Is zebrafish heart regeneration “complete”? Lineage-restricted cardiomyocytes proliferate to pre-injury numbers but some fail to differentiate in fibrotic hearts

Bertozzi, Alberto; Wu, Chi; Nguyen, Phong D.; Vasudevarao, Mohankrishna Dalvoy; Mulaw, Medhanie A.; Koopman, Charlotte D.; Boer, Teun P. de; Bakkers, Jeroen; Weidinger, Gilbert

doi:10.1016/j.ydbio.2020.12.004

Cited by 13 publications

(17 citation statements)

References 42 publications

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“…However, at day 21 after injury, we found similar amounts of PCNA positive CMs in eif4ebp3l overexpressing hearts and wild-type hearts. In agreement with previous studies, the average number of proliferating CMs was small, as CM proliferation peaks at seven dpci and approaches pre-injury levels at around 30 dpci [ 7 , 8 , 12 , 46 ]. Further studies are needed to evaluate if these observations are only limited to late cardiac regeneration or if eif4ebp3l affects heart regeneration at an earlier time point.…”

Section: Discussionsupporting

confidence: 92%

“…Second, at 21 dpci mRNA expression of eif4ebp3l increased in the injured area and reached baseline level of the healthy myocardium at 60 dpci. From day 21 after cryoinjury, the injured area is completely covered by vasculature [ 8 , 44 ] and most proliferative processes such as CM proliferation or proliferation of the endocardium are nearing their end [ 45 , 46 ]. For balancing cell proliferation and prevention of unrestrained, cancerous cell growth after injury, the up-regulation or general expression of anti-proliferative genes such as runx1 , is detected [ 47 , 48 ].…”

Section: Discussionmentioning

confidence: 99%

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eif4ebp3l—A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

Born

Andree

Frank

et al. 2022

IJMS

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The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

eif4ebp3l—A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

Born

Andree

Frank

et al. 2022

IJMS

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“…Zebrafish cardiac regeneration relies mainly on cell cycle re-entry of spared cardiomyocytes at the wound border [ 24 , 26 ]. Another important feature of zebrafish cardiac repair is that scarring is transient, since the ECM deposited in the wounded area within the first days post cryoinjury is gradually resorbed until little or no scar tissue remains in the regenerated heart [ 25 , 27 – 29 ]. Restoration of the healthy myocardium is supported also by revascularization of the injured area, an early response that starts at 15 hours post injury (hpi) with the formation of the first coronary vessel sprouts from the wound border and is completed by 4 days post injury (dpi) before the onset of cardiomyocyte proliferation [ 30 ].…”

Section: Resultsmentioning

confidence: 99%

“…The extent of the coronary plexus formed by endothelial cells at 4 dpi, measured as percentage of GFP + area inside the mCherry - injured area, was unaltered by Hdac1 inhibition (DMSO 16.26 ± 6.34%, n = 7, Mocetinostat 15.74 ± 5.62%, n = 8, p > 0.9999) ( Fig 7E and 7F ). Scar resorption was analyzed by Acid Fuchsin Orange G (AFOG) staining on hearts treated from 1 to 30 dpi ( Fig 7A ), a time point when typically cryoinjured hearts show only a fraction of the initial scar left [ 25 , 27 , 29 ]. Interestingly, the size of the remaining wound was only slightly, but not significantly bigger in the Mocetinostat-treated fish, compared to the DMSO control group at 30 dpi (DMSO 4.05 ± 4.85%, n = 8, Mocetinostat 5.59 ± 2.91%, n = 8, p = 0.3282) ( Fig 7G and 7H ), suggesting that, although cardiomyocyte proliferation was found decreased at the wound border zone at 7 dpi, injured hearts can, at least, initiate regenerative processes such as scar resorption or revascularization after Hdac1 inhibition.…”

Section: Resultsmentioning

confidence: 99%

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

et al. 2021

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In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.

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“…However, zebrafish take around 90 dpci to completely regenerate, including scar resolution (Fig. 1.13C) (Bertozzi et al, 2021). These data show that the cardiomyocyte numbers required to replace the lost tissue have already been generated by 30 dpci, and that these cells could be involved in digesting the transient matrix in order to repopulate the injured area.…”

Section: Cardiomyocyte Responsementioning

confidence: 86%

Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish

Allanki¹

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The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring. To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish. Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish. To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively. On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling. Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.

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Is zebrafish heart regeneration “complete”? Lineage-restricted cardiomyocytes proliferate to pre-injury numbers but some fail to differentiate in fibrotic hearts

Cited by 13 publications

References 42 publications

eif4ebp3l—A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

eif4ebp3l—A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish

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