogenic cell states. Ligand-receptor analysis and spatial transcriptomics predicted interactions between macrophages, T cells, and fibroblasts within spatially defined niches. CCR2 + monocyte and macrophage states were the dominant source of ligands targeting fibroblasts. Inhibition of IL-1β signaling to cardiac fibroblasts was sufficient to suppress fibrosis, emergence, and maturation of FAP + POSTN + fibroblasts. Herein, we identify a human fibroblast trajectory marked by FAP and POSTN expression that is associated with cardiac fibrosis and identify macrophage-fibroblast crosstalk mediated by IL-1β signaling as a key regulator of pathologic fibroblast differentiation and fibrosis.

Targeting the Immune-Fibrosis Axis in Myocardial Infarction and Heart Failure

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans

Background:
The human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low-input RNA sequencing have allowed definitions of cellular transcriptomes at single-cell resolution at scale, we have applied these approaches to assess the cellular and transcriptional diversity of the nonfailing human heart.
Methods:
Microfluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from 7 human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based on transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data.
Results:
We sequenced the transcriptomes of 287 269 single cardiac nuclei, revealing 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include 2 distinct groups of resident macrophages, 4 endothelial subtypes, and 2 fibroblast subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Intersection of our data set with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity.
Conclusions:
Using large-scale single nuclei RNA sequencing, we defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

Transcriptional and Cellular Diversity of the Human Heart

The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. We used single-cell RNA-sequencing (scRNA-seq) to perform genome-wide analysis of gene expression of the CCS at single-cell resolution. Overall design: Single cell transcriptional analysis of embryonic cardiac cells using 10x genomics 3' solution.

Heart Network

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