| Popis: |
Background: In contrast to mammals, zebrafish display a remarkable capability to heal heart injuries in a scar-free fashion and to replenish lost myocardial tissue. In zebrafish, upon cardiac damage spared cardiomyocytes located at the wound border de-differentiate and re-enter the cell cycle, thus promoting myocardial regeneration. Despite this striking potential, further research is still required to deeply investigate which signaling pathways are involved in zebrafish heart regeneration, and which molecular details orchestrate their function. In a recent work, our laboratory has shown that the Bmp (Bone Morphogenetic Protein) pathway is triggered as an injury-induced signal and it is essential for both de-differentiation and proliferation of border zone cardiomyocytes during zebrafish heart regeneration. Intriguingly, this evidence is in stark contrast with observations accumulated on infarcted adult mouse hearts, where Bmp loss-of-function limits apoptosis and infarct size. Thus, it appears that the endogenous Bmp signaling pathway exerts a pro-regenerative function during zebrafish heart regeneration, while it is detrimental for cardiac repair in mouse. In this context, we aimed at elucidating the molecular details that orchestrate the pro-regenerative Bmp signaling function in zebrafish heart regeneration. Comprehending how zebrafish retains regenerative potentials will help to discover therapeutic interventions suitable to mend infarcted hearts in humans. Results: We found that bmp2b, bmp4 and bmp7a, encoding Bmp ligands, are up-regulated in cardiomyocytes located at the wound border at 7 days post-injury (dpi), when cardiomyocyte proliferation is sustained. At 7 dpi, while in bmp2b or in bmp4 mutants cardiomyocyte proliferation is not affected, bmp7a mutants show reduced cardiomyocyte cell cycle re-entry. In a previous study, our laboratory had already shown that sustained bmp2b overexpression increases cardiomyocyte proliferation at 7 dpi. Strikingly, while sustained overexpression of bmp7a did not alter cardiomyocyte cell cycle progression, sustained bmp4 overexpression limited cardiomyocyte proliferation at 7 dpi. Apart from the zebrafish heart, in our hands the Bmp signaling pathway appeared to be active in other regenerative and non-regenerative cardiac contexts. While neonatal resected hearts retain a regenerative potential, adult medaka cryoinjured hearts are incapable to regenerate. Intriguingly, both models exhibited canonical Smad-mediated Bmp signaling activity in cardiomyocytes. In addition, we identified three promising Bmp downstream targets in border zone cardiomyocytes of zebrafish hearts: id2a, id2b and prdx1. After injury, their up-regulation was counteracted by short-term noggin3 overexpression, a widely used in vivo Bmp loss-of-function approach. Finally, additional preliminary data were focused on identifying the interaction between Bmp/Smad signaling and other injury-induced responses. We have shown that the activation of Bmp/Smad signaling might be under the control of the retinoic acid (RA) signaling pathway during heart regeneration. Moreover, Bmp signaling might modulate leukocyte recruitment to the injury site in response to zebrafish cardiac damage. Methods: To better dissect the role of Bmp2b, Bmp4 and Bmp7a, we made use of the following tools in the regenerative zebrafish model: • High-quality in situ hybridization experiments to localize bmp2b, bmp4 and bmp7a expression in regenerating hearts with temporal and spatial resolution. • Bmp ligand-specific mutant lines to dissect the in vivo physiological function of bmp2b, bmp4 and bmp7a during heart regeneration. • Bmp ligand-specific heat-shock inducible lines to conditionally drive ectopic expression of bmp2b, bmp4 and bmp7a during heart regeneration. We made use of genetic manipulation to inhibit the retinoic acid signaling pathway. We measured proliferation of cardiomyocytes based on PCNA expression and EdU incorporation, and we assessed Bmp signaling activation via accumulation of phosphorylated Smad 1/5/9 proteins in cell nuclei, a common readout for the canonical Smad-mediated Bmp signaling pathway. We used the L-Plastin marker to trace leukocyte recruitment in the heart. Conclusions: Our results show that the endogenous Bmp7a activity is essential for cardiomyocyte proliferation and for the activation of the canonical Smad-mediated Bmp signaling cascade during zebrafish heart regeneration. Intriguingly, in contrast to bmp2b, bmp4 overexpression affects cardiomyocyte proliferation, potentially acting in a cell-autonomous manner. Thus, stimulating the biosynthesis of different ligands can elicit opposing responses to heart injury in a naturally regenerating model like zebrafish. Furthermore, Bmp signaling appears to be active also in regenerating neonatal hearts and in non-regenerating medaka adult hearts. Investigating the role of Bmp signaling in multiple models, will help to broaden the understanding of its molecular action in different cardiac contexts. In our hands, Bmp loss-of-function analysis indicate that id2a, id2b and prdx1 might represent potentially direct, downstream targets of Bmp signaling during heart regeneration. Further studies will reveal epistatic interactions between Bmp signaling and its downstream targets. Finally, preliminary data suggest an interplay between the Bmp signaling pathway and other injury-induced signals. Additional research is required to dissect the interaction between Bmp signaling, retinoic acid signaling and the immune response in heart regeneration. |
