Recent insights into zebrafish cardiac regeneration
Andre´s Sanz-Morejo´n1,2 and Nadia Mercader1,2
Available online at www.sciencedirect.com
ScienceDirectIn humans, myocardial infarction results in ventricular
remodeling, progressing ultimately to cardiac failure, one of the
leading causes of death worldwide. In contrast to the adult
mammalian heart, the zebrafish model organism has a
remarkable regenerative capacity, offering the possibility to
research the bases of natural regeneration. Here, we
summarize recent insights into the cellular and molecular
mechanisms that govern cardiac regeneration in the zebrafish.
Addresses
1 Institute of Anatomy, University of Bern, 3012 Bern, Switzerland
2Centro Nacional de Investigaciones Cardiovasculares (CNIC),
28029 Madrid, Spain
Corresponding author: Mercader, Nadia (nadia.mercader@ana.unibe.ch)
Current Opinion in Genetics and Development 2020, 64:xx–yy
This review comes from a themed issue on Cell reprogramming,
regeneration and repair
Edited by Pentao Liu and Antonio Jacinto
https://doi.org/10.1016/j.gde.2020.05.020
0959-437X/ã 2020 The Author(s). Published by Elsevier Ltd. This is an
open access article under the CC BY license (http://creativecommons.
org/licenses/by/4.0/).
Introduction
Cardiovascular disease remains a dominant cause of death
worldwide and the burden of cardiomyopathies is pre-
dicted to increase substantially in the future [1]. Myocar-
dial infarction results from the formation of atheroscle-
rotic plaques and the blockage of coronary arteries, which
fail to deliver nutrients and oxygen to the myocardium,
causing the death of millions of cardiac cells. The replace-
ment of the damaged tissue by non-contractile scar tissue
protects the heart from wall rupture but ultimately leads
to pathologies such as adverse cardiac remodeling and
heart failure (reviewed in Ref. [2]).
For decades, the adult mammalian myocardium was
considered a post-mitotic tissue with very little to no
regenerative capacity [3]. Postnatal cardiac growth is
predominantly a result of cardiomyocyte hypertrophy
mediated by additional DNA synthesis without cytoki-
nesis, generating mononuclear polyploid and binucleated
diploid cardiomyocytes in humans and mouse, respec-
tively [4]. Remarkably, the neonatal mouse heart is able
to regenerate during a short period after birth [5]. Of
interest, a case reported complete functional recoverywww.sciencedirect.com after severe myocardial infarction in a human newborn
[6]. This observation might suggest that the transient
cardiac regenerative capacity in neonatal mice is con-
served, at least partially, in humans, and that a latent
regenerative capacity is actively suppressed during mat-
uration [7]. Accordingly, the exploration of how other
species retain cardiac regenerative capacity throughout
their lifespan continues to garner interest.
The zebrafish (Danio rerio) is one of the most relevant
models to study regenerative biology given its fascinating
capacity to regenerate most of its organs and tissues,
including the heart (reviewed in Ref. [8]). The biological
response to cardiac injury in the zebrafish requires the
orchestrated participation of multiple cell types involving
numerous molecular mechanisms that ultimately result in
the regeneration of the damaged tissue. Here, we sum-
marize recent discoveries on cardiac regeneration in the
adult zebrafish that provide mechanistic insights into how
this complex process is successfully achieved.
Cardiac regeneration in the zebrafish
The zebrafish heart shares numerous similarities with its
mammalian equivalent with regards to morphology, cel-
lular composition, genetic regulation and also embryonic
development (reviewed in Ref. [9]). During develop-
ment, cardiac progenitors derived from the first heart
field initially form a primordial heart tube. This structure
elongates and loops to form a two chambered embryonic
heart by the incorporation of cardiac progenitors from the
second heart field to the venous and arterial poles [10–13].
The adult cardiac muscle, or myocardium, is lined by an
endocardial layer facing the lumen and covered by an
epicardial layer. The zebrafish heart is two-chambered,
with the single atrium and ventricle connected by an
atrio-ventricular valve. Blood enters the heart through the
atrium, is pumped by the ventricle and is ejected into the
circulation through the bulbus arteriosus, a prominent
outflow tract. The myocardium can be subdivided into
three main layers: the inner trabecular layer, the primor-
dial layer and the outer cortical layer (Figure 1a–b00).
A seminal study by Poss and colleagues showed that upon
resection of 20% of the adult zebrafish ventricle, the lost
myocardium is replaced by newly functional cardiac
muscle, achieving regeneration by a virtually scar-free
process [14]. Later, further cardiac injury models were
developed, including ventricular cryoinjury and genetic
ablation. The ventricular cryoinjury induces cell death by
fast freezing part of the ventricle [15–17]. Cryoinjured
hearts are also able to regenerate, but regeneration occurs
concomitant with the transient deposition of a fibroticCurrent Opinion in Genetics & Development 2020, 64:1–7
2 Cell reprogramming, regeneration and repair
Figure 1
(a)
(c) (d) (e) (f) (g) (h)
(b) (b’) (b’’)
Current Opinion in Genetics & Development
Representation of cardiac regeneration in the adult zebrafish.
(a) Adult zebrafish heart anatomical position. (b) Overview of the uninjured zebrafish heart, comprising the atrium, ventricle and bulbus arteriosus.
The heart is covered and wired by the epicardium, lymphatic system, coronary arteries and nerves. (b0) Section of the zebrafish heart. Cardiac
valves separate the chambers. (b0 0) Zoomed region of (b0). Three myocardial layers can be identified: trabecular, primordial, and cortical
myocardium. The endocardium coats the lumen. The cortical layer is covered by the epicardium. Fibroblasts lie between the cortical and
trabecular myocardium. (c)–(h) Timeline of cardiac regeneration events upon cryoinjury. (c) Fast freezing of the ventricular apex leads to the
formation of the injury area. Necrotic and apoptotic cells trigger an inflammatory response characterized by the infiltration and activation of
neutrophils, monocytes, and macrophages, among others. Endothelial and epicardial cells are activated and infiltrate the injury area. (d) The acute
inflammation regresses and activated fibroblasts elicit a fibrotic response by depositing extracellular matrix (ECM). (e) Peak of cardiomyocyte
proliferation followed by migration along epicardial and endocardial cells. Treg cells home to the injured tissue. (f) The ECM remodels and
cardiomyocyte proliferation continues. (g) Fibroblasts undergo inactivation and the fibrotic scar regresses. (h) Complete regression of the fibrotic
scar and replenishment by functional myocardium. The cortical myocardial layer remains thickened and the primordial layer does not regenerate.
Abbreviations: at, atrium; ba, bulbus arteriosus; CM prolif, cardiomyocyte proliferation; cor, coronary arteries; cv, cardiac valves; ECM,
extracellular matrix; epi, epicardium; endo, cardiac endothelium; dpi, days post injury; fibro, fibroblast; ia, injury area; hpi, hours post injury; lymph,
the lymphatic system; Mw, macrophage; prim, primordial layer; trab, trabecular layer; v, ventricle.scar, which is ultimately resolved [15] (Figure 1c–h). The
third main injury model, genetic ablation of cardiomyo-
cytes, is currently based on the inducible and tissue-
specific expression of either diphtheria toxin A [18] or
nitroreductase, an enzyme that converts the prodrug
metronidazole into a cytotoxic metabolite that induces
cell death [19]. These methods, and others, have been
used extensively to interrogate cardiac regenerative
mechanisms in the zebrafish.
Cellular source of the regenerated
myocardium
Regarding heart regeneration, one central question to be
resolved is: where do new cardiomyocytes come from? The
current consensus is that newly formed cardiomyocytesCurrent Opinion in Genetics & Development 2020, 64:1–7 derive from preexistent differentiated cardiomyocytes
(Figure 1e). This hypothesis is strongly supported by
lineage tracing studies using the Cre-lox technology, in
which the cardiomyocytes from uninjured hearts were
irreversibly tagged using the cardiomyocyte-specific pro-
moter cmlc2 (myl7) [20,21]. Of note, myl7 starts to be
expressed in cardiomyocyte progenitor cells within the
anterior lateral mesoderm before cardiac looping [22].
Thus, not only fully differentiated cardiomyocytes express
myl7, a fact to considerwhen interpretingmyl7 fate mapping
studies during adult heart regeneration. In response to
injury, some cardiomyocytes, predominantly those located
in subepicardial regions and close to the injury border, re-
activate the expression of regulatory regions of gata4 [20]
and ctgfa [23] genes. More recently, the expression ofwww.sciencedirect.com
Insights into zebrafish cardiac regeneration Sanz-Morejo´n and Mercader 3sox10, a well-known neural crest marker, was shown to label
a subset of cardiomyocytes in the embryonic [24] and adult
[25] zebrafish heart. These cells proliferate preferentially
and contribute to the regenerated myocardium following
cardiac injury [26,27]. These findings might represent a
contribution of neural crest-derived cardiomyocytes to
cardiac regeneration or, alternatively, the activation of
specific neural crest genetic signatures within some prolif-
erating cardiomyocytes. In sum, the extent to which some
cardiomyocytes present a high regenerative capacity, and
which specific cellular and transcriptomic changes are
involved in this process, warrants further investigation.
Continuing this theme, there is evidence that cardiomyo-
cytes can partially switch their fate during regeneration
and rebuild different myocardial layers. For example,
ablation of embryonic ventricular cardiomyocytes can
be compensated by atrial cardiomyocytes [28]. Further-
more, clonal analysis in resected ventricles suggested that
cortical cardiomyocytes contribute to the regenerated
cortical layer, indicating a commitment to a particular
myocardial compartment [29]. More recently, trabecular
cardiomyocytes have been shown to also regenerate the
cortical layer, which reveals some degree of cardiomyo-
cyte plasticity [30] (Figure 1h). Whether cortical cardi-
omyocytes can contribute to the regenerated trabeculae is
currently unknown. Interestingly, the primordial layer of
the myocardium is not regenerated in cryoinjured hearts
[23] (Figure 1h). This observation, together with the
discovery that the regenerated cortical layer remains
thickened in resected and cryoinjured hearts [14,15],
(Figure 1h) and that ventricular wall contractility is not
completely reestablished [31], indicates that myocardial
regeneration is not fully achieved in the zebrafish.
The finding that adult cardiomyocytes in the zebrafish are
predominantly diploid [32] has long been regarded as a
possible explanation for their high proliferative potential.
Cardiomyocyte polyploidy is more frequent in non-regen-
erative than in regenerative species and represents a
barrier to proliferation [33,34]. Indeed, polyploidization
of cardiomyocytes is associated with the loss of cardiac
regenerative and reparative capacity in mice [5,35]. Nota-
bly, elegant genetic models have revealed that an increase
in cardiomyocyte ploidy reduces cardiac regenerative
capacity in zebrafish, pointing to a pivotal role for ploidy
in this process [36].
An important quest is the identification of endogenous
and exogenous molecules and environmental stimuli
inducing cardiomyocyte proliferation. The tyrosine-pro-
tein kinase receptor Erbb2 is one of the main mediators of
cardiomyogenesis during regeneration. One of its ligands,
Neuregulin 1 (Nrg1), is a potent cardiomyocyte mitogen
sharply induced in perivascular cells during cardiac regen-
eration [37]. Erbb2 signaling also acts downstream parti-
cipates of the effector cascade of vitamin D [38] orwww.sciencedirect.com hemodynamic forces [39] during cardiomyocyte prolifer-
ation. Erbb2 signaling mediates a switch from oxidative
phosphorylation to a glycolysis predominant metabolism
observed in proliferating cardiomyocytes [40]. Interest-
ingly, Erbb2 signaling has also been clearly associated to
heart regeneration in the neonatal mouse [41]. Additional
signaling pathways that influence cardiomyocyte prolif-
eration have been identified, including PPARd [42] and
vegfaa [43]. Whether these also interact with Erbb2 sig-
naling pathway is not known.
Extensive epigenetic remodeling precedes a regenerative
response in cardiomyocytes. The repression of sarcomeric
and cytoskeletal genes by H3K27me3-mediated epige-
netic silencing is a pre-requisite for cell cycle re-entry [44
]. Specific enhancers become activated during injury
response, as explored by histone H3.3 profiling [45].
Furthermore, transient cell membrane fusions in cardio-
myocytes [46] have been shown to play a role in myocar-
dial regeneration. Additional factors acting at the organ-
ismal level also influence cardiomyocyte proliferation
including swimming-induced exercise [47] and cardiac
preconditioning [48].
Overall, a tight temporal and spatial control of mitogenic
signals is crucial to promote cardiomyocyte proliferation
and heart regeneration. The coordinated participation of
other cell types, however, is necessary to successfully
achieve this complex process.
Immune system response
Following cardiac injury, there is an initial pro-inflamma-
tory phase in which necrotic cells trigger the activation
and infiltration of immune cells. These cells, both from
intra-cardiac and extra-cardiac origin, clear debris and
dead cells and remodels the extracellular matrix
(ECM) (Figure 1c–e). Several immune cell types partici-
pate in this process in a timely and spatially coordinated
manner (reviewed in Ref. [49]). For example, increased
neutrophil retention [50,51] or ablation of Treg cells [52]
lead to reduced organ regenerative capacity.
In mammals, cardiac-resident macrophages are the most
abundant immune cell populations in the heart and the
majority of them are derived from the yolk sac [53].
Depletion of macrophages leads to impaired heart regen-
eration in neonatal mice [54] and zebrafish [55].
Remarkably, a comparative analysis between zebrafish
and medaka (Oryzias latipes), also a teleost but unable to
regenerate the heart [56], revealed substantial differences
in the immune response upon cardiac injury [55]. For
instance, the stimulation of the Toll-like receptor in
medaka promoted immune cell recruitment, neovascu-
larization, neutrophil clearance, cardiomyocyte prolifera-
tion and scar resolution. Alternatively, delayed macro-
phage recruitment in zebrafish results in compromisedCurrent Opinion in Genetics & Development 2020, 64:1–7
4 Cell reprogramming, regeneration and repairneovascularization, neutrophil clearance, cardiomyocyte
proliferation and scar resolution [55].
The role of macrophages has been further defined by the
identification of pro-inflammatory macrophages expres-
sing tumor necrosis factor a (tnfa) at early stages upon
cardiac insult [57], in line with what was previously
reported during embryonic caudal fin regeneration [58].
Furthermore, pro-regenerative macrophages expressing
wilms tumor 1b (wt1b) show specific recruitment dynamics
and genetic signatures during heart regeneration [59].
Moreover, osteopontin-positive macrophages are impli-
cated in triggering a fibrotic response as well as fibrosis
regression [57]. Overall, a finely tuned temporal and
spatial control of inflammation is crucial for heart regen-
eration. Yet, the identification of additional immune cell
types and specific subpopulations involved in cardiac
regeneration in the zebrafish remains to be fully explored.
Cardiac endothelium, nerves and lymphatic
system
The cardiac endothelium is composed by two structures:
the coronary and the endocardial endothelium [60].
Angiogenic sprouting infiltrating the damaged tissue is
observed as early as 15 hours post injury (Figure 1c).
Inhibition of this process by overexpression of a vegfaa
dominant-negative isoform diminishes cardiomyocyte
proliferation and abrogates cardiac regeneration [61].
The peak of proliferation of endocardial cells surrounding
the damaged tissue occurs between 3 and 5 dpi, before
the cardiomyocyte proliferation peak rate at 7 dpi
(Figure 1d,e). In this context, the participation of Notch
[62] and Wnt [63] signaling in endocardial cells has been
described. Beyond their function in oxygenation and
nutrient delivery, regenerating coronaries serve as a scaf-
fold for cardiomyocytes to repopulate the injured area,
with the epicardial Cxcl12/Cxcr4 signaling axis playing an
important role in this process [64].
Cardiac innervation also influences the regenerative pro-
cess. Hypo-innervation of adult zebrafish heart leads to
reduced cardiomyocyte proliferative potential, abrogating
cardiac regeneration [65]. While the role of the lymphatic
system has long remained enigmatic in the regenerative
context, recent studies indicate its importance in fluid
drainage and inflammatory cells removal from the dam-
aged myocardium [66,67,68].
Overall, these results establish an essential role for the
endocardium, coronary endothelium, nerves and lym-
phatic system to support and promote cardiac regenera-
tion as a source of signals but also as a physical scaffold.
Fibrotic scar origin and fate
During cardiac regeneration, the epicardium and epicar-
dium-derived cells (EPDCs) contribute to the generation
of perivascular cells and fibroblasts, which are importantCurrent Opinion in Genetics & Development 2020, 64:1–7 for scar deposition and remodeling [69,70]. Indeed,
genetic ablation of tcf21+ epicardial cells reduces the
proliferative capacity of cardiomyocytes [71]. Collective
migration of epicardial cells is reliant on the generation of
polyploid epicardial leader cells at the migration front
[72]. Interestingly, epicardial cells secrete the ECM
substrates needed for their migration over the cardiac
surface [73]. The epicardium has also been suggested to
secrete trophic factors important for heart regeneration,
including mitogenic signals such as neuregulin 1 [37]. In
addition, EPDCs crosstalk with other cell types, medi-
ated for example by Neuropilin 1, a transmembrane
receptor whose ligands include platelet derived growth
factor (PDGF), fibroblast growth factor (FGF) and vas-
cular endothelial growth factor (VEGF), which mediates
epicardial activation and revascularization during regen-
eration [74].
Fibroblasts are the main source of collagen and other
ECM-proteins upon cardiac injury. The inactivation of
pre-existing cardiac fibroblasts, partly derived from the
embryonic epicardium, occurs during the scar resolution
phase [70] (Figure 1g,h). Moreover, cellular senescence
is observed at the injury site in the zebrafish and a correct
balance of senescent cells might be necessary for heart
regeneration [75,76]. Studies in neonatal mice showed
that fibroblast senescence is required for cardiac regener-
ation [76,77], and this needs to be confirmed in the
zebrafish model. Remarkably, genetic ablation of colla-
gen-producing cells upon heart injury is detrimental for
cardiomyocyte proliferation in the zebrafish [70]. The
composition and stiffness of the zebrafish cardiac ECM is
dynamic in composition and stiffness during injury reso-
lution [78] (Figure 1d–g). Yet, much remains to be
learned regarding which specific signals, components,
or physicochemical properties of zebrafish ECM influ-
ence heart regeneration.
Outlook and future perspectives
The last few years have yielded significant breakthroughs
in our understanding of the different cell types and cell
interactions influencing myocardial regeneration in the
zebrafish. We gained an improved perspective on how the
different cardiac structures contribute to heart regenera-
tion. We also learned that several cellular and molecular
mechanisms are conserved between zebrafish and neona-
tal mouse regeneration. Furthermore, the zebrafish has
also proven to be an excellent model to study cardiac
valve regeneration [79,80]. These findings represent an
important added value to the model, given that numerous
degenerative and congenital diseases known to affect
cardiac valves are important health concerns. With the
rapid development of omic
s-based approaches, databases
integrating available information – for example, [81] – will
be of immense benefit to the community. The functional
validation of how transcriptome and cellular changes are
integrated within different cell types and how thewww.sciencedirect.com
Insights into zebrafish cardiac regeneration Sanz-Morejo´n and Mercader 5outcome influences cardiac regeneration will become one
of the next big challenges in the field. In this regard, the
continued establishment of efficient technologies for
tissue-specific and cell type-specific genetic manipula-
tions will be ever more relevant. Finally, perfoming cross-
species analysis to define which results have a transla-
tional value will be important future steps towards unra-
velling the complicated processes of heart regeneration.
Conflict of interest statement
Nothing declared.
Acknowledgements
We thank the Mercader group members and Hector Sa´nchez-Iranzo for
critical reading of the manuscript. This work was supported by the
European Research Council ERC Consolidator Grant 819717 – TransReg
and the Swiss National Science Foundation grant ForceInRegeneration
310030L_182575. We apologize to our colleagues for omitting citations of
original reports due to space limitations.
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