Project description:In adult mice, “heart attack” or myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis) and function to drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents oedema and reduces inflammatory/fibrotic immune cell content to improve cardiac function. Given the importance of the adult cardiac lymphatics in macrophage clearance after injury, we investigated their role across the neonatal “regenerative window”. At post-natal day 1 (P1) neonatal mice fully regenerate their heart following MI, in a pro-regenerative macrophage-dependent manner, whereas equivalent injury at post-natal day 7 (P7) leads to scarring driven by pro-fibrotic macrophages. We hypothesised that lymphatics respond and function differently during this “window” to clear macrophage-specific subtypes, depending upon their requirement for regeneration versus fibrotic repair. Normal lymphatic growth and sprouting was evident in intact neonatal hearts until P16, with strain-dependent developmental differences. The response to injury revealed limited lymphangiogenesis (lymphatic vessel growth and sprouting) and minimal clearance of macrophages from P1 compared to P7 infarcted hearts, as determined by adoptive transfer, and monitoring of cleared macrophages to MLNs. This is coincident with maturation of lymphatic endothelial cell junctions across the neonatal period via transition from “zipper” (impermeable) to “button” (permeable) -type junctions and altered signalling between lymphatic endothelial cells and macrophages, including differential expression of the lymphangiocrine factor Reelin (RELN). Finally, in mice lacking the lymphatic endothelial receptor-1 (LYVE-1), that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels, magnetic resonance imaging (MRI) revealed a surprising impaired functional outcome in P1 mice 28 days post-MI. Given our observations that pro-regenerative macrophages at P1 are not trafficked, this suggested a distinct role for LYVE-1 in tissue-resident macrophages, consistent with its expression pattern in developing and post-natal hearts. Macrophage-specific deletion of Lyve1 during neonatal heart injury revealed impaired heart regeneration, characterised by persistent fibrosis, a reduced neovascular response and reduced function. ScRNA-seq of Lyve1 deficient CD45+ cells revealed a loss of tissue-resident macrophages and an increase in recruited monocytes which is predicted to increase inflammation and fibrosis, worsening the outcome post-MI. Collectively, this study reveals that cardiac lymphatics are developmentally compromised for clearance in early neonates, which enables retention of pro-regenerative tissue-resident macrophages, and that LYVE-1 plays an essential role in maintaining this population to promote heart regeneration and prevent inflammation and fibrotic repair.

Project description:Background - The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we construct a transcriptional atlas of multiple cardiac cell populations, which enables comparative analyses of the regenerative (neonatal) versus non-regenerative (adult) state for the first time. Methods - Cardiomyocytes, fibroblasts, leukocytes and endothelial cells from infarcted and non-infarcted neonatal (P1) and adult (P56) hearts were isolated by enzymatic dissociation and FACS. RNA sequencing (RNA-seq) was performed on these cell populations to generate a transcriptomic atlas of the major cardiac cell populations during cardiac development, repair and regeneration. In addition, we surveyed the epigenetic landscape of cardiomyocytes during post-natal maturation by performing deep sequencing of accessible chromatin regions using the Assay for Transposase-Accessible Chromatin (ATAC-seq) from purified cardiomyocyte nuclei (P1, P14 and P56). Results - Profiling of cardiomyocyte and non-myocyte transcriptional programs uncovered several injury responsive genes across regenerative and non-regenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts reverted to a neonatal state and re-activated a neonatal proliferative network following infarction. In contrast, cardiomyocytes failed to re-activate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during post-natal maturation. Conclusions – This work provides a comprehensive transcriptional resource of multiple cardiac cell populations during cardiac development, repair and regeneration. Our findings define a transcriptional program underpinning the neonatal regenerative state and identifies an epigenetic barrier to re-induction of the regenerative program in adult cardiomyocytes.

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A dataset of spatially resolved transcriptomics of post-natal cardiac development in mice

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