Introduction

In contrast to humans, zebrafish maintain the capacity to regenerate lost cardiac tissue throughout their adult life. This has been attributed to differences in metabolism, with the zebrafish relying mostly on glycolysis, allowing for cardiomyocytes to proliferate (Honkoop et al., 2019). In contrast, the mammalian heart mostly uses oxidative phosphorylation to maintain a high cardiac output. This results in the production of reactive oxygen species which can inhibit cardiomyocyte proliferation and is thought to block heart regeneration. However, using an inter- and intra-species comparative approach, we have found that OXPHOS is essential for long-term heart regeneration by promoting cardiomyocyte maturation following proliferation.

Methods

We cryoinjured the ventricles of seven wild-type zebrafish strains (AB, NA, SAT, TL, TU, WIK KCL) and Astyanax mexicanus and measured their regenerative characteristics at 1-, 7-, 21- and 90 days post-cryoinjury (Dpci). Additionally, we utilised bulk and single-cell (sc) RNAseq to identify the pathways and timeline of events during heart regeneration in our fish. 

Results 

Our data suggests that inter-strain differences exist in zebrafish heart regeneration with some strains (NA, TL) being capable of complete regeneration while others (SAT, TU) being regeneration-incompetent at 90Dpci. Surprisingly, correlating this data with our bulk RNAseq analysis identified OXPHOS as the top pathway which promotes regeneration. Furthermore, by re-analysing previously published scRNAseq datasets, we identified that it is the border zone cardiomyocytes that upregulate OXPHOS. Differential gene expression analysis identified that this upregulation might be fuelled by the malate aspartate shuttle (MAS) which was enhanced in the regeneration-competent strains. Indeed, pharmacological inhibition of both OXPHOS and the MAS ablated the regenerative potential of zebrafish but did not affect cardiomyocyte proliferation. 

Instead of the expected role for proliferation, we identified that it was the expression of embryonic myosins in the border zone cardiomyocytes that correlated with better regenerative outcome. This response appeared to be driven by increased OXPHOS gene expression. Indeed, pharmacological inhibition of both OXPHOS and the MAS led to reduced embcmhc expression but not proliferation, indicating that OXPHOS is necessary for cardiomyocyte re-differentiation. Our scRNAseq confirmed that OXPHOS is temporally separated from cardiomyocyte proliferation and is associated with the cardiomyocyte re-differentiation phase. Finally, we were able to identify that the beneficial role of OXPHOS is conserved and highly enriched in the regenerating surface fish hearts, whereas downregulated in the non-regenerative cavefish hearts, which show reduced cardiomyocyte re-differentiation.

Conclusion

We identified a surprising and unexpected beneficial role of OXPHOS and the MAS during heart regeneration. Our data suggests that OXPHOS is necessary to complete heart regeneration through promoting border zone cardiomyocyte re-differentiation by providing the energy required to sustain sarcomeric re-assembly. Importantly, this process is temporally distinct from cardiomyocyte proliferation, illustrating the existence of two phases in heart regeneration, a proliferative and maturation phase.