Single-cell chromatin accessibility landscape of cardiac non-myocytes identifies tissue repair program during heart regeneration

Figures

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  Fig. 1. Characterization of non-cardiomyocytes (Non-CMs) cellular composition and distribution during neonatal heart regeneration. a Schematic. showing apical resection (AR) or sham surgery performed after birth (P0) and subsequent days of sample collection for single-cell Assay for Transposase. Accessible Chromatin sequencing (scATAC-seq). b (left) Unsupervised clustering demonstrates seven distinct cell types shown in a Umap plot. Non-CMs. (n = 57,899) were obtained from 18 samples. (right) Bar plot of cell number among all cell types. c–e (left) Umap plots depicting scATAC-seq analysis in the. study, color-coded based on treatment or time origin. (right) uniform manifold approximation and projection (Umap) illustrating the distribution of cells in each sample at post-natal day 7 (P7) and post-natal day 14 (P14) timepoint. f Umap of all subclusters within each cell types. g The heatmap of ratio of cell. number among each sample of cell type clusters. The proportion normalized by the total cell number.

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  Fig. 2. Dynamics of Cebpd activation in neonatal cardiac fibroblasts (FBs) in response to injury. a Umap representation illustrating distinct sub-populations. of FBs analyzed through scATAC-seq. b Comprehensive distribution of FB sub-populations in each scATAC-seq sample, discerned following. sub-clustering analysis. The color code corresponds to the sub-populations represented in Umap (Panel a). c Heatmap depicting the expression patterns. of top-enriched genes for each FB sub-population, accompanied by Gene Ontology (GO) analysis illustrating the functional annotations of genes associated. with peaks. d Motif analysis revealing subtype-specific chromatin open regions in FBs. e Umap representation displaying the deviation score of. CCAAT/enhancer-binding protein (C/EBP) binding site accessibility across FB sub-populations. f Genescore profile of the C/EBP family within each FB. sub-population. g Violin plot illustrating the genescore distribution of the C/EBP family across all samples.

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  Fig. 3. Cebpd knockdown impairs neonatal cardiac regeneration and reduces FB activation. a Evaluation of Cebpd expression levels in response. to injury in the public dataset. b Bar plot illustrating GO terms associated with CEBPD binding chromatin regions. Quantitative Reverse Transcription-. polymerase Chain Reaction (qRT-PCR) analysis showcasing the expression levels of (c) Cebpd and (d) markers of activated FBs in TGF-β1-treated. conditions and TGF-β1-treated conditions with Cebpd knockdown. The scrambled-sequence shRNA (shScr) served as the non-targeting control group in. this experiment. Data are presented as means ± SD, n = 3; *p < 0.05, **p < 0.01. e Protein-level assessment of markers associated with activated FBs in. TGF-β1-treated conditions and TGF-β1-treated conditions with Cebpd knockdown. Data are presented as means ± SD, n = 3; *p < 0.05, **p < 0.01. f Representative images of FB cultures stained with 5-Ethynyl-2’-deoxyuridine (EdU) (red) and alpha-smooth muscle actin (α-SMA, green), indicating. activated FBs. Quantitative analysis was conducted on counts from multiple fields across three independent samples per group, with results representative. of three individual…

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  Fig. 4. AP-1 members are transiently activated in endothelial cells (ECs) during heart regeneration. a Umap plot illustrating major subsets captured. by scATAC-seq (n = 28,055). b Umap visualization of EC subclusters redefining ECs into capillary and non-capillary ECs. c Distribution of cell numbers. among samples within the EC subset. d Heatmap and GO analysis displaying the functional attributes of specific genes with heightened accessibility. in each EC population. e Umap representation of marker genes for each subtype. f (left) Comparative analysis of differential peaks between EC3 and. Non-EC3 populations. (right) GO analysis highlighting the functions of genes proximal to specific accessible regions in each group. g Bubble plot presenting. motif enrichment scores derived from EC3-specific accessible regions, ranked by enrichment score.

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  Fig. 5. Impact of AP-1 inhibition on EC proliferation and migration. a Analysis of clonal expansion in human umbilical vein endothelial cells (HUVECs). upon AP-1 inhibition (with T-5224 treatment). Two-tailed unpaired Student’s t test was employed, and data are presented as the mean ± SEMs, n = 3, *p < 0.0.05. b Immunostaining of (left) phosphorylated histone 3 (pH3), (middle) EdU, and (right) Ki67 in HUVECs subjected to AP-1 inhibition. Two-tailed. unpaired Student’s t test was utilized, and data are presented as the mean ± SEMs, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 20 μm. c Wound. scratch assay assessing the migration of HUVECs upon AP-1 inhibition. Two-tailed unpaired Student’s t test was employed, and data are presented as the. mean ± SEMs, n = 3; *p < 0.05. Scale bars, 100 μm. d Evaluation of angiogenesis by the formation of capillary-like tubes in HUVECs subjected to AP-1. inhibition in vitro. Two-tailed unpaired Student’s t test was utilized, and data are presented as the mean ± SEMs, n = 3; ***p < 0.001. Scale bars, 100 μm. e Schematic of the experimental design. Neonatal mice received daily injections of Vehicle or T-5224 (20 mg/kg) from P1 to P7 and were…