Huo et al. J Perioperative Science 2015, 2:1 http://www.perioperative-science.com/content/02/01

Journal of Perioperative Science REVIEW

OPEN ACCESS

Zebrafish models of heart development and cardiovascular diseases Zi-Rong Huo2, Lorraine Marshall1, Wei Zhou2,Zheng-Shang Ruan2, Bo Xu2, Bin He1,2, Xiao-Lei Xu1

Abstract In the past decade, zebrafish (Danio rerio) have been recognized by a variety of biological disciplines for their usefulness in animal models of organogenesis and various human diseases. The zebrafish has several advantages over mammalian models. For example, the experimental cost of using zebrafish is comparatively low: the embryos are transparent, develop externally, and have high fecundity, making them suitable for large-scale genetic screening. In this review, we focused on zebrafish usage in cardiovascular research. In zebrafish, many processes associated with early development, as well as those associated with the fundamental functions of the heart, appear to have been conserved, which supports its viability as an animal model for the cardiovascular system. Additionally, the zebrafish possesses strong myocardial cell regeneration capacity, offering unique research opportunities geared toward the development of cell-based therapies. With the advent of more sophisticated techniques, the zebrafish model may complement the mouse and make novel insights into heart development and cardiovascular diseases possible. Keywords: zebrafish; heart; development; cardiovascular disease ( J Perioper Sci 2015, 2:1)

Introduction

During

the past two decades, the zebrafish (Danio rerio) has become a popular vertebrate model due to the number of large-scale mutagenesis screens that have been conducted successfully with this animal [1]. Compared with other mammalian models, such as those involving mice, the zebrafish offers several advantages, which are summarized in table 1 [2]. Almost the entire genome of the zebrafish is sequenced, and its gene functions are highly From the1Department of Biochemistry and Molecular Biology, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN 55905, USA. 2 Department of Anesthesiology and SICU, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Kongjiang Road 1665, Shanghai 200092, China. Accepted for publication May 10, 2015. Reprints will not be available from the authors. Address correspondence to Bin He: [email protected], and Xiao-Lei Xu: [email protected].

Copyright © 2014 Journal of Periopertive Science

conserved compared with humans. The zebrafish organogenesis and related diseases because of its economic frugality, embryonic transparency, exuteral development, high fecundity and usefulness in large-scale genetic screens [3]. The advantages offered by the zebrafish make this teleost an appealing animal model in biology. The zebrafish recently became a subject of interest to various scientific communities. As a low vertebrate animal, the zebrafish has been used as a developmental and embryological model dating back to the 1930’s [4]. Although zebrafish cardiomyocytes are phenotypically immature, the stages of zebrafish heart development, as well as its fundamental heart functions, are very similar to those of other organisms, including the differentiation of myocardial and endocardial progenitors, the migration of these progenitors to the midline of the embryo, the formation of a linear heart tube, heart chambers and cardiac valves, the looping of the heart tube, and the development of the cardiac conduction system [5]. Fundamental heart functions are also similar between the zebrafish and humans [6]. Therefore, further studies

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Table 1. The advantages and disadvantages of cardiovascular research offered by zebrafish and mouse models Advantages

Disadvantages

1. A complete sequence of the zebrafish genome is 1. The zebrafish has only a prototypical heart, available, and many human genes are conserved the structure and function of which differ structurally and functionally in zebrafish significantly compared with mammals. (http://www.ncbi.nlm.nih.gov/genome/guide/zebr afish).

Zebrafish

2. Their short generation times, large numbers of 2. Their small size makes functional studies of eggs produced during each mating, and external zebrafish hearts challenging; therefore fertilizations make all stages of development physiological data are not easily collected. accessible. 3. The embryo’s transparency allows real-time 3. Zebrafish behavior is less complicated imaging of the process of cardiogenesis. compared with mammalian models. 4. Zebrafish efficiently absorb small molecular 4. Zebrafish are less reliable where the weight compounds directly from water, which development of insoluble drugs is concerned. makes this organism suitable for chemical screens.

Mouse

1. It is the most common animal model used for 1. Embryogenesis occurs over a much longer research, as mice have many anatomic, period in utero. Therefore, it is much less biochemical, physiologic and pathologic similarities accessible for the study of heart development. with humans, and their genetic structure is 90% homologous with that of humans. 2. Behavioral analyses of mice are more developed 2. The costs of feeding mice and performing than those pertaining to zebrafish, and the large-scale screening tests are higher quantitative criteria are more complete. compared with invertebrates and teleosts. 3. The mouse model is more reliable were the 3. The experimental cycle of the mouse is development of insoluble drugs is concerned. long; therefore, it may not be the best choice for large-scale screening tests.

utilizing the zebrafish model may be undertaken in order to explore the mechanisms and signaling pathways of cardiogenesis and a variety of heart diseases. Compared with mammals, the zebrafish has an excellent capacity for heart regeneration, even following amputation of up to 20% of the ventricle [7]. This finding offers researchers a new angle from which to study heart regeneration following injury or chronic ischemia, painting a more optimistic

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picture regarding the treatment of heart diseases such as heart failure. Of course, this invertebrate model has its limitations as well. For example, the structure and function of the heart are much more different between human and zebrafish compared with other mammal models. And the research on insoluble drugs may not enjoy the advantages of this model for its single way of medication administration. However, zebrafish provides us with a convenient ２

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way to construct a physiological and pathological model and for large-scale drug screening. Further mammal models and clinical trials are needed to make it more convincible. On this background, we review the researches of latest decades on various zebrafish models, especially focusing on the cardiovascular system from the perspective of heart development, cardiovascular diseases as well as relative drug screening.

Zebrafish model in heart development Zebrafish have long been used as an animal model in heart development, because of their transparent larva, short developmental times, external fertilization and inexpensive experiments. Although zebrafish cardiomyocytes are phenotypically immature compared with mammalian models such as mice or rats [8], zebrafish have played an indispensable role in cardiovascular development and genetics since the 1980s [9]. A time-saving model of heart development Compared with mammalian models such as mice or rat models, zebrafish have a shorter time spans of embryonic heart development and represent a prototypical form of vertebrate organogenesis. The heart is the first organ to form and function during vertebrate embryo development [10], although the time span of embryonic heart development varies depending on the species [5]. In the mouse embryo, on embryonic day 6.5 (E6.5), cardiac progenitor cells migrate in an anterior–lateral direction beneath the head folds and form two groups of cells on either side of the midline, where cardiac markers are first detected, indicating the start of heart development. The valve, a structure that prevents blood reflux from the ventricle to the atrium, forms on E15, marking the maturation of the embryonic heart [11]. Five hours post fertilization (hpf), cardiac progenitor cells have moved into the lateral marginal zone. By 105 hpf, the endocardial cushions have enlarged and differentiated into valve leaflets [12,13]. Heart development in zebrafish may be regarded as a miniature version of the process that occurs in mammals, albeit in a shorter time span, which largely decreases the length of the testing period. Journal of Perioperative Science

Additionally, there is a period during the early developmental stages when the heart is functional but not yet essential. The first heartbeat in the zebrafish embryo occurs before the establishment of circulation. Embryonic development may last for several days without circulation as a result of the organism’s small body size and large surface area, which allows oxygen to diffuse into the body [14]. This unique feature enables the analysis of mutants with cardiac defects for considerable periods of time. Conversely, a similar phenotype in a mouse model would result in early lethality and embryo reabsorption [15,16,17]. A transparent model of the morphological development of the heart The stages of heart development In order to uncover the mechanisms of heart development, several animal models have been used over the past several decades. The mouse is an established model for studying heart development and is widely used as an animal model in both physiology and pathology [18]. Compared with invertebrate animal models such as Drosophila and C. elegans, the mouse offers the following two essential advantages: 1) it has a continuous endothelial lining within the heart and vessels, and 2) it has developed a second chamber in the heart that generates high systemic blood pressure [5]. However, to gain better insight into the structure of the embryonic heart and specific stages of organogenesis, mice must be dissected, and their hearts must be sectioned in order to determine phenotypes [19]. The heart of the zebrafish is composed of only a single atrium and ventricle. Because the embryos are completely transparent, its cardiovascular system may be observed directly under a dissecting microscope [20,21]. Therefore, the zebrafish has a prototypical vertebrate heart and is widely accepted as a low-cost model for the dynamic observation of the development of the cardiovascular system. In zebrafish, many processes associated with the early development of the mammalian heart appear to have been conserved. There are two heart fields during embryogenesis. Early cardiac progenitor cells within the anterior mesoderm form the primary heart field, and the secondary heart field is derived from the pharyngeal mesoderm, located medial and anterior to the cardiac crescent. The progenitor cells migrate to the midline of the embryo and fuse to ３

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form a linear heart tube, shaping a stent for further development, a process that includes cardiac looping, valve formation and development of the conduction system [5,10,22]. There are several differences in the development of zebrafish hearts compared with those of mammals. For example, 1) the zebrafish heart consists of four chambers, the sinus venosus, atrium, ventricle, and bulbus arteriosus, which are connected in series: 2) there are no septations in either the ventricle or the atrium: 3) the zebrafish has no lung circulation with which to exchange desaturated venous blood for saturated blood. Instead, it has gill arches that absorb oxygen dissolved in water. Therefore, the zebrafish bears only a prototypical vertebrae heart that is regarded as a miniature of early embryonic hearts. The signaling pathways and transcriptional regulation of cardiogenesis The genetic program of the zebrafish is more similar to that of mammals than it is to invertebrates. The complete sequence of the zebrafish genome is available, and many human genes are conserved structurally and functionally, including the genes involved in heart development. The formation of the vertebrate heart is temporally and spatially controlled by complicated signals, as well as crosstalk between molecules in the primary heart field and secondary heart field. The genes encoding transcriptional activators are activated by a series of inductive signals, transcriptional activators such as bone morphogenetic protein (BMP), Notch, WNT and sonic hedgehog (SHH) [23,24,25]. These transcriptional factors are key regulators of genes that connect upstream signaling to muscle-specific genes, as well as genes that encode proteins involved in heart development. Some factors are involved with only one heart field, whereas others are involved with both. The transcription factor Tbox 5 (TBX5) is expressed only in the primary heart field [26,27]. The protein product of TBX1 is a central regulator of the cardiac outflow tract in the secondary heart field. GATA4 and NKX2.5 (NK2 homeobox 5) are central transcription factors in the primary heart field and the secondary heart field [26,28,29]. A promising model of the physiological functions of the heart Cardiac conduction system (CCS) Using conserved genetic markers, Bjarke et al. found that the conduction system of zebrafish adults Journal of Perioperative Science

is strikingly similar to that of a mammalian embryo [6]. In order to generate a coordinated beat, the heart relies on a complicated network of cardiomyocytes known as the cardiac conduction system (CCS). The system is conserved in mammals and birds, as shown in Figure 3. The CCS is required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling [30]. As with most mammals, the electrical signal initiates in the zebrafish sinoatrial (SA) node and travels through the atrium or atria to the AV node, a specialized tissue at which electrical signaling slows dramatically. The ventricular fast conduction system conducts the signal directly to the apex before it travels to the rest of the ventricle [31]. The physiological development of the CCS in zebrafish was recently tracked using calcium-sensitive dyes, as well as optical mapping [32,33]. The regular conduction system appears as soon as the heart starts beating, at 24 hpf. At 40 hpf, an AV conduction delay between the two forming chambers is noted [34]. Additionally, experiments using optogenetics have revealed that pacemaker activity starts diffusely around the venous pole. At 3 dpf, it becomes restricted to a specific area within the dorsal right quadrant of the SA ring [35]. The ventricular conduction system develops later, at approximately 72 hpf. Cardiac trabeculae, which predominantly line the ventricular outer curvature, begin to form at 5 dpf [36]. Membrane potential is observed, and velocities are markedly faster in the outer curvature than in the inner curvature [37,38]. At 96 hpf, conduction waves clearly pass from the AVC to the trabeculae, initially propagating to the apex before traveling to the base of the heart, which the waves reach by 21 dpf [39]. It was recently reported that the overall shapes of zebrafish action potentials (APs) are similar to those of humans. As is the case in mammals, zebrafish demonstrate functional acetylcholine-activated K+ channels in the atrium but not in the ventricle. Additionally, the AP upstroke is dominated by Na+ channels: L-type Ca2+ channels contribute to the plateau phase, and IKr channels are involved in repolarization. On the other hand, important differences also exist between zebrafish and mammals. For example, zebrafish exhibit strong Ttype Ca2+ currents in both atrial and ventricular cardiomyocytes, whereas T-type Ca2+ channels are expressed only in the developing heart or under pathophysiological conditions in most mammals, ４

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suggesting that adult zebrafish cardiomyocytes exhibit a more immature phenotype [40]. Heart regeneration The adult mammalian heart has a low capacity for regeneration and repair. As a consequence, the loss of cardiomyocytes as a result of intrinsic or extrinsic stress is difficult to overcome and renders affected individuals more susceptible to heart failure [5]. By contrast, zebrafish possess a robust capacity for myocardial cell regeneration. The underlying mechanisms of this phenomenon have been studied extensively, with the ultimate goal of reactivating this evolutionarily lost mechanism in humans in order to facilitate cell based cardiac repair [7,41]. Evidence supports the idea that regeneration initially involves the stimulation of the cells of the epicardium (the outer layer of the heart) in response to injury, cells that undergo an epithelial-tomesenchymal transition (EMT) [42]. Within days of the removal of the ventricular apex, embryonic gene programs are induced in cells throughout the entire chamber, as opposed to cells surrounding the local wound. Epicardial cells from both chambers increase their expression of the retinoic acidsynthesizing enzyme, Raldh2, and the subepicardial muscle cells adjacent to them stimulate the expression of the gata4 transcription factor [42,29]. Kazu et al. reported that cardiomyocytes throughout the subepicardial ventricular layer trigger the expression of the embryonic cardiogenesis gene, gata4, within a week of trauma, and cardiomyocytes subsequently proliferate and surround the injury site. Additionally, electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury [43]. A recent study that combined fluorescent reporter transgenes, fate-mapping and ventricle-specific genetic ablation systems suggested that differentiated atrial cardiomyocytes transdifferentiate into ventricular cardiomyocytes and contribute to zebrafish cardiac ventricular regeneration. Notch signaling is activated within the atrial endocardium following ventricular ablation [29]. Another report noted that regenerated heart muscle cells appear to originate from the proliferation of differentiated cardiomyocytes under the control of the gene product of polo-like kinase 1 (plk1) [44]. All of these experiments support the idea that the myocardium is regenerated through the dedifferentiation and proliferation of pre-existing cardiomyocytes, rather than from a progenitor stem Journal of Perioperative Science

cell population [40,41,28]. In vitro experiments have confirmed the capacity of zebrafish cardiomyocytes to proliferate and differentiate [45]. Cell cycle regulators such as Mps1 (monopolar spindle protein 1, a mitotic checkpoint kinase) are upregulated during zebrafish cardiac regeneration [7]. The ligand fgf17b in the myocardium, as well as receptors fgfr2 and fgfr4 in adjacent epicardialderived cells, is induced during regeneration [42].

Zebrafish diseases

models

of

cardiovascular

In addition to its uses in embryonic development, the zebrafish model has been studied for its ability to model various human cardiovascular diseases, including congenital malformations and adult-onset diseases. Many mutations have been identified in homologues of known genes that result in structural and functional heart diseases [46], offering researchers the opportunity to determine the specific functions of each gene. Congenital heart disease (CHD) Congenital heart malformations are the most common form of human birth anomalies. During the past decade, research utilizing zebrafish, chick, and mouse models has uncovered many key genetic pathways that control early cardiac patterning and differentiation. The contributions of the zebrafish model have been comprehensively reviewed [47]. Cardiovascular mutant phenotypes, including those associated with CHD, have been constructed using zebrafish models. We offer a brief view using the phases of embryonic heart development. During cardiac specification and differentiation, the cloche mutant and the faust mutant have delimited anterior cardiac fields, which results in the reduction of cardiac precursors [48,49]. In another mutant, hand2, the progenitor cells that express nkx2.5 (a cardiac transcription factor gene) fail to successfully differentiate into mature myl7 (a cardiac regulatory myosin light chain gene), resulting in cardiac defects [50]. Later, as the heart tube forms, other mutants prevent normal heart development. For example, miles apart (mil) and two of heart (toh)/sphingolipid transporter (spns2) are two types of cardia bifida mutants that have negative effects on sphingolipid signaling during progenitor cell migration [51,52]. Although epithelial polarity mutants such as heart and soul (has/prkci) still migrate to the midline, they do not transform into a linear heart tube [53]. During ５

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cardiac looping and chamber formation, half-hearted (haf/vmhc) mutants lack ventricular contractility due to an inability to form ventricular sarcomeres. Similarly, weak atrium (wea/myh6) mutants lack atrial contractility [30]. Mutations occurring during the process of atrioventricular canal formation and valve development cause other problems. For example, the jekyll mutation, a mutation of the UDP-glucose dehydrogenase (udgh) gene, causes turbulence between the atrium and ventricle [54]. Additionally, cardiac contractility mutants such sih and cardiofunk (cfk) exhibit not only reduced cardiac contractility but also damaged AV valves due to improper valve development [55]. Each of the mutations mentioned above causes various malformations and types of dysfunction related to CHD. However, these mutations also play significant roles in the signaling pathways involved in heart development. Adult-onset heart diseases In contrast to zebrafish embryos that have been used to study congenital heart diseases, adult zebrafish have also been utilized to study adult-onset heart diseases. Ischemic cardiomyopathy Prolonged ischemia causes irreversible necrosis of the heart muscle. In the adult mammalian heart, ventricular cardiomyocytes have a limited capacity to divide and to replace ventricular myocardium lost due to ischemia-induced infarcts [56]. Therefore, the loss of cardiomyocytes due to ischemia frequently leads to heart failure and death. However, zebrafish myocardial cells have the capacity to regenerate following amputation of as much as 20% of the ventricle; therefore, the zebrafish has been used as a model of ischemic cardiomyopathy [7,41]. Hypoxia following ventricular amputation actually plays a positive role in zebrafish heart regeneration [57]. However, as is the case in humans, hypoxia and reoxygenation also harm cardiomyocytes by causing cardiac oxidative stress and inflammation, myocardial cell death and proliferation [58]. A zebrafish heart infarct model subjected to cryoinjury recently demonstrated that cardiac cells enter the cell cycle and invade the infarcted areas. Fibrotic scar tissue is gradually eliminated via cell apoptosis and replaced with new myocardium over a period of two months. The accumulation of Vimentin-positive fibroblasts and expression of an extracellular matrix Journal of Perioperative Science

protein, Tenascin-C, are each associated with myocardial regeneration within the myocardialinfarct border zone [59,60]. In a recent study by Kun Wang et al., a long non-coding RNA (lncRNA), cardiac apoptosis-related lncRNA (CARL), suppressed mitochondrial fission and apoptosis by targeting miR-539 and PHB2, suggesting a new approach by which to treat apoptosis and myocardial infarction [61]. Cardiomyopathy Using positional cloning, mutants of titin and tnnt2 were identified as the first two embryonic cardiomyopathy zebrafish models [62,63]. A series of loss-of-function studies of known cardiomyopathy genes were subsequently published, including studies of actn2, mlc, rlc, cypher, and mlp, underscoring the value of the zebrafish in annotating known causative genes of cardiomyopathy [64-67]. Notably, the cardiac ilk and nexilin zebrafish mutants prompted investigations of the corresponding human genes in order to identify novel DCM-causative genes [68,69]. In spite of the success of this endeavor, the intrinsic limitations of fish embryos prevent them from being used more extensively to study cardiomyopathy. First, their short developmental times prevent fish embryos from faithfully recapitulating the pathogenesis of cardiomyopathy, which is typically characterized by age-dependent penetrance and gradual progression to overt heart failure in adulthood. Second, many types of cardiomyopathy are caused either by haploinsufficiency or by gain-of-function mechanisms, mechanisms that cannot be modeled via complete gene depletion. Some known cardiomyopathy causative genes, such as mybpc3, do not result in embryonic phenotypes. In fact, it is estimated that only 5-10% of the genome exhibits embryonic lethal phenotypes upon depletion [70]. Therefore, we developed the first two adult zebrafish models of cardiomyopathy [71,72]. Via the detailed characterization of tr265, an anemia mutant caused by a defective band 3 gene [73], we reported that the high output stress exerted by chronic anemia on the heart induces significant enlargement of the ventricular chamber. Hallmarks of cardiomyopathy, including reduced ejection fraction, muscular disarray, and fetal gene reactivation, were noted [71,74]. At the cellular level, we detected both cardiomyocyte hypertrophy and activated cardiomyocyte proliferation. We also reported that the injection of doxorubicin (DOX), a ６

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widely used anti-cancer drug that causes cardiomyopathy in human cancer patients and rodent models [75,76], induced ventricular enlargement in adult zebrafish [72]. As was the case with the anemia model, hallmarks of cardiomyopathy were detected, as well as cardiomyocyte hypertrophy at the cellular level. However, we also noted the activation of cardiomyocyte apoptosis and unchanged cardiomyocyte proliferation, findings suggestive of a different mechanism of pathogenesis from that of the anemia model. Cardiac Arrhythmia Cardiac arrhythmia is a common cardiovascular disease and may result from either inherited or acquired factors. Among all the precipitating factors of cardiac arrhythmia, electrolyte disorders are an important cause. During calcium cycling, the increased calcium concentration in the cytosol drives the contraction of the embryonic heart. To prepare for the next contraction, calcium is extruded from the intracellular space to either the extracellular space via the Na+/Ca2+ exchanger (NCX1) or into the sarcoplasmic reticulum via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In the tremblor (tre) mutant, in which the NCX1 (NCX1h) gene is disrupted, the ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias related to severe disruptions in the sarcomere assembly [77,78]. The knockout of SERCA2 activity by either morpholino-mediated translational inhibition or pharmacological inhibition results in embryonic death due to defects in cardiac contractility and morphology, but not in arrhythmia. Askcnh2 encodes a channel responsible for the rapidly activating delayed rectifier K+ current (I Kr). The loss of functional I Kr in embryonic hearts leads to ventricular cell membrane depolarization, an inability to generate action potentials (APs), and disrupted calcium release [79]. HERG (ether-à-go-go-related gene) is a poreforming subunit of the rapidly activating delayed rectifier K+ channel. Studies of its orthologue, Erg, in zebrafish demonstrated an evolutionarily conserved role of Erg in regulating heart rate and rhythm, underscoring the reliability of zebrafish as a model for testing cardiotoxicity [80]. The Popeye domain containing (Popdc) gene family is expressed in the heart, and null mutations of members of this gene family in zebrafish result in atrioventricular block. This phenotype is reminiscent of sick sinus Journal of Perioperative Science

syndrome (SSS) [81]. The experimental downregulation of gene expression in zebrafish recently identified 20 genes at 11 loci that are relevant to heart rate regulation and cardiac dysfunction [82]. Many FDA approved drugs may have known or underlying side effects on heart function. Zebrafish are a useful model with which to assess these unwanted side effects. Drug effects on cardiac function, including heart rate, rhythmicity, contractility and circulation are visually assessed in zebrafish. Mitoxantrone, terfenadine, clomipramine and thioridazine elicit bradycardia, abnormal atrial and ventricular (AV) ratios, decreased contractility and slow circulation in zebrafish [83]. Nanoparticle treatments cause concentration-dependent toxicity, including pericardial edema and cardiac arrhythmia, whereas Ag+ ions and stabilizing agents do not cause significant defects in developing embryos. Transmission electron microscopy (TEM) demonstrates that nanoparticles are distributed in the brain, heart, yolk and blood of embryos [84]. QT prolongation has gradually become a leading cause of heart failure during drug development. Prolongation of the QT interval indicates prolongation of the AV duration in a significant number of ventricular myocytes, which is associated with an increased risk of Torsade de Pointes (TdP), a serious heart arrhythmia that often leads to death [81]. Milan et al. have developed an automated, high-throughput assay for bradycardia in zebrafish embryos that correlates with QT prolongation in humans. They found that 22 of 23 drugs that cause QT prolongation in humans result in bradycardia in zebrafish [85].

Zebrafish models in preclinical drugs screening related to the cardiovascular system The zebrafish has long been acted as a wellestablished vertebrate model for preclinical tests on various drugs in cardiovascular diseases [86]. Actually, this invertebrate model has been proved to be potential to predict adverse drug effects and play an active role in early safety assessment of novel drugs [87]. Variation of the heart rhythm is commonly seen in various drugs. The genetic tractability and powerful fertility of the zebrafish will allow the observation of the heart rhythm [88]. Scientists ７

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reported that 18 out of 23 drugs known to cause QT prolongation and torsades de pointes in man also caused bradycardia in 3 d.p.f. zebrafish, while the remaining 4 false-negative drugs were likely to cause bradycardia through microinjection into the yolk sac. Further experiments on astemizole showed the increase in QTc to be concentration dependent [89]. As far as contractility of the heart, it is also been explored in in vivo zebrafish models. Chemotherapeutics such as doxorubicin are widely used in the treatment of common malignancies with the side effect of dose-dependent cardiotoxicity, which can result in significant cardiomyopathy and even congestive heart failure [90]. Besides traditional chemotherapeutic drugs, targeted drugs are more and more applied in malignant diseases. Like anthracyclines, tyrosine kinase inhibitors are prone to cause cardiovascular disease [91]. Sunitinib and sorafenib are common tyrosine kinase inhibitors, which may cause ventricular dilation and impaired cardiac function in zebrafish larvae [92]. These two examples suggested the potential usage of the zebrafish in preclinical screening of drugs that might influence the contractility of the heart. As an economical and effective animal model, zebrafish plays a significant role in the drug development regarding to vascular diseases as well. LDN-193189 is an optimized analog of dorsomorphin (a small-molecule inhibitor of BMP signaling). It may prevent the development of atheroma in low-density lipoprotein (LDL)-receptordeficient mice [93,94]. In addition, other disease models were formed by the zebrafish, such as AV malformations and tumor neovascularization, which will no doubt facilitate the development of relative drugs [95]. Absolutely, most of the drugs on trial should be soluble and absorbed by the skin of zebrafish. For those insoluble ones, zebrafish may not be the first choice. And in further pharmaceutical researches, mammalian models as well as clinical researches are indispensable.

widely used as an animal model to study embryonic development, physiological functions, disease pathogenesis and drug development. On the other hand, the differences between zebrafish and humans should not be neglected and mammalian models should be applied in subsequent experiments. However, as researchers are equipped with a unique collection of sophisticated techniques, zebrafish will continue to serve as an important vertebrate model that will facilitate the development of new therapies for human cardiovascular diseases.

Summary

[7]

The zebrafish has attracted increasing amounts of attention from scientists due to its specific advantages over other vertebrate animals, including those offered by its transparent embryo during early embryogenesis, as well as its strong reproductive capacity. For these reasons, the zebrafish has been

[8]

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Competing Interests All the authors deny any competing interests.

Acknowledgements This work was supported by the National Institutes of Health of USA (NIH HL81753 and HL107304) and the National Natural Science Foundation of China (81270003, 81470390 and 81100826).

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