Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration

Abstract

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (CL) treatment compromises neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first-week post injury (Lai et al., 2017). Here, we examined the molecular mechanisms underlying the cardiac repair of regenerative PBS-control hearts vs. non-regenerative CL-treated hearts. Bulk transcriptomic analyses revealed that CL-treated hearts exhibited disrupted inflammatory resolution and energy metabolism during cardiac repair. Temporal single-cell profiling of inflammatory cells in regenerative vs. non-regenerative conditions further identified heterogenous macrophages and neutrophils with distinct infiltration dynamics, gene expression, and cellular crosstalk. Among them, two residential macrophage subpopulations were enriched in regenerative hearts and barely recovered in non-regenerative hearts. Early CL treatment at 8 days or even 1 month before cryoinjury led to the depletion of resident macrophages without affecting the circulating macrophage recruitment to the injured area. Strikingly, these resident macrophage-deficient zebrafish still exhibited compromised neovascularization and scar resolution. Our results characterized the inflammatory cells of the zebrafish injured hearts and identified key resident macrophage subpopulations prerequisite for successful heart regeneration.

Defining mononuclear phagocyte distribution and behaviour in the zebrafish heart

“…Adult zebrafish are known to have populations of cTMs in the steady state and cardiac MNP populations expand following cardiac injury 24,29,62 . A recent study has highlighted the heterogeneity of resident cTMs and their involvement in promoting regeneration 42 echoing what has been described in neonatal mice 63 . However, we are yet to fully understand the spatial distribution of these cells in the heart, the roles played by distinct MNP lineages and to confirm strategies to identify these subpopulations.…”

“…Multiple studies have provided important insight into the ontogeny and maintenance of microglia 65,67,70,79 , but much of this hasn't been investigated in other tissues. Recent studies using single cell sequencing (scRNA-seq) are advancing our understanding of immune cell heterogeneity in the zebrafish heart and other organs [41][42][43]66,70 but much of this has been derived from mpeg1.1 expressing cells and is yet to be translated into new tools to define the distribution of these subpopulations. Our data indicates that using csf1ra and mpeg1.1 transgenic lines it is possible to identify four separate immune cell populations in the homeostatic heart and we have shown that these cells have distinct morphology, distribution, and behaviour in cardiac tissue.…”

“…However, much of the initial characterisation of these markers was performed in larval zebrafish and, although expression in tissue macrophages was confirmed, whether expression extends to monocytes and DCs has not been well defined. Recent technological advancements and accessibility to RNA sequencing technology has facilitated the molecular characterisation of MNPs [40][41][42][43] .…”

Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration

“…Through scRNA-seq analysis of border zone cells, we find a population of macrophages that express genes important for remodeling and degradation of ECM, in line with recent published studies that independently corroborate the presence of this ECM macrophage cluster in the regenerating zebrafish heart 30,62 . Notably, it has been recently shown that the resident macrophage population is essential for cardiac regeneration in zebrafish 29 , and that resident macrophage populations in the neonatal and adult mouse are essential for improving functional output following multiple types of cardiac injury and disease [63][64][65] . We suggest that one function of the resident macrophage population is to promote CM invasion and replenishment of collagen-containing injured tissue with CMs during regeneration.…”

“…The importance of immune cells in promoting the cardiac regeneration process has also been recognized in more recent years. In particular, macrophages have been shown to play an essential role in regeneration of zebrafish, salamander, and neonatal mouse hearts in response to injury [26][27][28][29] . Macrophages have been shown to directly contribute to and regulate the composition of scar/ECM in the cryoinjured heart 27,30,31 .…”