Zebrafish are able to fully regenerate their hearts through cardiomyocyte (CM) proliferation. Daily circadian rhythms regulate many processes including cell proliferation. As zebrafish differ from mammals in their ability to detect light with all cells in their body, this raises the question if there is a link between this difference in circadian rhythm and CM proliferation after injury. However, nothing is known in either species about a possible interplay.

To test this hypothesis, we characterised the circadian regulation of heart regeneration using zebrafish as animal models. As this is a completely unexplored field of research, we first confirmed if there is a clock oscillator in the fish heart by examining the rhythmic expression of a set of core clock genes including cry1a, per2, clock1, per1b and nr1d1 by qRT-PCR, confirming robust rhythmic expression of all these genes in the uninjured zebrafish heart. To test the importance of this circadian clock on heart regeneration, we performed cardiac cryo-injury on a mutant zebrafish line in which three core clock genes are knocked out (cry1a−/−; per2−/−; cry2a−/− triple knock-out zebrafish), called TKO zebrafish​. Importantly, light-dependent synchronisation of cellular clocks is impaired in the TKO​​. Wound size quantification at 7 and 21 days post cryo-injury (dpci) in TKO and wild-type (WT) control hearts showed reduced regeneration in the TKO. Moreover, CM proliferation was significantly reduced in the TKO consistent with inhibited regeneration. These data indicate that perturbing circadian rhythm negatively affects heart regeneration. To identify regeneration-dependent changes in circadian gene expression, we then performed bulk RNAseq on WT zebrafish control uninjured ventricles and at 7-8 days post cryo-injury (dpci), corresponding to the peak of CM proliferation​​. Ventricles were collected at 4-hour intervals for a total of 48 hours starting at 7dpci. Using the DryR algorithm​ we found that 322 gene exhibit a rhythmic expression pattern only after injury. Strikingly, gene ontology enrichment analysis revealed that this subset of genes is strongly enriched in functions linked to the cell cycle, indicating that the cell cycle is clock controlled during regeneration. As we know that fish heart regeneration is driven predominantly by proliferation of pre-existing CMs​, we next investigated if CM proliferation is rhythmic. 7dpci WT zebrafish ventricles were collected at 6-hour intervals over 24 hours and analysed for CM proliferation. We found that CM proliferation seems significantly increased near the wound at night (zt23) compared with the other time-points analysed. The rhythmicity of the CMs was further confirmed by analysis of p21 expression. P21 (cdkn1a) is most strongly upregulated in the proliferating wound border myocardium at zt17, while almost absent at zt5.  In summary, our data has uncovered that proliferation of zebrafish CMs is controlled by circadian rhythm, moreover, the disruption of the circadian rhythm impacts on the cardiac regenerative capacity in zebrafish. In the future, we would like to investigate if a distinct regulation of the circadian clock mechanism underlies the difference in proliferative capacity between species. Investigating these differences, we could identify novel targets for inducing CM proliferation after injury in patients.