- Open Access
Wnt/β-catenin signaling in heart regeneration
© Ozhan and Weidinger. 2015
- Published: 8 July 2015
The ability to repair damaged or lost tissues varies significantly among vertebrates. The regenerative ability of the heart is clinically very relevant, because adult teleost fish and amphibians can regenerate heart tissue, but we mammals cannot. Interestingly, heart regeneration is possible in neonatal mice, but this ability is lost within 7 days after birth. In zebrafish and neonatal mice, lost cardiomyocytes are regenerated via proliferation of spared, differentiated cardiomyocytes. While some cardiomyocyte turnover occurs in adult mammals, the cardiomyocyte production rate is too low in response to injury to regenerate the heart. Instead, mammalian hearts respond to injury by remodeling of spared tissue, which includes cardiomyocyte hypertrophy. Wnt/β-catenin signaling plays important roles during vertebrate heart development, and it is re-activated in response to cardiac injury. In this review, we discuss the known functions of this signaling pathway in injured hearts, its involvement in cardiac fibrosis and hypertrophy, and potential therapeutic approaches that might promote cardiac repair after injury by modifying Wnt/β-catenin signaling. Regulation of cardiac remodeling by this signaling pathway appears to vary depending on the injury model and the exact stages that have been studied. Thus, conflicting data have been published regarding a potential role of Wnt/β-catenin pathway in promotion of fibrosis and cardiomyocyte hypertrophy. In addition, the Wnt inhibitory secreted Frizzled-related proteins (sFrps) appear to have Wnt-dependent and Wnt-independent roles in the injured heart. Thus, while the exact functions of Wnt/β-catenin pathway activity in response to injury still need to be elucidated in the non-regenerating mammalian heart, but also in regenerating lower vertebrates, manipulation of the pathway is essential for creation of therapeutically useful cardiomyocytes from stem cells in culture. Hopefully, a detailed understanding of the in vivo role of Wnt/β-catenin signaling in injured mammalian and non-mammalian hearts will also contribute to the success of current efforts towards developing regenerative therapies.
All organisms have evolved means of repairing tissue loss after injury or disease. In most species, healing of epidermal wounds and other epithelia is an efficient repair process, whereas the ability to recuperate the damage in other tissues varies widely. Mammals (including humans) can repair injury of skeletal muscle, regenerate large parts of the liver, and repair damage to the epithelia of the kidney and the lung but have limited regenerative capacity in other organs . In contrast, other vertebrates, such as urodele amphibia (salamanders and newts) and certain teleost fish species, can completely regenerate lost limbs and tails and repair damage to the lens, the retina, and the central nervous system [2–5]. Importantly, zebrafish and newts can also replace lost heart tissue in adults. A thorough understanding of the cellular and molecular mechanisms of regeneration in urodele amphibia and fish is therefore very likely to be informative for the development of regenerative therapies in humans.
One of the pivotal signaling pathways regulating the regenerative process in many systems is the Wnt/β-catenin signaling pathway. In addition, Wnt/β-catenin signaling is activated in response to cardiac injury in adult mammals and plays important roles in hypertrophy and cardiac remodeling . Here, we review the cellular mechanisms underlying heart regeneration in lower vertebrates and the known functions of Wnt/β-catenin signaling in the cardiac injury response both in mammals and in non-mammalian vertebrates.
The adult mammalian heart has a very limited capacity to repair loss of cardiomyocytes (CMs) after infarction or cardiac overload disorders . In contrast, adult zebrafish can regenerate the heart in response to several injury paradigms, including surgical removal of myocardial tissue, cryoinjury, and genetic ablation of cardiomyocytes [3, 8–12]. Intriguingly, neonatal mice can regenerate the myocardium after partial surgical resection as well, but this ability is lost by 7 days after birth . During both zebrafish and neonatal mouse heart regeneration, differentiated CMs re-enter the cell cycle to proliferate and genetic lineage tracing experiments indicate that the majority of the newly forming myocardium is derived from pre-existing CMs [13–15]. Thus, zebrafish and neonatal mice regenerate lost myocardium not by differentiation of CMs from progenitor cells but by activating the re-entry of differentiated CMs into the cell cycle. In contrast, adult mammalian CMs have long been thought to be terminally differentiated and thus post-mitotic [16–18] and much of the growth of the postnatal mammalian heart occurs via enlargement of pre-existing CMs, i.e., hypertrophy [18, 19]. While some studies in adult mouse and human myocardial cells have suggested that certain CMs are not terminally differentiated and can reinitiate the cell cycle under physiological or pathological conditions [20–22], others have found little evidence for CM proliferation in the adult heart [23–25]. Indeed, using transgenic mice that facilitated unambiguous identification of CM nuclei, Soonpaa and Field found that only 1 out of 180.000 adult mouse ventricular CMs incorporate [3H] thymidine, and this increased to only 3 in 36.000 nuclei (0.0083 %) in the injured heart [23–25]. The consensus conclusion from these and other studies is that DNA synthesis is very rare in differentiated adult rodent CMs even after injury, and CM hypertrophy after injury appears to largely occur without increase in DNA content in the adult heart [24, 26].
Nevertheless, there is good evidence that some new CMs do form during adult mammalian life, including humans. A pulse of atmospheric carbon-14 (14C) was generated by nuclear bomb tests during the Cold War and rapidly declined after atmospheric tests were banned. Since 14C makes its way through the food chain into human cells, the 14C levels found in DNA of human CMs corresponds to the atmospheric levels at the time when these cells were born. Bergmann and colleagues have measured the 14C concentrations in DNA of human myocardial cells . They have found that in subjects born up to 22 years before the onset of bomb tests, 14C concentrations were elevated compared to the levels before the tests, indicating that myocardial cells contained DNA synthesized years after birth. Thus, human CMs are capable of renewal during adulthood . CM renewal, however, is very slow as estimated from this study; 1 % of the CMs are renewed per year at the age of 25 and only 0.45 % at the age of 75. Thus, approximately 45 % of all CMs are exchanged during a human lifetime while 55 % remain from neonatal stages .
While it is not possible to identify the cellular source of newly forming CMs in the adult human heart, several studies in mice have addressed whether CM renewal during homeostasis and the low-rate CM replacement after cardiac injury are due to proliferation of existing CMs or due to CM differentiation from progenitor cells [26, 28–33]. Unfortunately, the conclusions drawn by several studies based on genetic lineage tracing of CMs or cardiac progenitor cells differ considerably. While most reports using different means to track the fate of differentiated CMs conclude that renewal of CMs during homeostasis is due to proliferation of existing CMs , one study concluded that progenitor cells contribute to CM formation after cardiac injury, since the progeny of differentiated CMs get diluted with other cells in injured hearts . In contrast, other studies using lineage tracing of differentiated CMs and multi-isotope imaging mass spectrometry concluded that the limited CM replacement after injury is due to CM proliferation [29, 32]. However, c-kit-positive cardiac progenitor cells have been reported to contribute significantly to CM production after cardiac injury as well . In contrast, another study found that c-kit-positive cells form negligible numbers of CMs both during homeostasis and after injury . The dissimilar results obtained by these studies are a reminder of the fact that albeit genetic lineage tracing tools are the most powerful way to address questions of cellular lineage, each tool needs to be very critically evaluated. In particular, it is possible that some Cre lines are not specifically expressed in a particular cell type; Cre expression could cause toxicity, and the fact that most tools fail to label all cells of a particular cell population could result in failure to appreciate that the labeled population is actually heterogeneous. Yet, while further studies are needed to clarify how CMs are formed in the adult mammalian heart, the consensus is that the rate of CM formation is too low to result in significant myocardial regeneration after heart injury .
A possible reason for the failure of adult mammalian CMs to sufficiently proliferate in response to cardiac insult might be their increased DNA content. Most adult mammalian CMs contain more than two sets of chromosomes. Starting at 4 days post birth, rodent CMs grow and become binucleated with each nucleus remaining diploid . In contrast, most human CMs maintain a single nucleus, which however increases its DNA content to tetraploidy or even higher ploidy [34–36]. Binucleation, as seen in rodents, likely represents an impediment to successful mitosis, although some indirect evidence for the proliferation of binucleated CMs has been reported as well . Polyploidy of mononucleated cells might activate cell cycle arrest , yet many animal species exist as polyploids ; thus, non-diploid DNA content should not represent a principal impediment for cellular proliferation, although it possibly does in CMs. Intriguingly, experimental manipulation of several cell cycle proteins as well as activation of a variety of signaling pathways including Notch, neuregulin1/ErbB4, Fgf-1, Pi3K, and Akt signaling can stimulate cell cycle re-entry in differentiated mature mammalian CMs [40–45]. Yet, not all CMs that are driven by these regimes into DNA synthesis undergo cytokinesis, and at least for the case of neuregulin, it is clear that only mononucleated mouse CMs, which represent less than 10 % of adult CMs, could be stimulated. Nevertheless, constitutive activation of cyclin A2 and activation of neuregulin1/ErbB4, PI3K, and Akt have also been shown to promote cardiac repair and heart function upon myocardial infarction [41, 43, 46]. Thus, while adult mammalian hearts fail to activate a regenerative program of CM proliferation in response to myocardial injury, adult CMs appear to have no intrinsic block preventing their proliferation. This raises the possibility that therapeutic alteration of the signaling environment of the injured heart could activate compensatory proliferation of CMs.
Less is known about potential roles of Wnt/β-catenin signaling in the regulation of CM proliferation. Treatment of neonatal or adult rat CMs in culture with the Gsk3 inhibitory small molecule BIO, which, among other effects, results in stabilization of β-catenin, can induce cell proliferation and mitosis . Evidence that Wnt/β-catenin signaling regulates CM proliferation in vivo comes from a genetic study in mouse. Inactivation of the Hippo pathway, which restrains cell proliferation and thus controls organ size in Drosophila, in the embryonic mouse heart results in development of enlarged hearts which display increased numbers of CMs and increased CM proliferation . Intriguingly, β-catenin levels are upregulated in such hearts and deletion of one copy of β-catenin can rescue the heart overgrowth phenotype. Thus, Wnt/β-catenin signaling might promote CM proliferation and is kept in check by the Hippo pathway. However, since Hippo and β-catenin were conditionally deleted in nkx2.5+ cardiomyocyte precursor cells early during heart development in this study, it remains possible that these pathways act on precursor cell proliferation or differentiation and do not directly control CM proliferation.
Heart injuries caused by myocardial infarction, pressure/volume overload, inflammatory diseases, cardiomyopathy, chronic hypertension, or congenital heart disease mostly affect the ventricles and lead to complex responses including scarring, fibrosis, and CM hypertrophy in mammals, which are generally referred to as ventricular remodeling. These changes cause a decline in the ventricular performance and contractile function, which can result in heart failure.
In addition to its role during cardiac development, Wnt/β-catenin signaling has also been implicated in regulation of cardiac remodeling and injury responses in mammals. While there is a body of work suggesting roles for Wnt/β-catenin signaling in protection of CMs from apoptosis and regulation of CM hypertrophy, there is little evidence supporting a direct role of the pathway in these processes. Furthermore, several of the Wnt pathway components shown to play a role in the cardiac injury response, in particular Gsk3 and sFrps, have additional functions unrelated to Wnt signaling, which perplex the situation.
Several studies have shown that the expression of Wnt ligands and of the feedback regulators Dkk1 and Dkk2 is induced in response to injury of the mouse heart [76–78]. Aisagbonhi and colleagues described induction of Wnt-2, Wnt-4, Wnt-10b, and Wnt-11 5 days post permanent left anterior descending (LAD) coronary artery occlusion in whole heart samples , while Duan and colleagues reported that Wnt-1 is robustly induced 2 days post injury and sustained in entire hearts in response to transient LAD occlusion, whereas Wnt-4 and Wnt-7a are only upregulated transiently at later stages . It is surprising that these two studies only agree on Wnt-4 as an injury response gene. Further gene expression studies performed on transiently and permanently occluded hearts in parallel will be needed to clarify whether these discrepancies are due to the different injury models used. β-catenin levels have also been reported to increase in rat hearts starting 1 day post pressure overload induced by thoracic aortic constriction (TAC) . Although functional roles of Wnt/β-catenin signaling during cardiac repair have been studied since about a decade, the sites of Wnt pathway activation in the injured heart have been analyzed only recently [76, 78, 80]. Using the Axin2-LacZ mouse, which expresses the marker LacZ under control of regulatory elements of Axin2, which is considered a universal Wnt feedback target gene, and thus serves as useful readout of β-catenin signaling activity, upregulation of pathway activity was reported after permanent LAD in endothelial cells, Sca-1 and c-kit-positive progenitors, fibroblasts, and smooth muscle cells; LacZ+ cells were mainly found in the infarct border zone starting at 7 days post injury (dpi), peaking at 14 dpi . In addition, LacZ+ leukocytes started to accumulate at 3 dpi. Using the TopGAL mouse, upregulation of pathway activity could be detected 4 days after permanent LAD in subepicardial endothelial cells . One week after injury, myofibroblasts expressing the TopGAL reporter accumulated in the infarct area, and genetic lineage tracing showed that these were derived from endothelial cells that appeared to undergo an endothelial-to-mesenchymal transition (EndMT) . In contrast, TopGAL activity was reported in response to ischemia-reperfusion caused by transient LAD ligation primarily in the epicardium [76, 78, 80]. Since the different Wnt reporter mouse lines have been reported to display contrasting expression patterns in some tissues , a definitive determination of active sites of Wnt/β-catenin signaling in the injured mammalian heart will likely require the side-by-side comparison of different reporter mice using identical injury models and detection methods plus determination of endogenous target gene expression in isolated cardiac cell populations.
Although the studies by Aisagbonhi et al. and Duan et al. identified different primary sites of Wnt/β-catenin signaling induction in response to permanent LAD (subepicardial endothelium) and transient LAD (the epicardium), they surprisingly both define a functional role of Wnt/β-catenin signaling in promoting fibroblast formation from these cells in a process of endothelial or epithelial-to-mesenchymal transition (EndMT and EMT respectively) [76, 78]. In cultured endothelial cells, treatment with the Gsk3 inhibitor BIO, which induced direct β-catenin target genes and activity of the transcriptional Wnt reporter Topflash, was sufficient to induce EndMT and myofibroblast formation as evidenced by reduction of endothelial markers and induction of smooth muscle actin . Likewise, Wnt-1 overexpression promoted EMT and fibroblast differentiation of cultured epicardial cells, while β-catenin deletion in epicardial cells reduced epicardial expansion and EMT in response to injury and resulted in reduced cardiac function . Furthermore, β-catenin deletion in cardiac fibroblasts reduced their number in ischemia-reperfusion injured hearts and likewise caused cardiac dysfunction. Together, these data indicate that Wnt/β-catenin signaling promotes fibrosis in response to injury via inducing the transition to a mesenchymal state of both endothelial and epicardial cells; fibrosis, in turn, appears to be required to stabilize the wound and prevent cardiac dilation. Interestingly, Wnt/β-catenin signaling activation has also been reported in fibrotic diseases of the lung, liver, and kidney and Wnt/β-catenin signaling was found to be sufficient and essential for differentiation of fibroblasts into myofibroblasts and production of collagen by these cells [82, 83]. It will be very interesting to test whether Wnt/β-catenin signaling plays different roles in systems like the zebrafish or postnatal mice that activate a regenerative program rather than fibrosis in response to injury.
Whether CMs activate Wnt/β-catenin signaling at later stages after infarction requires further investigation: while LacZ reactivity has been reported in heart regions containing CMs in the Axin2-LacZ mice, the identity of these cells was not confirmed by marker gene expression and the LacZ, which is targeted to the nucleus in these mice, was found in the cytoplasm in the presumptive CMs, raising questions about the validity of these results . Furthermore, no CM expression was reported in the TopGAL mice [76, 78].
Thus, while activation of the β-catenin-dependent pathway in CMs in response to myocardial infarction in vivo has not been shown, functional evidence exists for an involvement of Wnt/β-catenin signaling in injury or stress-induced CM hypertrophy. However, conflicting data have been reported on whether β-catenin is required or actually inhibits CM hypertrophy. On one hand, several studies support a hypertrophy-promoting role for Wnt/β-catenin signaling. Conditional, CM-specific depletion of β-catenin in adult mice was found to impair CM hypertrophy in response to pressure overload induced by thoracic aortic constriction, while non-conditional transgenic overexpression of a dominant-negative Lef transcription factor in CMs throughout embryonic development resulted in CM hypotrophy . Furthermore, transgenic overexpression of Gsk3 (which among other effects might inhibit β-catenin signaling) suppressed CM hypertrophy in response to stress . β-catenin was found to be stabilized in cultured CMs in response to hypertrophic stimuli (phenylephrine or endothelin-1) due to inactivation of Gsk3 activity, but interestingly not in a Wnt pathway-dependent manner but rather via phosphorylation of Gsk3 at serine 9 by Protein kinase B (PtB) . β-catenin knockdown also reduced phenylephrine-induced CM hypertrophy in cultured cells, possibly since upregulation of the fetal gene anf in response to phenylephrine is directly regulated by Lef1/β-catenin . Contradicting a role for Wnt/β-catenin signaling in the promotion of hypertrophy are two studies using conditional deletion of β-catenin. β-catenin deletion in CMs did not impair CM hypertrophy in response to angiotensin II infusion . On the contrary, mice expressing a constitutively active, stabilized β-catenin in CMs (achieved via conditional deletion of exon 3 of β-catenin, which codes for the domain phosphorylated by GSK3β) showed an abrogated hypertrophic response to angiotensin II . Furthermore, the same mouse β-catenin deletion and overexpression models showed no alterations in CM hypertrophy at 2 and 4 weeks after infarct . Currently, it remains unexplored whether the discrepancy between these and the above-mentioned studies showing a requirement for β-catenin for hypertrophy in response to thoracic aortic constriction can be explained by the different molecular responses induced by the different injury and stress models used.
Interestingly, treatment of cultured neonatal and adult mammalian CMs with the small Gsk3 inhibitor BIO, which results in β-catenin stabilization, has been found to be sufficient to induce CM proliferation . Non-conditional Gsk3 knockout mice lacking Gsk3 throughout development display a CM hyperproliferation phenotype without defects in CM size . Together with the data discussed above on the interaction of the Hippo and β-catenin pathways, these results indicate that Gsk3, possibly via reducing Wnt/β-catenin signaling, negatively regulates CM proliferation during embryonic development, a role that can be re-activated in adult CMs, at least in culture. This stands in contrast to the evidence supporting a positive role of Gsk3 inhibition and Wnt/β-catenin signaling in CM hypertrophy in the stressed adult myocardium. Additional conditional in vivo loss-of-function data will be required to clarify whether β-catenin function switches from CM proliferation-promoting in the embryo to hypertrophy-promoting in the adult.
Constitutive overexpression of sFrp-1 in mice has been shown to reduce infarct size and cardiac function after coronary artery ligation or cryoinjury . In contrast, sFrp-1 overexpression specifically in CMs did not affect infarct size or cardiac function in an ischemia-reperfusion (IR) model . Rather, sFrp-1 expressed in CMs reversed the cardio-protective effect of pre-conditioning prior to IR and thus worsened infarcts after pre-conditioning, which stands in contrast to the positive effects observed after systemic sFrp-1 expression. Whether these differences are due to the different injury models used in these studies or reflect a cardio-protective function of sFrp-1 in non-CMs remains unexplained. sFrp-1 was proposed to activate Gsk3 function in the latter study using CM-specific sFrp-1 expression, but not via inhibition of Wnt/β-catenin signaling but by counteracting phosphorylation and thus inhibition of Gsk3 by a Pi3-kinase-protein kinase B pathway during cardiac pre-conditioning [95, 96]. This pathway regulates β-catenin-independent cardio-protective effects of Gsk3. Likewise, no effects on β-catenin levels were seen after CM-specific sFrp overexpression, indicating that sFrp-1 likely does not modify Wnt/β-catenin signaling in this context.
Clearly, additional experiments using transcriptional readouts of β-catenin activity in culture and in vivo plus loss-of-function experiments will be required to clarify whether Wnt/β-catenin signaling is cardio-protective and whether sFrps have an endogenous role as modifiers of this function. A recent report on the function of the secreted Wnt inhibitor Dkk3 actually showed that Dkk3 knockout mice had greater infarcts and aggravated left ventricular function after infarct, while Dkk3 overexpression protected from infarction . This indicates that active Wnt/β-catenin would exacerbate cardiac injury and remodeling, while inhibition of β-catenin signaling by Dkk3 is cardio-protective.
Intriguingly, another Wnt signaling-independent function of sFrp has been uncovered in regulation of fibrosis in response to cardiac injury. Mammalian sFrp-2 has been found to enhance the function of the metalloproteinase Bmp1, which is a rate-limiting enzyme in the processing of pro-collagen during collagen fiber formation at physiological low concentrations (10–20 nM)  (Fig. 3, Wnt-independent route). Since collagen deposition is a hallmark of fibrosis occurring after cardiac injury, sFrp-2 might enhance fibrosis and thus worsen cardiac function after infarction. Indeed, fibrosis was found to be reduced and cardiac function to be improved in sFrp-2 knockout mice after coronary artery ligation . Remarkably, at high concentrations, sFrp-2 was found to have the opposite effect: it inhibited Bmp1 activity and thus collagen deposition, and injection of recombinant sFrp-2 into the infarcted area of rats after coronary artery ligation reduced fibrosis and improved cardiac function . Thus, sFrps can act through Wnt-dependent or Wnt-independent pathways and appear to positively or negatively regulate the cardiac response to different injury types in a context-dependent manner (Fig. 3).
As outlined above, conflicting data exist on the in vivo importance of cardiac progenitor cells for CM formation during adult homeostasis and in response to heart injury [22, 24]. Some evidence exists for a role of Wnt/β-catenin signaling in regulation of progenitor cell differentiation. Zelarayan et al. reported that conditional depletion of β-catenin from the adult mouse myocardium using a MHC-Cre driver line resulted in improved survival and left ventricular function 4 weeks after chronic LAD . The Cre driver was found to be active in a population of progenitor cells (as defined by absence of expression of cardiac Troponin T (cTnT) but presence of proliferation markers and the embryonic CM transcription factors Gata4 and Tbx5). β-catenin depleted infarcted hearts showed increased numbers of cTnT-positive CMs in subepicardial and subendocardial positions, which the authors proposed not to be due to increased survival, proliferation, or hypertrophy of existing differentiated CMs. Rather, they found that isolated progenitor cells from β-catenin depleted hearts showed increased tendency to differentiate into cTNT+ cells in coculture with CMs . Thus, β-catenin might inhibit differentiation of cardiac progenitor cells in the injured heart, consistent with the negative role of Wnt/β-catenin signaling in the later phases of heart development. Another recent study found that injection of recombinant Wnt3a protein into infarcted mouse hearts resulted in an increase in infarct size and worsened cardiac performance, and it was suggested that this is due to a negative influence of Wnt3a on proliferation of cardiac side population (sca1+, c-kit−, isl−) progenitor cells . The in vivo relevance of this finding however has not been assessed using loss-of-function experiments.
Human pluripotent stem cells (hPSCs), including hESCs and iPSCs, provide a potentially unlimited supply of progenitor cells that can differentiate into the vast majority of somatic cell types including CMs [100–102]. Temporal modulation of Wnt/β-catenin signaling via small molecule inhibitors or shRNA knockdown has been shown to affect the differentiation of hPSCs to CMs. Lian and colleagues found that short hairpin RNA (shRNA) knockdown of β-catenin during the early hPSC differentiation period arrested CM specification while small molecule mediated inhibition of GSK3 within the same period promoted it in multiple hPSC lines . Inhibition of Wnt/β-catenin signaling during later phases of cardiac differentiation, however, can promote cardiac differentiation in hPSCs and generate functional CMs [104–106] as it does during development [66, 68–72]. Wnt/β-catenin signaling has also been reported to contribute to CM differentiation in progenitor cells, other than hPSCs. Cardiogenol C, a small molecule that was shown to induce ES cells to differentiate into CMs , was sufficient to promote hair bulge progenitor cells (HBPCs) to transdifferentiate into CM-like cells most likely by activating the Wnt/β-catenin pathway through suppression of Kremen1 . Early treatment of human iPSCs with Bmp-4 followed by late treatment with small molecule Wnt inhibitors caused a remarkable increase in the total yield of biologically functional CMs, suggesting that modification of Bmp-4 and Wnt/β-catenin signaling pathways in human iPSCs might create an efficient platform to produce new CMs after cardiac injuries . Similarly, the Cardionogen family of chemicals, which inhibit β-catenin-dependent transcription in murine ES cells and zebrafish embryos, modulated cardiogenesis in a biphasic manner and, while failing to induce CM differentiation during the initial ES cell differentiation period, increased the number of CMs by expanding the cells in both systems . Moreover, another Wnt/β-catenin pathway inhibitor molecule, pyrvinium, was found to reduce adverse cardiac remodeling in mice infarcted by LAD after intracardiac injection, increase proliferation of differentiated CMs, and hence promote wound repair . While modulation of the Wnt/β-catenin pathway thus appears to promote the cardiac differentiation of hESCs and iPSCs and tissue repair following heart injury, it should be noted that the molecular mechanisms of these effects remain unclear.
In contrast to the limited regenerative ability of the adult mammalian heart, urodele amphibia and teleost fish retain regenerative capacity of the heart throughout adult life [3, 112]. Most of our knowledge about the mechanisms underlying heart regeneration comes from studies using zebrafish. While teleost fish have only a two-chambered heart (one ventricle, one atrium), the cellular composition, physiology, and development of the fish heart are well conserved with that of mammals. Due to a wealth of accumulated knowledge about heart development and the availability of many molecular, genetic, and genomic tools, zebrafish is an excellent model to study the cellular and molecular mechanisms of heart regeneration [113–115]. Furthermore, due to their short generation time, small size, and cheap husbandry, a high number of individual fish can be studied and transgenic and mutant lines can be produced quickly and at relatively low cost. The adult zebrafish heart is also easily accessible for surgical or other experimental manipulations, and the animals are highly tolerant to experimental cardiac injury .
Adult zebrafish hearts have been shown to display remarkable regenerative capacity in response to three types of injury: surgical removal of up to 20 % of the apex of the ventricle, cryoinjury-induced necrosis of up to 25 % of the apical ventricular region [9, 11, 116], and even mosaic ablation of up to 70 % of CMs caused by transgenic expression of diphtheria toxin . In all three injury regimes, lost CMs are being replaced within 30 to 120 days and no or little permanent collagen-rich scar tissue forms.
While assays for heart function, in particular assessment of cardiac hemodynamics using ultrasound, are difficult due to the small size of the zebrafish heart and thus not trivial to perform [117–119], there is increasing evidence for functional recovery during zebrafish heart regeneration. Myocardium at the apex of the ventricle, which presumably has formed anew during regeneration in response to ventricular resection, was found to be electrically coupled to the rest of the ventricle, indicating functional recovery . Likewise, coupling measured by optical mapping was found to be restored to pre-injury levels 45 days post genetic ablation of CMs, as was a lengthening in action potential duration occurring after ablation as measured at the tissue level . Electrocardiogram recordings indicate that prolonged QT intervals were measured during recovery after cryoinjury . However, another report found that regenerated hearts retained a prolonged QT interval after ventricular resection . Side-by-side comparison of different injury models in one study as well as measurements of the electrophysiological properties of individual CMs  will be instrumental in clarifying whether zebrafish can completely recover the electrophysiology of the heart. Measurements of cardiac performance based on echocardiography indicate that heart function does recover; yet this might take longer than morphological regeneration. Here, one study reported that heart function after cryoinjury recovered only within 180 days , while another study showed that pumping fraction based on relative fractional volume shortening recovered within 60 days in cryoinjured hearts, while ventricular wall motion remained altered even after 140 days . Overall, these data indicate that zebrafish can not only regenerate heart tissue architecture but also function, albeit it remains somewhat unclear how complete the functional regeneration is.
The cellular mechanisms activated after wounding and underlying heart regeneration appear to be highly similar between the injury models. A few days after injury, transcriptional responses occur in the entire epicardium, including upregulation of genes normally expressed during development (including Wilms’ tumor 1b), which is followed by proliferation and expansion of the epicardium into a multilayered structure [9, 11, 12, 116, 124]. Interestingly, similar epicardial responses occur in regenerating neonatal mouse hearts . Genetic ablation of the epicardium has shown that it is essential for myocardial regeneration, but the nature of the molecular signals mediating its effects on CMs during zebrafish heart regeneration has not been elucidated . In infarcted mouse hearts, epicardial Wilms’ tumor 1 (wt1) expression can also be re-activated by experimental treatment with the peptide thymosin 4 and wt1-positive cells have been reported to represent multipotent progenitors that can give rise to CMs after injury, albeit at a low frequency . In contrast, in the regenerating zebrafish heart, genetic lineage tracing of epicardial cells identified by expression of tcf21 showed that epicardial cells give rise to perivascular cells but not CMs . Thus, naturally occurring myocardial regeneration in the zebrafish occurs without a cellular contribution of the epicardium. While regenerating CMs have initially been proposed to be derived from progenitor cells that do not have CM character , genetic lineage tracing data using the Cre-Lox system has rather indicated that the entire regenerated myocardium is derived from existing, differentiated CMs [14, 15]. In particular, a subepicardial population of CMs characterized by expression of a transgene driven by a gata4 promoter fragment appears to produce the bulk of the regenerated myocardial tissue [14, 129]. Thus, CMs dedifferentiate in response to injury, re-enter the cell cycle, and proliferate to replace the missing tissue.
Although a large body of data shows that Wnt/β-catenin signaling plays important roles in mammalian cardiac remodeling and regeneration processes occurring in response to heart injury, its potential role in regulation of zebrafish heart regeneration remains to be tested. We have previously found that a transgenic reporter of Wnt/β-catenin signaling is upregulated in the injured ventricle after ventricular resection . Besides, a novel Wnt target gene simplet (smp), which we have identified to be necessary for β-catenin-dependent signal transduction , is also transcriptionally activated in the zebrafish heart after amputation injury . Thus, while Wnt/β-catenin signaling appears to be linked to heart regeneration in zebrafish, our efforts to test its function in heart regeneration using transgenic lines allowing for heat shock-inducible overexpression of Wnt modifiers have been confounded by the realization that heart regeneration can be impaired by the stress inflicted by heat shocking the fish (data not shown). Thus, these tools, which we have successfully used to uncover an essential function for Wnt/β-catenin signaling in the regulation of zebrafish fin regeneration [130, 132], are not ideal for the analysis of heart regeneration. Likely, studies into the role of Wnt/β-catenin signaling in zebrafish heart regeneration will benefit from further technological advances allowing for alternative methods of inducible manipulation of gene expression [133, 134].
Heart regeneration is a complex process that only occurs in lower vertebrates, but not in mammals. Several processes such as dedifferentiation, proliferation, redifferentiation, and patterning should take place in concert in a highly regulated manner. Wnt/β-catenin signaling is crucial for orchestration of the regeneration process in other systems [132, 135]. Therefore, further studies will likely also uncover a role for Wnt/β-catenin signaling in lower vertebrate heart repair. Whether its role will be similar or different to the rather complex functions that Wnt/β-catenin signaling plays after mammalian heart injury will be interesting to study. Regenerating organisms such as zebrafish provide an excellent tool to address these issues, and regenerative therapeutic applications will surely benefit from a more widespread use of this model.
We thank Caghan Kizil for designing the figures and critically reading and commenting on the manuscript.
- Ozhan G, Weidinger G. Restoring tissue homeostasis: Wnt signaling in tissue regeneration after acute injury Wnt signaling in development and disease: molecular mechanisms and biological functions. In: Hoppler SP, Moon RT, editors. Wnt signaling in development and disease: molecular mechanisms and biological functions. Hoboken, New Jersey: Wiley-Blackwell; 2014. p. 459.Google Scholar
- Stoick-Cooper CL, Moon RT, Weidinger G. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes Dev. 2007;21(11):1292–315.PubMedGoogle Scholar
- Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science. 2002;298(5601):2188–90.PubMedGoogle Scholar
- Young W. Spinal cord regeneration. Cell Transplant. 2014;23(4–5):573–611.PubMedGoogle Scholar
- Kizil C et al. Adult neurogenesis and brain regeneration in zebrafish. Dev Neurobiol. 2012;72(3):429–61.PubMedGoogle Scholar
- Bergmann MW. WNT signaling in adult cardiac hypertrophy and remodeling: lessons learned from cardiac development. Circ Res. 2010;107(10):1198–208.PubMedGoogle Scholar
- Laflamme MA, Murry CE. Heart regeneration. Nature. 2011;473(7347):326–35.PubMed CentralPubMedGoogle Scholar
- Raya A et al. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc Natl Acad Sci U S A. 2003;100 Suppl 1:11889–95.PubMed CentralPubMedGoogle Scholar
- Chablais F et al. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol. 2011;11:21.PubMed CentralPubMedGoogle Scholar
- Gonzalez-Rosa JM, Mercader N. Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish. Nat Protoc. 2012;7(4):782–8.PubMedGoogle Scholar
- Schnabel K et al. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One. 2011;6(4):e18503.PubMed CentralPubMedGoogle Scholar
- Wang J et al. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development. 2011;138(16):3421–30.PubMed CentralPubMedGoogle Scholar
- Porrello ER et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331(6020):1078–80.PubMed CentralPubMedGoogle Scholar
- Kikuchi K et al. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature. 2010;464(7288):601–5.PubMed CentralPubMedGoogle Scholar
- Jopling C et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 2010;464(7288):606–9.PubMed CentralPubMedGoogle Scholar
- Goldstein MA, Claycomb WC, Schwartz A. DNA synthesis and mitosis in well-differentiated mammalian cardiocytes. Science. 1974;183(4121):212–3.PubMedGoogle Scholar
- Burton PB et al. An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes. Dev Biol. 1999;216(2):659–70.PubMedGoogle Scholar
- Li F et al. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol. 1996;28(8):1737–46.PubMedGoogle Scholar
- Yoshizumi M et al. Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts. J Clin Invest. 1995;95(5):2275–80.PubMed CentralPubMedGoogle Scholar
- Anversa P, Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res. 1998;83(1):1–14.PubMedGoogle Scholar
- Anversa P, Nadal-Ginard B. Myocyte renewal and ventricular remodelling. Nature. 2002;415(6868):240–3.PubMedGoogle Scholar
- Beltrami AP et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001;344(23):1750–7.PubMedGoogle Scholar
- Cheng W et al. Down-regulation of the IGF-1 system parallels the attenuation in the proliferative capacity of rat ventricular myocytes during postnatal development. Lab Invest. 1995;72(6):646–55.PubMedGoogle Scholar
- Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 1997;272(1 Pt 2):H220–6.PubMedGoogle Scholar
- Soonpaa MH et al. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J Clin Invest. 1997;99(11):2644–54.PubMed CentralPubMedGoogle Scholar
- Senyo SE, Lee RT, Kuhn B. Cardiac regeneration based on mechanisms of cardiomyocyte proliferation and differentiation. Stem Cell Res. 2014;13(3 Pt B):532–41.PubMedGoogle Scholar
- Bergmann O et al. Evidence for cardiomyocyte renewal in humans. Science. 2009;324(5923):98–102.PubMed CentralPubMedGoogle Scholar
- Hsieh PC et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med. 2007;13(8):970–4.PubMed CentralPubMedGoogle Scholar
- Ali SR et al. Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proc Natl Acad Sci U S A. 2014;111(24):8850–5.PubMed CentralPubMedGoogle Scholar
- Ellison GM et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 2013;154(4):827–42.PubMedGoogle Scholar
- Malliaras K et al. Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart. EMBO Mol Med. 2013;5(2):191–209.PubMed CentralPubMedGoogle Scholar
- Senyo SE et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature. 2013;493(7432):433–6.PubMed CentralPubMedGoogle Scholar
- van Berlo JH et al. c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature. 2014;509(7500):337–41.PubMed CentralPubMedGoogle Scholar
- Adler CP. Relationship between deoxyribonucleic acid content and nucleoli in human heart muscle cells and estimation of cell number during cardiac growth and hyperfunction. Recent Adv Stud Cardiac Struct Metab. 1975;8:373–86.PubMedGoogle Scholar
- Adler CP, Friedburg H. Myocardial DNA content, ploidy level and cell number in geriatric hearts: post-mortem examinations of human myocardium in old age. J Mol Cell Cardiol. 1986;18(1):39–53.PubMedGoogle Scholar
- Olivetti G et al. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol. 1996;28(9):2005–16.PubMedGoogle Scholar
- Naqvi N et al. A proliferative burst during preadolescence establishes the final cardiomyocyte number. Cell. 2014;157(4):795–807.PubMed CentralPubMedGoogle Scholar
- Ganem NJ, Pellman D. Limiting the proliferation of polyploid cells. Cell. 2007;131(3):437–40.PubMedGoogle Scholar
- Otto SP. The evolutionary consequences of polyploidy. Cell. 2007;131(3):452–62.PubMedGoogle Scholar
- Campa VM et al. Notch activates cell cycle reentry and progression in quiescent cardiomyocytes. J Cell Biol. 2008;183(1):129–41.PubMed CentralPubMedGoogle Scholar
- Bersell K et al. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell. 2009;138(2):257–70.PubMedGoogle Scholar
- Engel FB et al. FGF1/p38 MAP kinase inhibitor therapy induces cardiomyocyte mitosis, reduces scarring, and rescues function after myocardial infarction. Proc Natl Acad Sci U S A. 2006;103(42):15546–51.PubMed CentralPubMedGoogle Scholar
- Kuhn B et al. Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nat Med. 2007;13(8):962–9.PubMedGoogle Scholar
- Pasumarthi KB et al. Targeted expression of cyclin D2 results in cardiomyocyte DNA synthesis and infarct regression in transgenic mice. Circ Res. 2005;96(1):110–8.PubMedGoogle Scholar
- Chaudhry HW et al. Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium. J Biol Chem. 2004;279(34):35858–66.PubMedGoogle Scholar
- Cheng RK et al. Cyclin A2 induces cardiac regeneration after myocardial infarction and prevents heart failure. Circ Res. 2007;100(12):1741–8.PubMedGoogle Scholar
- MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009;17(1):9–26.PubMed CentralPubMedGoogle Scholar
- Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810.PubMedGoogle Scholar
- Willert K, Nusse R. Wnt proteins. Cold Spring Harb Perspect Biol. 2012;4(9):a007864.PubMed CentralPubMedGoogle Scholar
- Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149(6):1192–205.PubMedGoogle Scholar
- Angers S, Moon RT. Proximal events in Wnt signal transduction. Nat Rev Mol Cell Biol. 2009;10(7):468–77.PubMedGoogle Scholar
- Stamos JL, Weis WI. The beta-catenin destruction complex. Cold Spring Harb Perspect Biol. 2013;5(1):a007898.PubMed CentralPubMedGoogle Scholar
- Behrens J et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature. 1996;382(6592):638–42.PubMedGoogle Scholar
- Kimelman D, Xu W. beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene. 2006;25(57):7482–91.PubMedGoogle Scholar
- MacDonald BT, He X. Frizzled and LRP5/6 receptors for Wnt/beta-catenin signaling. Cold Spring Harb Perspect Biol. 2012;4(12):a007880.PubMed CentralPubMedGoogle Scholar
- Kikuchi A, Yamamoto H. Regulation of Wnt signalling by receptor-mediated endocytosis. J Biochem. 2007;141(4):443–51.PubMedGoogle Scholar
- Yamamoto H, Komekado H, Kikuchi A. Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin. Dev Cell. 2006;11(2):213–23.PubMedGoogle Scholar
- Özhan G et al. Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains. Dev Cell. 2013;26(4):331–45.PubMedGoogle Scholar
- Kafka A, Basic-Kinda S, Pecina-Slaus N. The cellular story of dishevelleds. Croat Med J. 2014;55(5):459–67.PubMed CentralPubMedGoogle Scholar
- Kizil C et al. Simplet/Fam53b is required for Wnt signal transduction by regulating beta-catenin nuclear localization. Development. 2014;141(18):3529–39.PubMedGoogle Scholar
- Cadigan KM, Waterman ML. TCF/LEFs and Wnt signaling in the nucleus. Cold Spring Harb Perspect Biol. 2012;4(11):a007906.PubMed CentralPubMedGoogle Scholar
- Taelman VF et al. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell. 2010;143(7):1136–48.PubMed CentralPubMedGoogle Scholar
- Kagermeier-Schenk B et al. Waif1/5T4 inhibits Wnt/beta-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization. Dev Cell. 2011;21(6):1129–43.PubMedGoogle Scholar
- Semenov M, Tamai K, He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J Biol Chem. 2005;280(29):26770–5.PubMedGoogle Scholar
- Cruciat CM, Niehrs C. Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb Perspect Biol. 2013;5(3):a015081.PubMed CentralPubMedGoogle Scholar
- Gessert S, Kuhl M. The multiple phases and faces of wnt signaling during cardiac differentiation and development. Circ Res. 2010;107(2):186–99.PubMedGoogle Scholar
- Lickert H et al. Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell. 2002;3(2):171–81.PubMedGoogle Scholar
- Ueno S et al. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc Natl Acad Sci U S A. 2007;104(23):9685–90.PubMed CentralPubMedGoogle Scholar
- Klaus A et al. Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc Natl Acad Sci U S A. 2007;104(47):18531–6.PubMed CentralPubMedGoogle Scholar
- Naito AT et al. Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc Natl Acad Sci U S A. 2006;103(52):19812–7.PubMed CentralPubMedGoogle Scholar
- Kwon SM, Alev C, Asahara T. The role of notch signaling in endothelial progenitor cell biology. Trends Cardiovasc Med. 2009;19(5):170–3.PubMedGoogle Scholar
- Paige SL et al. Endogenous Wnt/beta-catenin signaling is required for cardiac differentiation in human embryonic stem cells. PLoS One. 2010;5(6):e11134.PubMed CentralPubMedGoogle Scholar
- David R et al. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat Cell Biol. 2008;10(3):338–45.PubMedGoogle Scholar
- Tseng AS, Engel FB, Keating MT. The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem Biol. 2006;13(9):957–63.PubMedGoogle Scholar
- Heallen T et al. Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size. Science. 2011;332(6028):458–61.PubMed CentralPubMedGoogle Scholar
- Aisagbonhi O et al. Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition. Dis Model Mech. 2011;4(4):469–83.PubMed CentralPubMedGoogle Scholar
- Barandon L et al. Reduction of infarct size and prevention of cardiac rupture in transgenic mice overexpressing FrzA. Circulation. 2003;108(18):2282–9.PubMedGoogle Scholar
- Duan J et al. Wnt1/betacatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. EMBO J. 2012;31(2):429–42.PubMed CentralPubMedGoogle Scholar
- Haq S et al. Stabilization of beta-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth. Proc Natl Acad Sci U S A. 2003;100(8):4610–5.PubMed CentralPubMedGoogle Scholar
- Oerlemans MI et al. Active Wnt signaling in response to cardiac injury. Basic Res Cardiol. 2010;105(5):631–41.PubMed CentralPubMedGoogle Scholar
- Al Alam D et al. Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair. PLoS One. 2011;6(8):e23139.PubMed CentralPubMedGoogle Scholar
- Akhmetshina A et al. Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. Nat Commun. 2012;3:735.PubMed CentralPubMedGoogle Scholar
- Laeremans H et al. Wnt/frizzled signalling modulates the migration and differentiation of immortalized cardiac fibroblasts. Cardiovasc Res. 2010;87(3):514–23.PubMedGoogle Scholar
- Antos CL et al. Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2002;99(2):907–12.PubMed CentralPubMedGoogle Scholar
- Chen X et al. The beta-catenin/T-cell factor/lymphocyte enhancer factor signaling pathway is required for normal and stress-induced cardiac hypertrophy. Mol Cell Biol. 2006;26(12):4462–73.PubMed CentralPubMedGoogle Scholar
- Zhang CG et al. beta-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes. Biochem Biophys Res Commun. 2009;390(2):258–62.PubMedGoogle Scholar
- Baurand A et al. Beta-catenin downregulation is required for adaptive cardiac remodeling. Circ Res. 2007;100(9):1353–62.PubMedGoogle Scholar
- Zelarayan LC et al. Beta-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation. Proc Natl Acad Sci U S A. 2008;105(50):19762–7.PubMed CentralPubMedGoogle Scholar
- Kerkela R et al. Deletion of GSK-3beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation. J Clin Invest. 2008;118(11):3609–18.PubMed CentralPubMedGoogle Scholar
- Schumann H et al. Expression of secreted frizzled related proteins 3 and 4 in human ventricular myocardium correlates with apoptosis related gene expression. Cardiovasc Res. 2000;45(3):720–8.PubMedGoogle Scholar
- Kobayashi K et al. Secreted frizzled-related protein 2 is a procollagen C proteinase enhancer with a role in fibrosis associated with myocardial infarction. Nat Cell Biol. 2009;11(1):46–55.PubMed CentralPubMedGoogle Scholar
- Mangi AA et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med. 2003;9(9):1195–201.PubMedGoogle Scholar
- Mirotsou M et al. Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. Proc Natl Acad Sci U S A. 2007;104(5):1643–8.PubMed CentralPubMedGoogle Scholar
- Esteve P et al. Secreted frizzled-related proteins are required for Wnt/beta-catenin signalling activation in the vertebrate optic cup. Development. 2011;138(19):4179–84.PubMedGoogle Scholar
- Barandon L et al. Involvement of FrzA/sFRP-1 and the Wnt/frizzled pathway in ischemic preconditioning. Circ Res. 2005;96(12):1299–306.PubMedGoogle Scholar
- Tong H et al. Phosphorylation of glycogen synthase kinase-3beta during preconditioning through a phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. Circ Res. 2002;90(4):377–9.PubMedGoogle Scholar
- Bao MW et al. Dickkopf-3 protects against cardiac dysfunction and ventricular remodelling following myocardial infarction. Basic Res Cardiol. 2015;110(3):481.Google Scholar
- He W et al. Exogenously administered secreted frizzled related protein 2 (Sfrp2) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction. Proc Natl Acad Sci U S A. 2010;107(49):21110–5.PubMed CentralPubMedGoogle Scholar
- Oikonomopoulos A et al. Wnt signaling exerts an antiproliferative effect on adult cardiac progenitor cells through IGFBP3. Circ Res. 2011;109(12):1363–74.PubMed CentralPubMedGoogle Scholar
- Takahashi K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.PubMedGoogle Scholar
- Thomson JA et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–7.PubMedGoogle Scholar
- Yu J et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917–20.PubMedGoogle Scholar
- Lian X et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A. 2012;109(27):E1848–57.PubMed CentralPubMedGoogle Scholar
- Mehta A et al. Phasic modulation of Wnt signaling enhances cardiac differentiation in human pluripotent stem cells by recapitulating developmental ontogeny. Biochim Biophys Acta. 2014;1843(11):2394–402.PubMedGoogle Scholar
- Lian X et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc. 2013;8(1):162–75.PubMed CentralPubMedGoogle Scholar
- Cao N et al. Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013;23(9):1119–32.PubMed CentralPubMedGoogle Scholar
- Wu X et al. Small molecules that induce cardiomyogenesis in embryonic stem cells. J Am Chem Soc. 2004;126(6):1590–1.PubMedGoogle Scholar
- Yau WW et al. Cardiogenol C can induce mouse hair bulge progenitor cells to transdifferentiate into cardiomyocyte-like cells. Proteome Sci. 2011;9(1):3.PubMed CentralPubMedGoogle Scholar
- Ren Y et al. Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells. J Mol Cell Cardiol. 2011;51(3):280–7.PubMed CentralPubMedGoogle Scholar
- Ni TT et al. Discovering small molecules that promote cardiomyocyte generation by modulating Wnt signaling. Chem Biol. 2011;18(12):1658–68.PubMed CentralPubMedGoogle Scholar
- Saraswati S et al. Pyrvinium, a potent small molecule Wnt inhibitor, promotes wound repair and post-MI cardiac remodeling. PLoS One. 2010;5(11):e15521.PubMed CentralPubMedGoogle Scholar
- Oberpriller JO, Oberpriller JC. Response of the adult newt ventricle to injury. J Exp Zool. 1974;187(2):249–53.PubMedGoogle Scholar
- Bakkers J, Verhoeven MC, Abdelilah-Seyfried S. Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis. Dev Biol. 2009;330(2):213–20.PubMedGoogle Scholar
- Thisse C, Zon LI. Organogenesis–heart and blood formation from the zebrafish point of view. Science. 2002;295(5554):457–62.PubMedGoogle Scholar
- Stainier DY. Zebrafish genetics and vertebrate heart formation. Nat Rev Genet. 2001;2(1):39–48.PubMedGoogle Scholar
- Gonzalez-Rosa JM et al. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development. 2011;138(9):1663–74.PubMedGoogle Scholar
- Sun L et al. In vivo cardiac imaging of adult zebrafish using high frequency ultrasound (45–75 MHz). Ultrasound Med Biol. 2008;34(1):31–9.PubMed CentralPubMedGoogle Scholar
- Huang CC, Su TH, Shih CC. High-resolution tissue Doppler imaging of the zebrafish heart during its regeneration. Zebrafish. 2015;12(1):48–57.PubMedGoogle Scholar
- Kang BJ et al. High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts. J R Soc Interface. 2015;12(103):20141154.PubMedGoogle Scholar
- Yu F et al. Electrocardiogram signals to assess zebrafish heart regeneration: implication of long QT intervals. Ann Biomed Eng. 2010;38(7):2346–57.PubMed CentralPubMedGoogle Scholar
- Nemtsas P et al. Adult zebrafish heart as a model for human heart? An electrophysiological study. J Mol Cell Cardiol. 2010;48(1):161–71.PubMedGoogle Scholar
- Hein SJ et al. Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury. PLoS One. 2015;10(4):e0122665.PubMed CentralPubMedGoogle Scholar
- Gonzalez-Rosa JM et al. Use of echocardiography reveals reestablishment of ventricular pumping efficiency and partial ventricular wall motion recovery upon ventricular cryoinjury in the zebrafish. PLoS One. 2014;9(12):e115604.PubMed CentralPubMedGoogle Scholar
- Lepilina A et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell. 2006;127(3):607–19.PubMedGoogle Scholar
- Wang J, et al. Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling. Nature. 2015. doi:10.1038/nature14325.
- Smart N et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature. 2011;474(7353):640–4.PubMed CentralPubMedGoogle Scholar
- Kikuchi K et al. tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development. 2011;138(14):2895–902.PubMed CentralPubMedGoogle Scholar
- Kikuchi K et al. Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev Cell. 2011;20(3):397–404.PubMed CentralPubMedGoogle Scholar
- Gupta V et al. An injury-responsive gata4 program shapes the zebrafish cardiac ventricle. Curr Biol. 2013;23(13):1221–7.PubMed CentralPubMedGoogle Scholar
- Stoick-Cooper CL et al. Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development. 2007;134(3):479–89.PubMedGoogle Scholar
- Kizil C et al. Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration. Dev Biol. 2009;325(2):329–40.PubMedGoogle Scholar
- Wehner D et al. Wnt/beta-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal Fin. Cell Rep. 2014;6(3):467–81.PubMedGoogle Scholar
- Knopf F et al. Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish. Proc Natl Acad Sci U S A. 2010;107(46):19933–8.PubMed CentralPubMedGoogle Scholar
- Hans S et al. Temporally-controlled site-specific recombination in zebrafish. PLoS One. 2009;4(2):e4640.PubMed CentralPubMedGoogle Scholar
- Reuter H et al. Beta-catenin-dependent control of positional information along the AP body axis in planarians involves a teashirt family member. Cell Rep. 2015;10(2):253–65.PubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.