Contrary to mammals, zebrafish can regenerate their hearts after a significant loss of their cardiomyocytes. Taking into account the high incidence of myocardial infarction in humans understanding heart regeneration in zebrafish might provide clues to enhance our limited regenerative abilities. Now, a recent paper from the laboratory of Ken Poss reports on the important role of neuregulin1 (nrg1) t o induce the endogenous heart regeneration program in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25830562).
Nrg1 had been previously identified as having multiple roles in cardiovascular biology in both mammals and zebrafish, although some contradictory reports existed respect to the role of nrg1 on cardiomyocytes proliferation. Therefore, in this paper, the authors addressed the role of nrg1 on the strong regenerative response of the zebrafish heart. Whereas nrg1 expression was rarely detected in uninjured hearts, after the genetic ablation of about 50% of the cardiomyocytes there was a significant upregulation of nrg1 that peaked at 7 days post amputation (dpa), a stage in which cardiomyocytes proliferation also peaks. Double stainings determined that most nrg1 positive cells in the postnatal ventricular wall came from the tcf21+ epicardial derived perivascular cell compartment. Rare overlap of nrg1 and markers of vascular endothelial cells and cardiomyocytes was observed.
Nrg1 signals through the Erbb2 and Erbb4b receptors. The pharmacological inhibition of these receptors decreased cardiomyocytes proliferation during regeneration. On the other hand, gain-of-function experiments showed that overexpressing nrg1 in cardiomyocytes resulted in a strong increase of cardiomyocyte proliferation near the injury site, clearly suggesting an important role of nrg1 signalling on cardiomyocyte proliferation during heart regeneration. Next, the authors sought to determine the effects of overexpressing nrg1 in uninjured hearts. After 7 days of treatment (dpt) they observed a marked increase in cardiomyocyte proliferation especially within the ventricular wall. Over time, this increased proliferation resulted in obvious changes in cardiac anatomy. Thus, the ventricular wall thickened by 76% at 7 dpt, by 265% at 14 dpt and by 459% after 30 dpt. Measurements of cardiomyocyte cell number and size suggested that this enormous thickening of the ventricular wall was mainly due to an increase in cell number. The authors referred to it as nrg1-induced cardiac hyperplasia (iCH). Next, the authors analysed the long-term effects of iCH on uninjured adult hearts. On average after 6 months of treatment those hearts showed a 2.1-fold greater ventricular section area. An increased cardiomyocyte proliferation was still evident at this late stage although the authors also observed some mild fibrin and collagen deposition in some regions of the ventricular wall that were not seen just after 30 dpt.
By measuring physiological parameters and cardiac function the authors did not find any cardiac dysfunction at 3 months of iCH. However, at 8-9 months the animals showed a reduction in swimming endurance, a test based on measuring the ability of the fish to swim against increasing water currents, that is an assay of cardiac function sensitive to cardiac damage or heart failure. Therefore, these results indicated a deleterious effect of nrg1 continuous overexpression over a long period of time.
In a last set of experiments, the authors investigated whether iCH depended on similar mechanisms to those triggered by injury-induced regeneration. During normal development the cortical muscle in the ventricular wall form from a small number of large cardiomyocyte clones with discernable boundaries between them. In contrast, during regeneration there is an increase of cardiomyocyte proliferation at the injury site that generates a mixed conglomeration of small clones in the regenerated ventricular wall. The authors observed that the thickening of the ventricular wall during iCH in uninjured hearts contained mixed small clones something reminiscent of an injury-induced regenerative response. When analysing the molecular signatures of iCH the authors first saw an upregulation of gata4 (an embryonic cardiogenic transcription factor) in the outermost layer of cortical muscle at 7dpt. Previous results had shown that after apex amputation gata4 is upregulated in cardiomyocytes near the injury and is essential for regeneration. Moreover, they observed that after iCH there was a reduction of cmlc2 (cardiomyocyte marker) expression in the cortical muscle and a sarcomere disorganization, which would resemble the dedifferentiation process that occurs during regeneration. Another genes implicated in regeneration, such as tgfb3, fn1 (fibronectin synthesis) and raldh2 (retinoic acid signalling), were also upregulated after iCH. Finally, iCH also induced the formation of a major vascular network throughout the expanded ventricular wall. Overall, all these results suggest that nrg1 is sufficient to induce and maintain a global heart regeneration program involving several cell types.
In summary, the authors report here nrg1 as a potent activator of the heart regeneration program in zebrafish. In the future, it will important to determine how is nrg1 upregulated during regeneration as well as its downstream targets.