Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide 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 that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Single-cell transcriptomics for the assessment of cardiac disease.

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.

Pathogenic variants damage cell composition and single cell transcription in cardiomyopathies.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

Characterization of a common progenitor pool of the epicardium and myocardium.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

Human pluripotent stem cell-derived cardiomyocytes as a target platform for paracrine protection by cardiac mesenchymal stromal cells.

Ischemic heart disease remains the foremost cause of death globally, with survivors at risk for subsequent heart failure. Paradoxically, cell therapies to offset cardiomyocyte loss after ischemic injury improve long-term cardiac function despite a lack of durable engraftment. An evolving consensus, inferred preponderantly from non-human models, is that transplanted cells benefit the heart via early paracrine signals. Here, we tested the impact of paracrine signals on human cardiomyocytes, using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the target of mouse and human cardiac mesenchymal stromal cells (cMSC) with progenitor-like features. In co-culture and conditioned medium studies, cMSCs markedly inhibited human cardiomyocyte death. Little or no protection was conferred by mouse tail tip or human skin fibroblasts. Consistent with the results of transcriptomic profiling, functional analyses showed that the cMSC secretome suppressed apoptosis and preserved cardiac mitochondrial transmembrane potential. Protection was independent of exosomes under the conditions tested. In mice, injecting cMSC-conditioned media into the infarct border zone reduced apoptotic cardiomyocytes > 70% locally. Thus, hPSC-CMs provide an auspicious, relevant human platform to investigate extracellular signals for cardiac muscle survival, substantiating human cardioprotection by cMSCs, and suggesting the cMSC secretome or its components as potential cell-free therapeutic products.

Spatial protein analysis in developing tissues: a sampling-based image processing approach.

Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry-based information from diverse mammalian tissue images. Our open-source image analysis platform, called 'SilentMark', can cope with three-dimensional noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors, graphics cards or training datasets and can be run even with just several hundred megabytes of memory. This makes it possible to use the method as a Web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell-derived embryoid bodies and mouse embryonic heart, and relate protein localization to tissue geometry. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Detecting cardiac contractile activity in the early mouse embryo using multiple modalities.

The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodeling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodeling is controlled but also the origin of congenital heart defects (CHDs). Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.