Investigating macrophage-lymphatic vessel interactions in neonatal mouse model of heart regeneration

Myocardial infarction (MI) triggers an immune response, whereby phagocytic cells remove dead tissue and assist with the subsequent remodelling and repair of the infarcted heart. In adult mice, MI activates cardiac lymphatics, which function to drain the build-up of interstitial fluid (oedema) and traffic macrophages to mediastinal lymph nodes (MLNs), reducing inflammatory/fibrotic cell content and improving cardiac output. Mice at postnatal day 1 (P1) fully regenerate their heart following MI in a pro-regenerative macrophage-dependent manner, whereas similar injury at P7 leads to scarring driven by pro-fibrotic macrophages. The role of cardiac lymphatics in the regenerative capacity of neonatal mice remains unexplored. Therefore, we hypothesised that lymphatics respond and function differently following MI during this regenerative window (P1 to P7), to clear macrophage specific subtypes depending upon their requirement for regeneration (P1) or fibrotic repair (P7).
To understand the spatiotemporal changes that take place in the cardiac lymphatic vasculature following birth, we initially quantified the expansion of the vascular network along the sub-epicardium. This revealed lymphatic growth and sprouting until P16, and strain-dependent developmental differences. We then investigated the maturation status of lymphatic endothelial cell junctions, which suggested a potential transition from “zipper” (impermeable) to “button”-type (permeable) junctions during the first two weeks of life. In addition, we examined the lymphangiogenic response and the trafficking efficiency of cardiac lymphatics after surgically induced MI. Using 3D light-sheet and confocal imaging; we found that VEGFR3-expressing lymphatics have limited lymphangiogenic response in P1 compared to P7 hearts 7 days after MI. To assess trafficking of macrophages to MLNs, we performed adoptive transfer of adult splenic hCD68-eGFP labelled monocytes into the myocardium of P1 and P7 recipient mice undergoing coronary artery ligation. Imaging of MLNs from these animals indicated a less efficient clearance of GFP-labelled cells from P1 compared to P7 hearts 7 days after MI . To further investigate differences in immune cell trafficking in P1 versus P7 hearts and their association with regeneration/repair, we made use of mice lacking Lyve1 that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels in adult mice. Unexpectedly, MRI analysis of Lyve1-/- mice revealed impaired cardiac regeneration after P1 MI, while no changes were observed in cardiac function after P7 MI compared to the respective intact controls. Lastly, to gain insight into the molecular underpinnings of lymphatic endothelium-macrophage interactions in P1 versus P7, we generated unbiased single cell RNA sequencing datasets from samples collected at different time-points after MI. A summary of the initial computational analyses, as well as future approaches are discussed here.
The results of this project show that cardiac lymphatics continue to grow and mature postnatally and support the hypothesis that cardiac lymphatics respond and function less efficiently after P1 MI, compared to P7, in line with a need to retain pro-regenerative macrophages in the neonatal heart versus clearance of pro-inflammatory/fibrotic macrophages 7-days later. Further analysis is required to uncover the molecular mechanisms that lead to this differential response.

Keywords: myocardial infarction
heart attack
macrophages
heart regeneration
cardiac lymphatics