Fig. 7: Spatiotemporal interactions of the myocardial niches.

a, UMAP of scRNA-seq data for CM subtypes from day 1–56 post-lesion. Post-lesion enriched subtypes are highlighted in bold. b, DEGs for each subtype. Dot size indicates fraction of cells expressing the gene. Dot color indicates normalized expression level. c, CM subtype proportion across days 1–56 and sham. d, Quantification of aggregated G2M gene expression across cells (Methods) on logarithmic scale. Statistics used were one-way ANOVAs. Box center, median. Box upper and lower bounds, 25 and 75 percentiles. Whisker maxima, 75 percentile + 1.5 interquartile. Whisker minima, 25 percentile − 1.5 interquartile. n = 1,170, 14, 16, 2,260, 452, 87 and 139 nuclei. e, Annotated spatial maps of CM subtypes on day 7 with detailed annotation scheme, and quantifications across days 7 and 28. f, Representative IF staining images of LAD day 7 heart RZ sections with PCM1, Mki67 and αSMA. Mki67 labels proliferating nuclei, and αSMA labels de-differentiating CMs. n = 2 experiments. g, Comparison of pseudotime trajectory from homeostatic to the de-differentiated state in adult versus neonatal CMs post-lesion⁴. h, Spatial neighborhoods (NiCo interaction scores) of de-differentiating CMs, ILC2 and T_reg cells on day 7. Neighborhood scores, NiCo regression coefficients. Error bars, s.e. of the coefficient estimates. i, De-differentiating CM niche visualization on days 7 and 28, visualized by the spatial transcriptomic data (top) and IF staining (bottom, LAD day 7 RZ), respectively. For the IF staining, de-differentiating CMs (Myoglobin⁺), ECs (CD31⁺) and ILC2 (Gata3⁺) were detected. n = 2 experiments. j,k, IF of day 7 LAD in IZ-BZ for ILC2 (KIT⁺GATA3⁺BMP-7⁺) (j) and de-differentiating CMs (sarcomeric actinin⁺ aSMA⁺) (k). n = 2 experiments. WGA labels cell membranes. Arrows point to IL-1R1⁺ de-differentiating CMs. l, Primary P7-CM culturing (Methods). Cells were exposed to ligands for 48 h. Representative IF images of ligand-exposed CMs. CMs (sacromeric actinin⁺) with Mki67⁺AuroraB⁺DAPI⁺ nuclei exhibit cell cycle activity. White arrows point to cytokinetic morphology indicating CM cell division. m, Image quantifications of CMs with cell cycle activity (left) and undergoing cytokinesis (right) for data in l. n, qRT–PCR of Actc1 (progenitor marker) and Myh6 (mature marker) of cultured CMs. Actc1-to-Myh6 expression ratios (as a progenitor state score) are shown. From left to right, n = 5, 7, 7, 6 and 6 biological replicates. o, Schematic diagram of human cardiac slice culture (Methods). p, Representative images showing loss of sacromeric structures in the BIT-exposed CMs for human cardiac slice culture. n = 2 experiments. q, Representative images showing Mki67⁺ CMs and NMs in BIT-treated slices, but not in BSA-treated slices. White dotted lines highlight cell boundaries. n = 2 experiments. r, Quantification of Mki67⁺DAPI⁺ nuclei in CMs (sarcomeric actinin⁺ cells). n = 8 biological replicates. Statistics used were unpaired two-tailed t-tests for two experimental groups (m,n,r). NS, not significant. Error bars show s.d. centered at mean.

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