Immune and stromal remodeling underlies radiation-induced heart injury: insights from single-cell transcriptomics

Figure 1. The single-cell landscape of rat heart and PBMC before and after irradiation. (A) Workflow of experiment design; (B,C) UMAP for all clusters and cell types of the heart (n = 38,941 cells); (D,E) UMAP for all cluster and cell types of PBMC (n = 41,097 cells); (F) The relative proportion of each cell cluster between groups is indicated. CN_H: the heart tissue samples from the control rat, one simple; RIHI_H: the heart tissue samples from the irradiated rat, two replicates; (G) The relative proportion of each cell cluster across 2 groups is indicated. CN_P: the PBMC samples from the control rat, one sample; RIHI_P: the PBMC samples from the irradiated rat, two replicates; (H) Heatmap showing the differentially expressed genes of cell types ordered by hierarchical clustering.

Figure 2. Endothelial cell profiling and transcriptional remodeling after irradiation. (A,B) UMAP for cell types of RIHI and control heart, including AECs, CapECs, VECs, Endocardial Cells, and LECs; (C) Heatmap of marker genes defining endothelial subsets; (D) Representative top 10 GO terms enriched in different EC types; (E) The relative proportion of each cell cluster between groups; (F) The representative top 10 GO terms enriched in different EC types in RIHI samples; (G) Heatmap showing upregulated genes in CapECs post-irradiation; (H) Heatmap of MHC-I and MHC-II pathway genes across endothelial subclusters pre- and post-irradiation; (I) Western blot validation of MHC-II in endothelial cells before and after irradiation; (J) Pseudotime trajectory analysis of endothelial cells; (K) Ligand–receptor interaction analysis highlighting chemokine-mediated communication patterns in control (CN) and irradiated (RIHI) conditions.

Figure 3. Fibroblast remodeling and ferroptosis-related signatures after irradiation. (A,B) UMAP for cell types of RIHI and control heart, fibroblasts 1–4 (n = 919 cells); (C) Heatmap of marker genes defining fibroblast subsets; (D) Dotplot showing the representative top 10 GO terms enriched in different fibroblast types; (E) The relative proportion of each cell cluster between groups; (F) Module scoring of signature genes in all fibroblasts pre- and post-irradiation; (G) Heatmap of ferroptosis-related genes across fibroblast subclusters pre- and post-irradiation; (H) Boxplot of total iron, antioxidant process, and peroxidation process in fibroblast subclusters; (I) Violin plots showing the expression of Gpx4, SLC3a2, and Sesn2 in fibroblast subclusters pre-and post-irradiation. The p-value corresponds to the paired t-test; (J) Flow cytometric analysis of intracellular Fe2+ levels in endothelial cells before and after irradiation.

Figure 4. Macrophage remodeling and trajectory dynamics after irradiation. (A,B) UMAP for cell types of RIHI and control heart, macrophages 1_3 (990 cells); (C) Heatmap of canonical markers for macrophage subtype identification; (D) Violin plots showing the expression of Fabp4, Fcna, and Spp1 in macrophages 1_3; (E) Heatmap showing the scaled expression level of signature genes in macrophages 1_3; (F) Heatmap showing the relative change in the expression level of signature genes in all macrophages pre- and post-irradiation. (G) Heatmap showing the relative change in the expression level of signature genes in each macrophage subcluster pre- and post-irradiation; (H) The relative proportion of each cell cluster between groups; (I) Unsupervised transcriptional trajectory of macrophages 1_3. The color from heavy to light represents development from beginning to end; (J) Heatmap of pseudotime-associated genes; (K) Heatmap of transcription factors driving macrophage state transitions. The color key from blue to red indicates relative expression levels from low to high.

Figure 5. Neutrophil remodeling and functional programs after irradiation. (A,B) UMAP for cell types of RIHI and control heart, neutrophils1_3 (n = 1,904 cells); (C) Heatmap of marker genes defining neutrophil subsets; (D) Violin plots showing representative marker genes across neutrophil subclusters; (E) The representative top 10 GO terms enriched in different neutrophil cell types; (F) The relative proportion of each cell cluster between groups; (G) The representative top 10 GO terms enriched in neutrophil_1 and neutrophil_2 in RIHI samples; (H) The heatmap exhibits the average regulon activities of transcription factors in different neutrophil cell types; (I) The expression of Nfatc1 target genes. The color key from blue to red indicates relative expression levels from low to high.

Figure 6. T and NK cell remodeling and functional states after irradiation. (A,B) UMAP for cell types of RIHI and control heart, including DPT, IFN_T, NK, NKT, NaiveT, CD4HelperT, and CD8Teff cells (n = 10,174 cells); (C) Heatmap of marker genes for T and NK cell subtype identification; (D) The relative proportion of each cell cluster between groups; (E) Boxplot showing the changes in the average expression of signature genesets in T and NK subclusters pre- and post-irradiation; (F) Violin plots showing the expression of Gzmk, Tgfb1, Ccl5, and Fabp4 in T and NK subclusters pre- and post-irradiation. The p-value corresponds to the paired t-test; (G) Trajectory of T and NK cells. The color from heavy to light represents development from beginning to end; (H) Heatmap of pseudotime-associated genes indicating state-specific transcriptional programs; (I) Heatmap of transcription factors and regulators enriched in distinct T/NK cell subsets. The color key from blue to red indicates relative expression levels from low to high.

Figure 7. Single-cell landscape of B cells in RIHI and control rat. (A,B) UMAP for cell types of RIHI and control heart, including NaiveB1_7 (n = 5,003 cells); (C) Heatmap of canonical marker genes defining B-cell subsets; (D) The representative top 10 GO terms enriched in NaiveB1_3; (E) The relative proportion of each cell cluster between groups; (F) Heatmap showing the relative change in the expression level of signature genes of MHC-I and MHC-II in all B cells pre- and post-irradiation; (G) Pseudotime trajectory analysis of B cells. The color from heavy to light represents development from beginning to end.