Spatiotemporal Atlas of Heart Development Reveals Blood-Flow-Dependent Cellular, Structural, Metabolic, and Spatial Remodeling

Figures

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  Figure 1. Spatiotemporal single-cell and spatial transcriptomics atlas of the heart under perturbed hemodynamics. (A) Schematic overview of sample collection and surgical perturbation. Heart samples were collected at multiple developmental stages and processed for single-cell or spatial transcriptomic profiling. Hemodynamic perturbations were introduced by left atrial ligation (LAL) or right atrial ligation (RAL). (B) Workflow of scRNA-seq analysis. Cell type annotation was performed hierarchically, beginning with coarse classification followed by granular subtypes. RNA velocity analysis was carried out independently within each coarse cell type. (C) Overview of cell type and plasticity score mapping. Cell types were assigned to spatial spots using cell2location based on the dominant contributing cell type. Plasticity scores were computed for each spot as the product of the cell type proportion and its corresponding cell type plasticity score. (D) Schematic of manual region annotation with Napari for left ventricle (LV) and right ventricle (RV) analysis. (E) Schematic of subdomain identification for valve neighborhood analysis with recursive GraphST.

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  Figure 2. Cellular dynamics during normal heart development. (A) Cell type map (top) and plasticity score map (bottom) of fibroblasts across developmental time points (Day 4, 5, 6, 7 and 12). (B) Cell type map (top) and plasticity score map (bottom) of epithelial cells across five time points (Day 6, 7,10 and 12). (C) Line plot of proportional changes in aCM-immature and aCM-mature across developmental time points (Day 4, 5, 6, 7 and 12). Representative maps of cell type and plasticity score shown for Day 4, Day 6, and Day 12. (D) Cell type map (top) and plasticity score map (bottom) of vCMs, CMs, and avCM across timepoint Day 4, 6, and 10. (E) Cell type map of endothelial cells at Day 7 and 10, displaying endocardial cells and VECs (top), CECs (bottom). (F) Zoomed in a spatial map around the ventricular shoulder. (E) Zoomed in a spatial map around the valve. LAV: left atrioventricular valve, RAV: right atrioventricular valve

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  Figure 3. Identification of LOX expressing LAL-enriched cell types and Their Preferential Localization in the RV. (A) UMAP plot of cardiomyocytes from scRNA-seq data, colored by cell type. The vCM-LAL cluster is outlined with a dashed line (left). Spatial maps of vCMs in normal (top right) and LAL (bottom right) hearts across developmental time points. (B) UMAP plot of endothelial cells from scRNA-seq data, colored by cell type. The FN1+ Endo-LAL cluster is outlined with a dashed line (left). Spatial maps of non-CECs in normal (top right) and LAL (bottom right) hearts across developmental time points. (C) Spatial expression of LOX in normal (left top), LAL (left bottom), and RAL (right) heart across collected developmental time points.

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  Figure 4. Alteration of Heart Ventricle Architecture and Signs of Lagged Maturation in LAL Heart (A) Schematic overview of RV and LV annotation and layer position calculation in normal and LAL hearts. (B) Distribution plots showing layer-wise changes in non-vascular endothelial cell and vCM proportions from outer to inner layers of the LV and RV in LAL hearts. (C) Spatial maps of vCM-immature populations at Day 10 and 12 in normal (top) and LAL (bottom) hearts. (D) Line plots of the proportion of aCMs and valve cells across developmental time points (Day 4, 5, 6, 7 and 12) in both normal and LAL conditions.

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  Figure 5. Molecular characterization of LV and RV in normal and LAL Heart. (A) Boxplots of module scores for “Aerobic respiration” and “Proton motive force-driven ATP synthesis” at Day 10 and 12 LVs. Overlapping leading-edge genes from both time points were used for module score calculation. Full GSEA results are available in Table S3. (B) Boxplots of module scores for “Mitochondrial translation” and “Hydrogen peroxide metabolic process” at Day 10 LVs. Full GSEA results are available in Table S3. (C) Bar plot of normalized enrichment scores (NES) showing the top two gene sets enriched in LVs and two gene sets associated with normal heart function enriched in normal LVs at Day 12. Full GSEA result available in Table S3. (D) Boxplots of module scores for “Regulation of store operated calcium channel activity” on Day 10 RVs and “Sodium ion transport” on Day 12 RVs. Full GSEA results are available in Table S3.

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  Figure 6. Neighborhood Analysis of Valve Region Revealing Plasticity in LAL Valve. (A) Schematic overview of the neighborhood analysis in valves. Recursive GraphST was applied to define the valve region and its sub-domains. For each sub-domain, plasticity scores were calculated alongside cell type composition. Pie charts represent sub-domains, with slices colored according to cell type composition and positioned according to the plasticity score of the corresponding sub-domain. (B) Normal heart neighborhood dynamics of the valve region across developmental time points (Day 6, 7, 10, and 12). From left to right: spatial plots colored by plasticity score; spatial plots colored by sub-domains; dots colored by sub-domains; and pie charts for each sub-domain colored by cell type composition, positioned according to the plasticity score of each sub-domain. (C) LAL heart neighborhood dynamics of the valve region across developmental time points (Day 6, 7, 10, and 12). From left to right: spatial plots colored by plasticity score; spatial plots colored by sub-domains; dots colored by sub-domains; and pie charts for each sub-domain colored by cell type composition, positioned according to the…