Transcriptional and Cellular Diversity of the Human Heart

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

Endothelial β‐arrestins regulate mechanotransduction by the type II bone morphogenetic protein receptor in primary cilia

Abstract Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how β‐arrestins, intracellular proteins that regulate agonist‐mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to β‐arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of β‐arrestin 1 and β‐arrestin 2. We found that β‐arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of β‐arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. β‐arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, β‐arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR‐II), whose disruption similarly led to an impaired endothelial shear response. β‐arrestins also regulated Smad transcription factor phosphorylation by BMPR‐II. Mice with endothelial specific deletion of β‐arrestin 1 and β‐arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial β‐arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR‐II and contribute to vascular development

Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm

Elucidating the gene regulatory networks that govern pharyngeal arch artery (PAA) development is an important goal, as such knowledge can help to identify new genes involved in cardiovascular disease. The transcription factor Tbx1 plays a vital role in PAA development and is a major contributor to cardiovascular disease associated with DiGeorge syndrome. In this report, we used various genetic approaches to reveal part of a signalling network by which Tbx1 controls PAA development in mice. We investigated the crucial role played by the homeobox-containing transcription factor Gbx2 downstream of Tbx1. We found that PAA formation requires the pharyngeal surface ectoderm as a key signalling centre from which Gbx2, in response to Tbx1, triggers essential directional cues to the adjacent cardiac neural crest cells (cNCCs) en route to the caudal PAAs. Abrogation of this signal generates cNCC patterning defects leading to PAA abnormalities. Finally, we showed that the Slit/Robo signalling pathway is activated during cNCC migration and that components of this pathway are affected in Gbx2 and Tbx1 mutant embryos at the time of PAA development. We propose that the spatiotemporal control of this tightly orchestrated network of genes participates in crucial aspects of PAA development

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures