Multiple cryoinjuries modulate the efficiency of zebrafish heart regeneration

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.