Heart development is a continuous process involving significant remodeling during embryogenesis and neonatal stages. To date, several groups have used single-cell sequencing to characterize the heart transcriptomes but failed to capture the progression of heart development at most stages. This has left gaps in understanding the contribution of each cell type across cardiac development. Here, we report the transcriptional profile of the murine heart from early embryogenesis to late neonatal stages. Through further analysis of this dataset, we identify several transcriptional features. We identify gene expression modules enriched at early embryonic and neonatal stages; multiple cell types in the left and right atriums are transcriptionally distinct at neonatal stages; many congenital heart defect-associated genes have cell type-specific expression; stage-unique ligand-receptor interactions are mostly between epicardial cells and other cell types at neonatal stages; and mutants of epicardium-expressed genesWt1andTbx18have different heart defects. Assessment of this dataset serves as an invaluable source of information for studies of heart development.

Endothelial cells line the inner surface of vasculature and play an important role in normal physiology and disease progression. Although most tissue is known to have a heterogeneous population of endothelial cells, transcriptional differences in organ specific endothelial cells have not been systematically analyzed at the single cell level. The Tabula Muris project profiled mouse single cells from 20 organs. We found 10 of the organs profiled by this Consortium have endothelial cells. Unsupervised analysis of these endothelial cells revealed that they were mainly grouped by organs, and organ-specific cells were further partially correlated by germ layers. Unexpectedly, we found all lymphatic endothelial cells grouped together regardless of their resident organs. To further understand the cellular heterogeneity in organ-specific endothelial cells, we used the heart as an example. As a pump of the circulation system, the heart has multiple types of endothelial cells. Detailed analysis of these cells identified an endocardial endothelial cell population, a coronary vascular endothelial cell population, and an aorta-specific cell population. Through integrated analysis of the single cell data from another two studies analyzing the aorta, we identified conserved cell populations and molecular markers across the datasets. In summary, by reanalyzing the existing endothelial cell single-cell data, we identified organ-specific molecular signatures and heart-specific subpopulations and molecular markers. We expect these findings will pave the way for a deeper understanding of vascular biology and endothelial cell-related diseases.

The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we have analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.

Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been fully studied, which hinders their wide spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart diseases with chamber defects. We show that our systems generate chamber-specific organoids comprising of the major cardiac cell types, and we use single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we developed a machine learning label transfer approach leveraging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebstein’s anomaly associated genetic variant in NKX2-5, and we successfully recapitulated the disease’s atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.

An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3^P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3^P209L-eGFP mice. The mutation renders hBAG3^P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3^P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.