Identifying Transcription Factors Distinguishing Regenerating Cardiomyocytes in the Zebrafish Heart Using Single-Cell Transcriptomics

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Mammalian hearts exhibit limited regeneration, whereas zebrafish hearts present a remarkable capacity to regenerate upon injury through cardiomyocyte (CM) proliferation. Thus, understanding genes and especially transcription factors (TFs) enabling zebrafish CM regeneration may open new avenues for human heart repair. Herein, we injured zebrafish hearts by apex resection (AR) and found that the number of cycling CMs peaked around 7–14 days post-injury (dpi). Single-photon confocal imaging of 3D zebrafish hearts confirmed cycling cardiac cells throughout the heart at 7 dpi, while flow cytometry estimated that 1–2% of CMs were cycling. High-resolution single-cell RNA sequencing identified a CM cluster exclusively present after AR, whereas differential gene expression profiling identified genes defining this AR-specific CM cluster. Yet, overexpression of a single gene hit, Anxa2a, in mouse CMs did not override the inability of mammalian CMs to re-enter cell cycling. Instead, we outlined predicted upstream TFs responsible for the observed gene expression profile in the AR-specific CMs and, by proof-of-concept, overexpressed two predicted TF hits, Insm1 (Insulinoma-associated protein 1) and Tead1 (TEA Domain transcription factor 1) in mouse CMs. Notably, both Insm1 and Tead1 doubled the number of cycling mouse CMs, and despite that this induction was modest, CyclinB1 and Aurkb levels increased, pointing towards mouse CM division rather than a stimulation of polyploidy. In summary, we provide a new network of TFs predicted to regulate an AR-specific CM population in zebrafish that may be further explored to unravel zebrafish heart regeneration and eventually utilized for inducing CM proliferation in the mammalian heart.