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Epicardial Tcf21 facilitates cardiomyocyte dedifferentiation and heart regeneration in zebrafish

Miglė Kalvaitytė-Repečkė, Sofija Gabrilavičiūtė, Kotryna Kvederavičiūtė, Leonard Burg, Edita Bakūnaitė, Kenneth D. Poss, Darius Balciunas

Posted on: 13 September 2025

Preprint posted on 17 May 2025

Epicardial pathways to a regenerating heart: A Zebrafish model ~ The authors use a zebrafish model of cryoinjury along with a hydroxytamoxifen (4-HT)-inducible CreER-based recombinase system to evaluate the role of Tcf21 in heart regeneration.

Selected by Theodora Stougiannou

Categories: cell biology

“Epicardial pathways to a regenerating heart: A Zebrafish model”

Background: After myocardial injury in the zebrafish and under physiological conditions, quiescent epicardial cells activate throughout the heart, a response that peaks by day 3 after injury. These cells then cover the injured area, a response occurring by day 7 after injury. Cardiomyocytes in the border zone also protrude, or even actively migrate into the injured area; this involves cardiomyocyte dedifferentiation and proliferation [1] [2]. The epicardium is involved in this regeneration process, secreting appropriate signaling factors and serving as a source of diverse cell types such as vascular smooth muscle cells and fibroblasts [3]. Secreted factors include proteins such as fibronectin and collagen whilst the epicardium-derived cells (EPDC) enter the subepicardial tissue and transdifferentiate into the previously mentioned cell types [4].

The authors make use of a hydroxytamoxifen (4-HT)-inducible CreER-based recombinase system to knock-out (KO) the expression of Tcf21 in embryo and adult zebrafish. In short, a conditional or floxed tcf21 allele has been generated in this study, whose expression can be disrupted upon administration of 4-HT and induction of the CreER recombinase [5]. Myocardial injury has been recapitulated via a cryoinjury model. Cryoinjury generally involves use of a cryoprobe cooled with liquid nitrogen to induce tissue injury [6] ~ in this case, the zebrafish are anaesthetized and the heart exposed via thoracotomy, which is later brought into contact with the probe [7].

Fish can generally withstand tissue injury/cell death of ~ 25% of total ventricular myocardium after cryoinjury [8]

The effects of inducible tcf21 KO on the physiological cardiac regeneration after cryoinjury in adult animals are evaluated in this study, by comparing animals with Tcf21 ON and animal with Tcf21 OFF. Heart regeneration is evaluated via a scoring system that comprises 3 levels, ranging from impaired regeneration, partial regeneration to complete regeneration [2].

Effects of conditional tcf21 KO (after 4-HT administration):

  1. Expression of tcf21 normally increases threefold after ischemic cardiac injury ~ in transgenic animals treated with 4-HT to induce conditional tcf21 KO, expression is reduced to almost undetectable levels.
  2. Effects on heart regeneration (Cardiac tissue regeneration analysis):
    1. Epicardial cells are present in the injured area in all animals
    2. Partial regeneration or impaired regeneration in 9 out 17 zebrafish ventricles in Tcf21 OFF animals ~ decrease in numbers of dedifferentiated cardiomyocytes in the area of injury, decreased cardiomyocyte protrusions towards the site of injury, increased levels of collagen, fibrin in the injured cardiac tissue.
    3. Complete regeneration in 12 out of 13 zebrafish ventricles (regeneration contiguous) in Tcf21 ON animals.
  3. Effects on heart regeneration (Differentially expressed genes [DEG] analysis):
    1. ~ 1,175 DEG in Tcf21 ON versus Tcf21 OFF hearts, with downregulation of ~ 375 genes related to chemotaxis (chemokine ligand genes such as ccl34a.4, ccl34b.4, cxcl11.6, cxcl11.1, ccl36.1)
    2. ~ 1,175 DEG in Tcf21 ON versus Tcf21 OFF hearts, with upregulation of ~ 800 genes related to organization of the actin cytoskeleton, development of skeletal muscle fibers, heart contraction
    3. Tcf21 thus supresses genes related to the actin cytoskeleton, muscle fiber development, upregulates genes involved in chemotaxis and cardiac regeneration

Why this work is interesting: Exploring the role of Tcf21 in heart regeneration in the adult zebrafish has not been as straightforward until recently, as the conditional inactivation of genes in adult zebrafish harbours some challenges. The authors use a conditional system for this purpose to inactivate Tcf21 in adult zebrafish. While cardiomyocyte dedifferentiation and proliferation have been shown as a mechanism of post-infarct myocardial regeneration, this study highlights the contribution of the epicardium in this process as well.

 

References:

[1] Zuppo DA, Tsang M. Zebrafish heart regeneration: Factors that stimulate cardiomyocyte proliferation. Seminars in Cell & Developmental Biology 2020;100:3–10. https://doi.org/10.1016/j.semcdb.2019.09.005.

[2] Kalvaitytė-Repečkė M, Gabrilavičiūtė S, Kvederavičiūtė K, Burg L, Bakūnaitė E, Poss KD, et al. Epicardial Tcf21 facilitates cardiomyocyte dedifferentiation and heart regeneration in zebrafish 2025:2025.05.15.654216. https://doi.org/10.1101/2025.05.15.654216.

[3] Cao J, Poss KD. The epicardium as a hub for heart regeneration. Nat Rev Cardiol 2018;15:631–47. https://doi.org/10.1038/s41569-018-0046-4.

[4] Weinberger M, Simões FC, Gungoosingh T, Sauka-Spengler T, Riley PR. Distinct epicardial gene regulatory programs drive development and regeneration of the zebrafish heart. Developmental Cell 2024;59:351-367.e6. https://doi.org/10.1016/j.devcel.2023.12.012.

[5] Kim H, Kim M, Im S-K, Fang S. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res 2018;34:147–59. https://doi.org/10.5625/lar.2018.34.4.147.

[6] Chablais F, Ja&#378 A, wi&#324, ska. Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury. JoVE (Journal of Visualized Experiments) 2012:e3666. https://doi.org/10.3791/3666.

[7] González-Rosa JM, Mercader N. Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish. Nat Protoc 2012;7:782–8. https://doi.org/10.1038/nprot.2012.025.

[8] Wu C-C, Weidinger G. Zebrafish as a Model for Studying Cardiac Regeneration. Curr Pathobiol Rep 2014;2:93–100. https://doi.org/10.1007/s40139-014-0042-2.

 

 

Tags: cardiovascular, cell biology, heart regeneration, myocardial infarction, zebrafish

doi: https://doi.org/10.1242/prelights.41407

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Author's response

Miglė Kalvaitytė-Repečkė, Darius Balciunas shared

  • Have you observed any immune cell infiltrate during preparation of tissue sections for the experiment?

We stained heart sections at 3 days post cryoinjury (dpci) using L-plastin, which is a pan-leukocytic marker. We observed positive cells in the entire heart and the injury area under both conditions (Tcf21 ON and Tcf21 OFF). However, we did not conduct further analyses or quantification at that stage. It would be beneficial to perform additional experiments to characterize specific immune cell populations and evaluate their responses to injury in the tcf21 knockout background.

  • Based on other similar research and the results of your own experiments, would you say that activation of immune mechanisms contributes at all to the greater capacity for cardiac regeneration observed in zebrafish? Or, alternatively, is it solely a matter of zebrafish cardiomyocytes being capable of cell cycle entry and proliferation due to other mechanisms and unique zebrafish cardiomyocyte characteristics, compared to mammals for example?

In our study, we observed that cardiomyocyte proliferation was activated under both conditions, along with cardiomyocyte dedifferentiation, as indicated by the presence of embryonic myosin.

These results suggest that while zebrafish cardiomyocytes possess an intrinsic ability to dedifferentiate and re-enter the cell cycle, this capability alone is insufficient for successful regeneration. Additional mechanisms, particularly those mediated by non-cardiomyocyte cell types, including immune and epicardial cells, are necessary to create a pro-regenerative environment.

Recent studies by others (for examples, see Constanty et al., 2025; Wei et al., 2023) underscore the essential role macrophages play in regeneration and demonstrate that macrophage-induced extracellular matrix remodeling at the wound border is necessary for cardiomyocyte protrusion into the injured area. Furthermore, prior research has highlighted the close association between epicardial and immune cells (Stevens et al., 2016; Weinberger et al., 2020; Bruton et al., 2022).

Building on these findings and our own results, we believe that Tcf21 in epicardial cells may initiate signaling pathways that recruit and/or activate immune cells, which then support cardiomyocyte protrusion into the injury site and heart regeneration.

Differences in immune response following injury among species appear to be one of the key factors that contribute to variation in regenerative capacity. In this context, immune-modulating therapies could represent a promising strategy to promote cardiac regeneration in non-regenerating species, such as adult mammals (see Lai et al., 2019; Simoes and Riley, 2022).

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