|Abstract||Unlike mammals, zebrafish can achieve scar-free healing of heart injuries, which is associated with dedifferentiation and cell cycle re-entry of cardiomyocytes. However, it is unknown to which extent cardiomyocytes lost to injury are restored, since only indirect evidences of myocardial regeneration exist thus far. In addition, while Wnt signaling has been shown to induce fibrosis and prevent production of new cardiomyocytes in the infarcted murine heart, involvement of this pathway in zebrafish cardiac regeneration has not been yet investigated. Hence, we set out to provide the first absolute quantification of cardiomyocyte numbers before and after injury in zebrafish, as well as to address whether Wnt signaling impinges on cardiomyocyte proliferation and wound healing.
Firstly, we used stereological methods to quantify the number of cardiomyocytes on heart sections at 3, 14, 30 and 90 days post cryoinjury. Based on histological markers, we investigated the morphology of the regenerated myocardium. Secondly, we performed quantitative PCR and in situ hybridization to check whether Wnt signaling is active, and which Wnt ligands are expressed in the injured heart. Finally, we employed genetic tools for Wnt/β-catenin manipulation and measured proliferation and dedifferentiation in cardiomyocytes, as well as wound resorption, upon inhibition of the pathway.
We found that injured zebrafish hearts did regenerate to the pre-injury number of cardiomyocytes already within four weeks. Mathematical modeling indicated that all cardiomyocytes that enter the cell cycle at the wound border actually proliferate. The regenerated myocardium does not display signs of altered morphology compared to uninjured hearts. Thus, we establish that zebrafish can indeed completely and faithfully regenerate the ventricular myocardium. Surprisingly, full cardiomyocyte regeneration also occurred in hearts that retained scars, indicating that cardiomyocyte regeneration is more efficient than scar-free healing. In addition, we found that Wnt/β-catenin signaling is active in regenerating hearts both in cardiomyocytes and in endothelial cells. Blockade of the pathway impairs cardiomyocyte dedifferentiation and proliferation, likely in a cell autonomous manner. However, Wnt signaling has no significant effect on scar removal. Finally, we found significant upregulation of the Wnt9a ligand in endothelial cells after injury.
Our results show that zebrafish cardiomyocyte regeneration is efficient and complete, and it does not require complete scar removal. Wnt/β-catenin is an important player in this process, since it is required to promote cell cycle re-entry and dedifferentiation of pre-existing cardiomyocytes.||dc.description.abstract