Recent insights into zebraﬁsh cardiac regeneration

In 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. tbx5a can contribute to rebuild the cortical injury. devel- ﬁeld-derived cardiomyocytes the genetic ablation of the ﬁrst heart ﬁeld. Altogether the results reveal plasticity of cardiomyocyte fate during injury This study describes that upon injury, cardiomyocytes undergo extensive epigenome remodeling, revealed by H3K27me3-mediated silencing, that is required for their proliferation. This represents one of the ﬁrst reports regarding the importance of epigenetic adaptations during heart regeneration in the zebraﬁsh. This study describes that wt1b + macrophages show distinctive genetic signatures and recruitment dynamics within regenerating tissues in the zebraﬁsh. The results also show that Wt1b is required for heart regeneration. The authors explore how epicardial and endocardial cues inﬂuence the coronary revascularization necessary for cardiac regeneration in the zebraﬁsh. They propose that regenerating coronaries and epicardium can be used as a scaffold for regenerating cardiomyocytes. This in how disruption The dissect the of the cardiac lymphatic system and show an important contribution this system to ﬁbrosis resolution This study explores the developmental origin of the lymphatic vasculature in the heart and its role during adult cardiac regeneration. The results show that cardiac cryoinjury, but not apical resection, elicits a robust lymphangiogenic response. This suggests that cardiac regenerative mechanisms might be injury context dependent.


Introduction
Cardiovascular disease remains a dominant cause of death worldwide and the burden of cardiomyopathies is predicted to increase substantially in the future [1]. Myocardial infarction results from the formation of atherosclerotic 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 replacement 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 cytokinesis, generating mononuclear polyploid and binucleated diploid cardiomyocytes in humans and mouse, respectively [4]. Remarkably, the neonatal mouse heart is able to regenerate during a short period after birth [5]. Of interest, a case reported complete functional recovery after severe myocardial infarction in a human newborn [6]. This observation might suggest that the transient cardiac regenerative capacity in neonatal mice is conserved, at least partially, in humans, and that a latent regenerative capacity is actively suppressed during maturation [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 summarize 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, cellular composition, genetic regulation and also embryonic development (reviewed in Ref. [9]). During development, 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 primordial layer and the outer cortical layer (Figure 1a-b 00 ).
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 fibrotic scar, which is ultimately resolved [15] (Figure 1c-h). The third main injury model, genetic ablation of cardiomyocytes, is currently based on the inducible and tissuespecific 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 cardiomyocytes 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 promoter 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 consider when interpreting myl7 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, reactivate the expression of regulatory regions of gata4 [20] and ctgfa [23 ] genes. More recently, the expression of 38 Cell reprogramming, regeneration and repair

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. (b 0 ) Section of the zebrafish heart. Cardiac valves separate the chambers. (b 00 ) Zoomed region of (b 0 ). 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.  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-inflammatory 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- . Overall, a finely tuned temporal and spatial control of inflammation is crucial for heart regeneration. 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 scaffold 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 process. 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 damaged myocardium [66 ,67 ,68 ].
Overall, these results establish an essential role for the endocardium, coronary endothelium, nerves and lymphatic system to support and promote cardiac regeneration as a source of signals but also as a physical scaffold.

Fibrotic scar origin and fate
During cardiac regeneration, the epicardium and epicardium-derived cells (EPDCs) contribute to the generation of perivascular cells and fibroblasts, which are important 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, mediated for example by Neuropilin 1, a transmembrane receptor whose ligands include platelet derived growth factor (PDGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), which mediates epicardial activation and revascularization during regeneration [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 regeneration [76,77], and this needs to be confirmed in the zebrafish model. Remarkably, genetic ablation of collagen-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 resolution [78] (Figure 1d-g). Yet, much remains to be learned regarding which specific signals, components, or physicochemical properties of zebrafish ECM influence 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 regeneration. We also learned that several cellular and molecular mechanisms are conserved between zebrafish and neonatal 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 omics-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 the outcome 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 manipulations will be ever more relevant. Finally, perfoming crossspecies analysis to define which results have a translational value will be important future steps towards unravelling the complicated processes of heart regeneration.

Conflict of interest statement
Nothing declared.

23.
Pfefferli C, Ja zwi nska A: The careg element reveals a common regulation of regeneration in the zebrafish myocardium and fin. Nat Commun 2017, 8:15151. The authors identify that an enhancer of ctgfa, named careg, labels highly proliferative cardiomyocytes surrounding the injury area during heart regeneration. The same element is also active in the regenerating caudal fin, suggesting the presence of a common regenerative response between different tissues/organs. Additionally, the careg element also labels the primordial myocardium, which fails to regenerate upon cardiac cryoinjury.

25.
Abdul-Wajid S, Demarest BL, Yost HJ: Loss of embryonic neural crest derived cardiomyocytes causes adult onset hypertrophic cardiomyopathy in zebrafish. Nat Commun 2018, 9:4603. This study describes the contribution from sox10-positive neural crest (NC) derived cardiomyocytes to the developing zebrafish heart. The genetic ablation of this population leads to severe hypertrophic cardiomyopathy, suggesting an important role for NC-derived cardiomyocytes in heart growth.

26.
Tang W, Martik ML, Li Y, Bronner ME: Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish. eLife 2019, 8. The authors describe that neural crest derived cardiomyocytes contribute to cardiac regeneration in the zebrafish.    . This study describes that wt1b + macrophages show distinctive genetic signatures and recruitment dynamics within regenerating tissues in the zebrafish. The results also show that Wt1b is required for heart regeneration.