Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, like zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here we show that the homeo-box containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA-sequencing we find that Prrx1b is activated in epicardial-derived cells (EPDCs) where it restricts TGF-β ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal EPDCs and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart.
RationaleThe human heart loses millions of cardiomyocytes after an ischemic injury, but is unable to regenerate the lost tissue. Instead, the injured human heart is repaired by pro-fibrotic fibroblasts that form a large permanent scar. In contrast, the injured zebrafish heart regenerates efficiently without the formation of a permanent scar. While fibroblasts have been shown to be indispensable for zebrafish heart regeneration, very little is known about the mechanisms balancing the fibrotic and regenerative response. A better understanding of these mechanisms could lead to the discovery of novel therapeutic strategies to reduce fibrosis and promote heart regeneration. ObjectiveTo identify novel mechanisms that regulate the balance between cardiac fibrosis and scarfree regeneration. Methods and ResultsUsing a genetic approach, we first show that zebrafish prrx1b loss-of-function mutants display reduced cardiomyocyte proliferation and impaired heart regeneration. Using a lineage tracing approach, we show that Prrx1b is expressed in tcf21+ epicardial-derived cells localizing around and inside the injured area. Next, we used a single cell RNA-sequencing approach on sorted tcf21+ cells isolated from injured prrx1b-/-and wild-type hearts and identified two distinct fibroblast populations. With combined bioinformatic and histological analysis we found that prrx1b-/-hearts contain an excess of pro-fibrotic fibroblasts that produce TGF-β ligands and collagens, while fewer pro-regenerative Nrg1-expressing fibroblasts are formed. Furthermore, by injecting recombinant NRG1 in prrx1b-/-fish we were able to rescue their cardiomyocyte proliferation defect. Finally, using cultured human fetal epicardial cells and siRNA mediated knock-down of PRRX1 we found that PRRX1 is required for NRG1 induction in human epicardial-derived cells. ConclusionsPrrx1b in the injured heart restricts fibrosis and stimulates regeneration by directing epicardialderived cells towards a pro-regenerative Nrg1-producing fibroblast state.restrictive Prrx1 expression in a confined region in and around the injury area at 14dpi ( Fig.2F). Similar to what we observed at earlier stages, Prrx1 expression was only detected in a subset of mCherry+ cells. At 30dpi, only a few mCherry/Prrx1+ cells were detected, of which the majority was located inside the remaining injury area. Taken together, these results indicate that upon cardiac injury, Prrx1 expression is induced in a subpopulation of EPDCs, which are initially localised at the epicardium and found more dispersed in the injury area at later stages. Proliferation and invasion of EPDCs is unaffected in prrx1b-/-heartsSince cardiac injury induces the proliferation of EPDCs (Lepilina et al. 2006) and Prrx1b is expressed in this cell type we decided to investigate whether prrx1b plays a role in this process. To do so, we used the Tg(tcf21:CreERT2; ubi:loxP-EGFP-loxP-mCherry) line to identify EPDCs in wild-type and prrx1b-/-hearts and used PCNA expression to identify proliferating cells ( Fig.S4A,B). We observed ...
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