Fig. 5: Single-cell RNA sequencing analyses of the cardiac organoids.

A UMAP dimensionality reduction of total cells in control and CI samples. Clusters were annotated based on canonical cell type markers. B Heatmap depicting top 30 genes in each cluster in (A) (logFC threshold = 0.3, min.pct = 0.3, adjusted p-value < 0.05). The number of DEGs in each cluster relative to all other clusters is indicated below the heatmap. Color bars indicate cell type (top) and condition (bottom). C Cardiomyocytes were sub-clustered. UMAP dimensionality reduction of cardiomyocytes in control and CI populations. D Relative frequency of each cardiomyocyte cluster in control and CI organoid populations. E Heatmap depicting top 30 genes in each cardiomyocyte cluster (logFC threshold = 0.3, min.pct = 0.3, adjusted p-value < 0.05). The number of DEGs in each cluster relative to all other clusters is indicated below the heatmap. Color bars indicate cluster(top) and condition (bottom). F Pathway enrichment analysis (gProfiler, GO: Biological Processes) was performed using DEGs upregulated in cardiac injury vs. control in each cardiomyocyte cluster. Heatmap depicts the enrichment score (-log₁₀ of adjusted p-value) scaled across clusters for each pathway individually. G Pathway scores were generated using genes annotated for each term in the GO: Biological Processes database. Violin plots show single cell expression of each cumulative pathway score in each cluster for control and CI populations. H A cardiomyocyte maturation score was generated using 8 maturation signature genes²⁰. Heatmap displays the maturation signature score in each cardiomyocyte cluster in the control sample. One-way ANOVA was performed for each cluster compared to the lowest scoring cluster (CM2 and CM6). I An in vivo human heart failure signature was generated using the top 25 DEGs upregulated in ventricular cardiomyocytes in coronary heart failure vs. normal hearts (single cell dataset published in Wang et al.). Each cell in all clusters of control and CI organoids were scored using this signature and displayed in the heatmap. A two-sided unpaired t-test was performed for each pairwise comparison (cardiac injury vs. control in each cluster). For differential expression analysis (B, E, F, H), Wilcoxon Rank Sum Test (unpaired, two-sided) was performed, and p-values were adjusted for multiple comparisons using Bonferroni correction. SMC smooth muscle cells, CM cardiomyocyte, DEG differentially expressed genes. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.