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Huo et al. J Perioperative Science 2015, 2:1
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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
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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.
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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
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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
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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,
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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
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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
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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
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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
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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
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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
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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]
Journal of Perioperative Science
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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