Renewal of embryonic and neonatal-derived cardiac-resident macrophages in response to environmental cues abrogated their potential to promote cardiomyocyte proliferation via Jagged-1–Notch1

Figure 1. Stromal cell composition of the mouse heart under steady states during ontogeny. (A) Scheme of the study design and workflow. n = 2 (one male and one female mouse were included at every point. More than 10,000 cells were collected at indicated points). (B) Cells were reduced dimensionally by principal component analysis, clustered based on a graph-based clustering approach, spectral t-SNE was used to reduce to 2-dimension for visualization. Each point depicts a single cell, and different color represents each cluster. (C) Gene-expression Heatmap of top 10 marker genes for each cell cluster. (D) t-SNE plot of single cells isolated from the heart of varying ages. Cells were colored according to their time point of origin. (E) Relative fraction of each cluster was given by the number of cells at a different time point and scaled to 100%. (F) t-SNE plot of cardiac stromal cells composition after unsupervised clustering. (G) Dot plot of marker genes identifying cell type. Individual dots are sized to reflect the proportion of cells and color to reflect the average expression levels of each marker gene across all cells. E18D, P7D, P4W and P1Y mean embryonic Day 18, post-natal 7 days, 4…

Figure 2. CRMs properties at different time points during ontogeny. (A) Unsupervised sub-cluster showed four distinct cell subsets in a t-SNE map. (B) Heatmap of top 10 marker genes in down-sampling of more than 100 cells for each cluster. (C) Violin plots of the marker genes expression across five subsets. (D) Distribution characteristics of different subsets at different time during ontogeny based on scRNA-seq analysis. (E) Four CRM subsets existed successively. Macrophages were identified by the expression of CD45 and CD64 in hearts, and separated into four subsets based on Ly6C, CX3CR1, and MHCII, as determined by flow cytometry. At every point, six to eight mice were included. (F) The dynamic changes of MP1–4 frequencies at different points. E18D, P7D, P4W and P1Y mean embryonic Day 18, post-natal 7 days, 4 and 8 weeks, and 1-year-old, respectively.

Figure 3. Four CRM subsets existed successively and exhibited different functions during ontogeny. (A) Selected categories from GO enrichment analysis of DEGs are expressed as –log10P value adjusted for multiple comparisons. (B) Immunofluorescence staining of Ki67 (left) and Aurora B (right) in cTNT positive neonatal mouse cardiomyocytes directly co-cultured with CRMs from mouse hearts of different ages (None, E18D, P7D, P4W, P1Y). The representative images are shown above and statistical analysis shown below. X axis presents different CRMs treatment. (C) Immunofluorescence staining of Ki67 and Aurora B in neonatal mouse cardiomyocytes co-cultured with CRMs from embryonic or neonatal mice directly or indirectly. The representative images are shown left and statistical analysis shown right. (D) Neonatal mouse cardiomyocytes were directly co-cultured with CRMs from embryonic and neonatal mouse hearts by cell–cell contact, CRMs were stained by F4/80, and the cardiomyocytes were stained by cTNT. For B and C, the data were repeated 3–5 times, similar data were obtained, the representative images are shown. ∗P < 0.05. E18D, P7D, P4W and P1Y mean embryonic Day 18, post-natal 7 days, 4 and 8…

Figure 4. CRMs derived from embryonic or neonatal mice facilitated the cardiomyocytes proliferation via Jagged-1–Notch. (A) The analysis of biologically relevant ligand–receptor interacting between CRMs from embryonic or neonatal mice and cardiomyocytes. (B) Notch1-4 expression was analyzed. RNA was extracted from purified neonatal mouse cardiomyocytes, and the gene expression of Notch1-4 was measured by RT-qPCR and normalized relative to β-actin (n = 4). (C) Jagged-1 expressions on CRMs of different ages mice (n = 4). (D) Immunofluorescence staining of Aurora B and Ki67 in cTNT positively neonatal mouse cardiomyocytes directly co-cultured with CRMs from embryonic or neonatal mice in the presence or absence of DAPT (n = 3). (E) qPCR analysis of Hes1 and Hey1 mRNA in neonatal mouse cardiomyocytes, results normalized to β-actin (n = 3). (F) Aurora B and Ki67 expression in neonatal mouse cardiomyocytes co-cultured with CRMs from embryonic or neonatal mice with or without pre-exposure to Jagged-1 antibody for 20 min (n = 4). (G) CRMs from embryonic or neonatal mice and P1Y mouse hearts were immediately transferred into the appropriate recipient mice by intra-myocardial injection on Day 1 after…

Figure 5. MP1 cluster, a powerful and diverse function subset, had different derivations. (A) Ordering CRMs along a cell conversion trajectory using Monocle analysis. CRMs were color-labeled by pseudotime or sub-cluster. Cell clustering of MP1 based on the state along pseudotime trajectory showed five distinct trajectory states. (B) Heatmap of marker genes expression in the indicated states of MP1. (C) Selected top GO enrichment categories of the DEGs between different states. 30–50 cells per category were included.

Figure 6. The replacement of MP1 by MP4 was driven by the cellular redox state. (A, B) Heatmap display DEGs expression level in each time point (A) or MP1–4 (B). (C) Heatmap of gene expression along the pseudotime trajectory of CRMs. For A–C, 30–50 cells per category were included. (D) Frequencies of MP1 and MP4 were determined by flow cytometry. CRMs derived from different age mice were treated by GSH or H2O2 for 48 h and then analyzed using flow cytometry (n = 4). (E) Intracellular ROS levels were measured by flow cytometry using DCFDA dye fluorescence in cells treated with or without H2O2. (F) The phenotypes of CRMs from embryonic or neonatal mice were detected after co-culturing with H2O2 or H2O2+GSH pretreated MCMs by flow cytometry. (G) Dil-labeled MP1s were detected on the 3rd after MI, the phenotype was identified by the expression of CD45, CD64, CX3CR1 and MHCII (n = 6). ∗P < 0.05, ∗∗P < 0.01. E18D, P7D, P4W and P1Y mean embryonic Day 18, post-natal 7 days, 4 and 8 weeks, and 1-year-old, respectively. MI and MCMs mean myocardial infarction and murine cardiomyocyte cell line, respectively.