Heart regeneration is restricted to the first week after birth in mice, and the underlying molecular mechanisms remain poorly understood. We compared the transcriptomes and epigenomes of the regenerative and nonregenerative mouse hearts over a 7-d time period after myocardial infarction. We show that injury of the regenerative heart triggers a unique immune response involving a macrophage-secreted factor, CCL24, that promotes regeneration. We also describe a developmental gene program that is active in the regenerative heart, within which we found an RNA-binding protein, IGF2BP3, that enhances regeneration. Our study reveals a detailed blueprint of the transcriptional basis of heart regeneration and represents a resource for the identification of genes that may facilitate cardiac repair in response to injury.

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

Heart disease is the leading cause of death worldwide (1). In response to a myocardial infarction (MI), the adult human heart can lose up to a billion cardiomyocytes (CMs) that cannot be replenished due to the inability of adult CMs to proliferate. Loss of CMs leads to diminished cardiac contractility, scar formation, and heart failure with potentially fatal complications (2). In contrast, the neonatal mouse heart can efficiently regenerate following apical resection as well as MI, but this capacity is lost as early as postnatal day 7 (P7) (3, 4). Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide new strategies for heart repair.

Neonatal heart regeneration is orchestrated by multiple cell types, including cardiac resident cells and immune cells that infiltrate the heart after injury (5). Macrophages promote heart regeneration via a paracrine effect (6, 7), and neural innervation has been shown to be required for neonatal heart regeneration (8). Although these studies underscore the importance of multiple cell types for neonatal heart regeneration, this process ultimately relies on the activation of CM proliferation following injury. It has been shown that a significant proportion of CMs in the neonatal heart remain proliferative but exit the cell cycle during the first week after birth (9, 10). The timing of CM cell cycle withdrawal is concurrent with the loss of regenerative capacity of the postnatal heart. Whether the ability for CMs to proliferate after injury relies on their retained proliferation potential or injury-induced environmental cues unique to regenerative hearts, or both, remains poorly understood.

Here, we examine the transcriptome and active chromatin landscapes in regenerative and nonregenerative mouse hearts, and their responses to cardiac injury during an initial 7-d time course. Our analyses identified genes and biological processes that are uniquely activated in injured regenerative hearts, among which the immune response is dramatically distinct between regenerative and nonregenerative hearts. From the secreted factors expressed in regenerative neonatal hearts, we found that C-C motif chemokine ligand 24 (CCL24) protein can promote CM proliferation. Furthermore, we showed that the embryonic cardiac gene program is retained during the neonatal regenerative time window, and identified insulin-like growth factor 2 messenger RNA-binding protein 3 (Igf2bp3), which encodes an RNA-binding protein, as a developmental regulator of postnatal CM proliferation. Our data suggest that neonatal heart regeneration is associated with specific injury responses and expression of cardiac developmental genes. The molecular decoding of cardiac regeneration provides numerous inroads whereby this process could be therapeutically manipulated to enhance cardiac function in response to injury.

Results

Transcriptomic Changes during Neonatal Heart Regeneration. To investigate the mechanistic basis of neonatal heart regeneration, we profiled changes in gene expression spanning the first 7 d post-MI injury in regenerative and nonregenerative mouse hearts at P1 and P8, respectively. P1 and P8 hearts were subjected to permanent ligation of the left anterior descending (LAD) artery or sham surgery; ventricles were collected at 1.5, 3, and 7 d postinjury (dpi) or postsham (dps); and gene expression was analyzed by RNA sequencing (RNA-Seq) (Fig. 1A). Trichrome staining revealed massive loss of CMs in both P1 and P8 hearts at 1.5 dpi (SI Appendix, Fig. S1A). However, P1 hearts showed progressive regeneration and recovery, with minimal fibrosis at 7 dpi. In contrast, P8 hearts exhibited extensive fibrosis, wall thinning, and ventricular dilation at 3 and 7 dpi (SI Appendix, Fig. S1A). For RNA-Seq analysis, heart tissue was harvested below the LAD ligation plane. Analysis of gene expression in hearts subjected to MI or sham surgery identified differentially expressed genes (DEGs) that showed either higher expression (MI-up genes) or lower expression (MI-down genes) in MI samples compared with age-matched sham control samples (SI Appendix, Fig. S1B). Examination by Venn diagram of MI-up genes in P1 and P8 hearts showed that the majority of the MI-up genes were unique to a specific time point. Moreover, of all of the 2,523 MI-up genes in P1 and P8 hearts, 621 genes were shared, 874 genes were unique to the P1 hearts, and 1,028 genes were unique to the P8 hearts (SI Appendix, Fig. S1C). Fig. 1. Transcriptomic analysis reveals distinct and stage-specific responses in regenerative and nonregenerative hearts. (A) Schematic illustration of experimental design and time points for sample collection. (B) Spearman correlation of RNA-Seq datasets showing the transcriptomic similarity among samples. Four distinct clusters were identified. (C, Left) Heatmap showing log10 (fold change [F.C.]) of genes induced by MI. Fold change was calculated by comparing the expression of each gene in MI samples over sham samples for each time point. MI-induced genes from each time point were merged and sorted into 6 groups based on the time point that has the highest fold change. (C, Right) Selected top enriched GO terms for MI-up genes from clusters 1 to 6. (D) Heatmap showing normalized enrichment scores of selected biological pathways that were significantly enriched from the GSEA. (E) Immunostaining of cTnT (purple), vimentin (red), and Ki67 (green) proteins on transverse sections at the level of suture from P1+1.5 dpi, P1+1.5 dps, P8+1.5 dpi, and P8+1.5 dps hearts. Nuclei were counterstained with Hoechst (blue). (Scale bar, 50 μm.) Hierarchical clustering of all of the samples based on their transcriptomic similarities segregated them into 4 clusters (Fig. 1B and SI Appendix, Fig. S1D). The P1+1.5 dpi sample formed its own cluster (cluster 3), reflecting the unique MI response in P1 hearts. Interestingly, the P1+3 dpi sample clustered with P1+1.5 dps and P1+3 dps controls (cluster 4), whereas the P1+7 dpi sample clustered with its corresponding sham control and all sham samples from P8 (cluster 1). This suggests that by 3 and 7 d after MI, the regenerative hearts are already similar to the uninjured hearts of the same age with respect to gene expression patterns. P8 hearts post-MI formed a distinct cluster (cluster 2). The uninjured P1 and P8 hearts clustered into different groups (cluster 4 and cluster 1, respectively), indicating transcriptional changes reflective of postnatal development of the heart (Fig. 1B). These findings indicated that the key transcriptional regulation of regeneration occurs in the initial response following cardiac injury, and that the P1 regenerative hearts rapidly revert to a gene expression pattern similar to that of uninjured control samples.

Distinct Injury-Responsive Pathways Associated with Neonatal Heart Regeneration. To explore the injury-responsive genes unique to the P1 heart, we clustered the MI-up genes based on the time point when they showed highest expression relative to age-matched sham controls. This clustering analysis identified 6 modules of genes that were preferentially up-regulated at specific time points following injury of P1 and P8 hearts (Fig. 1C). For the P1 time points, gene ontology (GO) analysis identified immune processes, such as the inflammatory response and leukocyte activation, as the top GO terms. This is consistent with published reports that the innate immune response, mediated by macrophages, is essential for neonatal heart regeneration (6, 11). In contrast, the top enriched GO terms for the P8 time points included cell cycle process at +1.5 dpi, cell migration and adhesion at +3 dpi, and extracellular matrix organization at +7 dpi, reflecting active fibrotic remodeling of the injured P8 hearts (Fig. 1C). Clustering of the MI-down genes also revealed time point-specific responses (SI Appendix, Fig. S1E). MI-down genes at P1+1.5 dpi were associated with metabolic processes, including fatty acid metabolism and tricarboxylic acid (TCA) cycle. In contrast, MI-down genes at P8 time points were related to ion transport and muscle contraction, reflecting the compromised cardiac function of injured, nonregenerative hearts (SI Appendix, Fig. S1E). We used gene set enrichment analysis (GSEA) to identify dynamically regulated biological processes in P1 and P8 hearts after injury (12) (Fig. 1D). Immune response pathways were highly enriched for both P1 and P8 time points. Intriguingly, the enrichment scores of the immune response pathways peaked in P1 hearts at 1.5 dpi, whereas in P8 hearts, the peak enrichment was observed at 3 dpi. In addition, P8 hearts showed strong enrichment of fibrotic remodeling processes at 3 and 7 dpi. Counterintuitively, cell cycle processes were induced in P8 hearts at 1.5 dpi, but were not induced in regenerative P1 hearts following injury. This may be attributed to the existing high levels of cell cycle-related gene expression at the regenerative stage when not all CMs withdraw from the cell cycle (9, 13). Indeed, our RNA-Seq data suggest that cell cycle-related genes are highly expressed at P1+1.5 and P1+3, regardless of MI or sham, and become down-regulated starting at P8 (P1+7 dps) when the regeneration capacity is lost (SI Appendix, Fig. S1F). In addition, immunohistochemistry of P1 and P8 hearts at 1.5 d after MI showed augmented cell cycle activation in fibroblasts of P8 hearts, whereas cell cycle activity was primarily detected in the CMs of P1 hearts (Fig. 1E). These data indicate that the endogenous cell cycle gene program is active in P1 CMs during heart regeneration. In contrast, the transient MI-induced cell cycle gene expression observed in the P8 hearts reflects fibroblast activation that contributes to cardiac fibrosis, as previously reported (14). Together, these findings reveal distinct injury-response processes and transcriptional dynamics in regenerative hearts.

Active Chromatin Regions Associated with Neonatal Heart Regeneration. To understand the regulatory mechanisms underlying the distinct transcriptomic changes in response to injury, active chromatin regions of P1 and P8 hearts were profiled by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for histone H3 lysine-27 acetylation (H3K27ac), which marks active enhancers and promoters (Fig. 1A). H3K27ac signals were detected at the genomic loci of cardiac marker genes, such as Myh6, Tnnt2, Nppb, and Nppa, at similar levels in replicate samples, validating data reproducibility (SI Appendix, Fig. S2 A–C). Comparative analysis of the H3K27ac peaks between MI and sham samples of P1 and P8 hearts revealed genomic regions that showed increased (MI-gained) or decreased (MI-lost) H3K27ac signals at each time point following injury. The genomic distribution of the 13,447 MI-gained peaks showed that the majority were intronic (48%) or intergenic (35%), with only 8% being mapped to promoters (Fig. 2A). Similar to the trend observed for the DEGs (SI Appendix, Fig. S1B), the numbers of MI-gained and MI-lost peaks decreased significantly across the P1 time points following injury (SI Appendix, Fig. S2D). However, the number of peaks continuously increased in P8 hearts over time following injury (SI Appendix, Fig. S2D). Fig. 2. Injury-induced active chromatin regions revealed by H3K27ac ChIP-Seq. (A) Pie chart showing the genomic distribution of MI-gained H3K27ac peaks. (B, Left) Heatmap showing all MI-gained H3K27ac peaks, grouped and rearranged by the time point when they have the highest fold change compared with the sham control. (B, Right) Top GO terms associated with each group of peaks and the corresponding -log10 P value. (C, Upper) H3K27ac ChIP-Seq tracks showing the intronic peak activity in the Fhl1 gene locus at P8+3 dpi and P8+3 dps. (C, Lower) In situ hybridization images showing Fhl1 mRNA expression in transverse sections from P8+3 dpi and P8+3 dps hearts at the suture level. (Scale bar, 500 μm.) (D, Upper) H3K27ac ChIP-Seq tracks showing the Tgm2 enhancer peak activity at P8+3 dpi and P8+3 dps. (D, Lower) In situ hybridization images showing Tgm2 mRNA expression in transverse sections from P8+3 dpi and P8+3 dps hearts at the suture level. (Scale bar, 500 μm.) (E) Heatmap showing TF binding motifs preferentially enriched in MI-gained enhancers at each time point. For P8+3 dpi and P8+7 dpi, the top 10 enriched TF binding motifs are shown. To identify MI-gained H3K27ac peaks unique to the regenerative heart, we clustered the peaks based on the time point when they showed the highest signal relative to the sham controls. This clustering analysis identified 6 modules of MI-gained H3K27ac peaks that were specific to each time point following injury for P1 and P8 hearts (Fig. 2B). Analysis of genomic regions in each group using genomic region enrichment of annotations tool revealed biological processes regulated by their associated genes (15). General immune responses, including inflammation and leukocyte activation, were highly enriched at P1+1.5 dpi (module 1), P8+3 dpi (module 5), and P8+7 dpi (module 6). This is consistent with the immune response dynamics observed in the GSEA (Fig. 1D). In addition, peaks in the P8+7 dpi group (module 6) were associated with fibrotic remodeling processes and represent the most pronounced injury response, as evidenced by the total number of peaks (Fig. 2B). Notably, vascular developmental processes were preferentially enriched at P1+3 dpi (module 2), highlighting the unique chromatin regions that respond to injury of the neonatal heart (Fig. 2B). In summary, these findings show that the epigenomic H3K27ac chromatin landscape responds differently to injury in regenerative and nonregenerative hearts, and highlight the unique dynamics of active chromatin landscape in regenerative hearts. To determine whether the gene expression changes were attributable to changes in active chromatin, we examined whether there was a positive correlation between MI-gained H3K27ac peaks and the expression of their nearest genes (SI Appendix, Fig. S2E). Overall, genes associated with MI-gained H3K27ac peaks showed substantially higher expression levels in the MI samples compared with sham controls in RNA-Seq. Similarly, genes associated with MI-lost H3K27ac peaks showed markedly lower expression in the MI samples compared with sham controls. The positive correlation between the H3K27ac peaks and gene expression suggests transcriptional regulation at the epigenomic level. We found the GO terms derived from MI-up gene modules (Fig. 1C) and H3K27ac peak modules (Fig. 2B) to be mostly inclusive, with some discrepancy. This disparity could be caused by (1) overrepresentation of various active chromatin-associated genes due to enhancer redundancy, (2) delayed transcriptional response after enhancer activation, or (3) inaccurate mapping of target genes whose enhancers act at a long distance through chromatin looping. To validate that the MI-gained chromatin regions are associated with MI-up genes after injury, we selected 2 MI-gained H3K27ac peaks and examined the expression of their nearest genes using in situ hybridization. The first peak is located in the intronic region of the Fhl1 gene. Following MI of P1 and P8 hearts, this genomic region was activated at multiple time points, with the highest fold change at P8+3 dpi (Fig. 2C). Fhl1 was also an MI-up gene at this time point, as revealed by our RNA-Seq data. In situ hybridization of mouse heart sections at the same time point showed substantial Fhl1 up-regulation in regions surrounding the infarct, compared with the sham controls (Fig. 2C). The second candidate region is located ∼20 kb upstream of the Tgm2 gene. H3K27ac deposition on this region and Tgm2 gene expression were both significantly induced at P8+3 dpi, compared with P8+3 dps (Fig. 2D). In situ hybridization showed intense Tgm2 expression in the border zone as well as the right ventricular wall after MI (Fig. 2D). Together, the transcriptome and active chromatin profiles revealed various biological processes that are temporally activated after injury during heart regeneration and cardiac remodeling. We further performed motif analysis to identify enhancer-associated transcription factors (TFs) controlling gene expression during heart regeneration. We first excluded H3K27ac peaks in promoter regions, and surveyed for TF-binding motifs that were preferentially enriched in MI-gained enhancers (referred to as MI-gained motifs) at each time point. This revealed TF-binding motifs that were significantly enriched in MI-gained enhancers at 1 or more time points examined, which were clustered based on the time point with highest enrichment to identify TF motifs that are unique to regenerative P1 hearts compared with nonregenerative P8 hearts (Fig. 2E). We found motifs such as nuclear factor κB (NFκB), Foxh1, and Stat5 to be preferentially enriched at P1+1.5 dpi; these motifs may play important roles during neonatal heart regeneration (16). Interestingly, we found motifs for class I NFκB (p50, p52) to be specifically enriched in P1+1.5 dpi hearts, whereas class II NFκB (p65, Rel) motifs were preferentially enriched in P8+3 dpi hearts (17, 18). This suggests that different classes of NFκB and their target genes may have opposing effects on heart regeneration. Consistent with the motif enrichment in the MI-gained enhancers, genes encoding some of the enriched TFs, such as Nfkb2 (p52), Gata3, Bcl6, Zfp692, Cebpa, Nfe2, Rel, Spi1 (PU.1), Nfatc3, Jun (AP1), Runx1, and multiple members of the IRF family (Irf1, Irf5, Irf7, and Irf9) were also up-regulated after MI (log2 fold change > 0.5) (SI Appendix, Fig. S2F). Although activation of the TF-directed gene network can be achieved by posttranslational modifications of TFs, such as phosphorylation and acetylation (19, 20), our data suggest that the transcriptional activation of these TFs can also contribute to the changes in the active chromatin landscape following injury.