Multiple cryoinjuries modulate the efficiency of zebrafish heart regeneration

Abstract

Zebrafish can regenerate their damaged hearts throughout their lifespan. It is, however, unknown, whether regeneration remains effective when challenged with successive cycles of cardiac damage in the same animals. Here, we assessed ventricular restoration after two, three and six cryoinjuries interspaced by recovery periods. Using transgenic cell-lineage tracing analysis, we demonstrated that the second cryoinjury damages the regenerated area from the preceding injury, validating the experimental approach. We identified that after multiple cryoinjuries, all hearts regrow a thickened myocardium, similarly to hearts after one cryoinjury. However, the efficiency of scar resorption decreased with the number of repeated cryoinjuries. After six cryoinjuries, all examined hearts failed to completely resolve the fibrotic tissue, demonstrating reduced myocardial restoration. This phenotype was associated with enhanced recruitment of neutrophils and decreased cardiomyocyte proliferation and dedifferentiation at the early regenerative phase. Furthermore, we found that each repeated cryoinjury increased the accumulation of collagen at the injury site. Our analysis demonstrates that the cardiac regenerative program can be successfully activated many times, despite a persisting scar in the wounded area. This finding provides a new perspective for regenerative therapies, aiming in stimulation of organ regeneration in the presence of fibrotic tissue in mammalian models and humans.

Persistent Ventricle Partitioning in the Adult Zebrafish Heart

“…Immunofluorescence analyses of heart sections were performed essentially as previously described [ 40 ]. Briefly, entire larvae or dissected adult hearts were fixed in 2% paraformaldehyde overnight at 4 °C.…”

Zebrafish as a tractable model of human cardiovascular disease

“…Inducible genetic ablation models, where cell type-specific promoters drive expression of either diptheria toxin (DTA), or nitroreductase (Ntr, an enxyme which converts Metronidazole into a cytotoxic agent) provides further mechanistic insights into how different cell types contribute to cardiac regeneration [85][86][87][88][89] . Interestingly, while the regenerative response to cryoinjury is robust, scar resorption diminishes with repeated injuries and after 6 cryoinjuries hearts fail to resolve fibrotic tissue 90 . While this demonstrates that the heart can regenerate after multiple insults (the ability to regenerate cardiomyocytes themselves does not appear to be impacted after multiple injuries), it suggests there is a limit to the ability to replace the fibrotic tissue with new cardiomyocytes.…”

Using Zebrafish as a Disease Model to Study Fibrotic Disease

“…For instance, after six applications, the heart presented with uncomplete fibrotic tissue resolution and increased accumulation of collagen at the wound site. The phenotypic outcome was secondary to an enhanced recruitment of neutrophils and decreased proliferation and dedifferentiation of cardiomyocytes [65].…”