Download Fishing for the genetic basis of cardiovascular disease

PRIMER
Disease Models & Mechanisms 2, 18-22 (2009) doi:10.1242/dmm.000687
Fishing for the genetic basis of cardiovascular
disease
Disease Models & Mechanisms DMM
Tillman Dahme1, Hugo A. Katus1 and Wolfgang Rottbauer1,*
Cardiovascular disease (CVD) has recently overtaken infectious disease to become
the biggest global killer. Genetic factors have emerged as being of major
importance in the pathogenesis of CVD. Owing to disease heterogeneity, variable
penetrance and high mortality, human genetic studies alone are not sufficient to
elucidate the genetic basis of CVD. Animal models are needed to identify novel
genes that are involved in cardiovascular pathology and to verify the effect of
suspected disease genes on cardiovascular function. An intriguing model organism
is the zebrafish danio rerio. Several features of the zebrafish, such as a closed
cardiovascular system, transparency at embryonal stages, rapid and external
development, and easily tractable genetics make it ideal for cardiovascular
research. Moreover, zebrafish are suitable for forward genetics approaches, which
allow the unbiased identification of novel and unanticipated cardiovascular genes.
Zebrafish mutants with various cardiovascular phenotypes that closely correlate
with human disease, such as congenital heart disease, cardiomyopathies and
arrhythmias, have been isolated. The pool of zebrafish mutants, for which the causal
gene mutation has been identified, is constantly growing. The human orthologues
of several of these zebrafish genes have been shown to be involved in the
pathogenesis of human CVD. Cardiovascular zebrafish models also provide the
opportunity to develop and test novel therapeutic strategies, using innovative
technologies such as high throughput in vivo small molecule screens.
Cardiovascular disease (CVD) is the most
common cause of death worldwide (Lopez
et al., 2006). When CVD involves structural
heart development it is called congenital
heart disease, a condition that, today, is usually diagnosed before or shortly after birth.
If cardiac function is affected, the disease is
classified either as cardiomyopathy affecting contractile function or as arrhythmia,
subsuming conditions with altered electrical excitability or conductance that lead to
disturbed heart rhythm. CVD affecting cardiovascular function can arise at all ages. A
substantial part of CVD is either directly
caused by gene mutations (monogenic), or
is at least modified by genetic variation
(polygenic, multifactorial). However, human
genetics has not been able to elucidate fully
the genetic basis of CVD owing to disease
heterogeneity, variable penetrance, often a
late onset of symptoms and high mortality,
1
all of which inhibit family linkage analyses.
This underscores a need for in vivo animal
models for CVD, which could help to overcome this dilemma.
Over the last decade, the zebrafish has
become a well-established model organism
in biomedical research. As a vertebrate, it
depends on a closed cardiovascular system,
unlike other genetic model organisms such
as worms and flies. In zebrafish, venous
blood is collected in the sinus venosus,
from where it flows into the atrium and then
through the atrioventricular canal into the
ventricle before being pumped through the
bulbus arteriosus into the ventral aorta.
Since fish do not have lungs, there is no pulmonary circulation; instead, part of the
blood from the ventral aorta flows through
the branchial arches, where oxygen is taken
up by the gills (Fig. 1). The advantages of
the zebrafish over mammals as a model
Department of Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
*Author for correspondence (e-mail: [email protected])
18
organism include its external development,
the large number of progeny and their rapid
development, and the ease of genetic
manipulation. Additional features of the
zebrafish make it an intriguing model
organism, especially for cardiovascular
research. First, zebrafish embryos are transparent, which allows direct observation of
the beating heart and circulating blood in
vivo by simple light microscopy. Second,
during the first week of zebrafish development, sufficient oxygen can be delivered by
diffusion enabling even those zebrafish with
severe cardiovascular abnormalities to
develop throughout embryogenesis and into
the larval stages. Several techniques allow
for detailed and quantitative phenotyping of
zebrafish heart mutants, such as determination of the cardiac ventricular and atrial
shortening fraction, analysis of blood flow
velocity and the recording of electrocardiograms (Fig. 2). Histology, histochemistry, ultrastructural analyses by electron
microscopy, and whole-mount antisense
RNA in situ hybridization are just a few
more examples of methods that can be
applied to obtain a detailed characterization
of zebrafish mutants. Moreover, the generation of transgenic zebrafish lines expressing fluorochromes under the control of various promoters has launched the novel field
of whole-animal in vivo fluorescence
microscopy.
Saturation mutagenesis screens have
been conducted in zebrafish since 1996 and
have led to a wealth of heart mutants, several of which have already been characterized in detail and positionally cloned.
Reverse genetics by targeted gene inactivation using morpholino-modified antisense
oligonucleotides has added many more in
vivo models, which are now being used to
understand cardiovascular disease genes.
Several approaches, such as the establishment of tilling libraries by ENU-mutagenesis (Sood et al., 2006), viral insertional
mutagenesis (Amsterdam and Hopkins,
2004) and, very recently, the targeted inactivation of genes using engineered zinc-findmm.biologists.org
PRIMER
Disease Models & Mechanisms DMM
Zebrafish as a model for CVD
Fig. 1. Zebrafish structural characterization. (A) Zebrafish embryo at 72 hours postfertilization (hpf).
Owing to its transparency, the heart can be found just anterior to the yolk sac. (B) The heart of a zebrafish
embryo visualized at 72 hpf by simple light microscopy. An atrial and a ventricular chamber can be seen.
The heart chambers are filled with blood, mainly made up by erythrocytes. The outlines of the atrium and
the ventricle are marked with a dotted line for better visualization. (C) In a sagittal histologic section, the
atrioventricular (AV)-canal can be seen connecting the atrial and ventricular chambers.
ger nucleases (Doyon et al., 2008; Meng et
al., 2008), are adding to the diversity of genespecific zebrafish models.
Here, we focus on zebrafish models of
human CVD that are derived from chemical mutagenesis screens. One important
aspect about forward genetics is that it
is an unbiased, non-hypothesis-driven
approach. Many genetic pathways that are
important in cardiac pathology have been
revealed by zebrafish mutants that might
not otherwise have been suspected to contribute to heart disease. Some of the genes
identified in zebrafish heart mutants have
already been confirmed in human genetics
studies, in a case-control design, by candidate gene approach. However, one has to
keep in mind that zebrafish models usually
carry recessive mutations, whereas the
majority of inheritable CVDs are transmitted in humans by an autosomal dominant
mode of inheritance, meaning that the
zebrafish phenotypes may differ substantially from the corresponding human disease phenotype. As a result, the zebrafish
phenotype provides a hint towards the diseased module (heart development, heart
function, heart rhythm), rather than a direct
translation of the human disease.
Congenital heart disease
With a frequency of nearly 1%, heart defects
are the most common congenital anomalies
Disease Models & Mechanisms
in humans. Siblings have a high probability
of sharing congenital heart disease, indicating a genetic contribution, but in most
cases there is no Mendelian inheritance.
The zebrafish offers the possibility to dissect pathways controlling heart development; components of these pathways can
then be analyzed in human patients.
For example, the zebrafish mutant heartstrings (hst) is caused by a mutation in the
transcription factor tbx5, which leads to
abnormalities in cardiac differentiation and
fin development and, when mutated in
humans, causes Holt-Oram syndrome with
cardiac septation defects and limb abnormalities (Garrity et al., 2002; Mori and
Bruneau, 2004). This shows that even
though zebrafish do not have an atrial or
ventricular septum, both of which are
affected in the human disease, the respective gene still fulfills essential functions in
zebrafish heart development.
slipjig (sli) mutants harbor a mutation in
the zebrafish homologue of the transcription factor Foxn4, and display structural
atrioventricular canal malformation accompanied by atrioventricular conduction
defects. Foxn4 is expressed in the atrioventricular canal and binds to a tbx2 enhancer
domain to drive transcription of tbx2b in the
atrioventricular canal (Chi et al., 2008).
Atrioventricular canal defects are rather
frequent in humans but, genetically, are
poorly understood. Future studies will show
whether Foxn4 also plays a role in human
atrioventricular canal defects.
One particularly interesting zebrafish
mutant is gridlock (grl), which carries a
mutation in the hey2 gene leading to
impaired blood flow in the tail owing to
arterial blockade in the anterior trunk.
This makes it a suitable model for the
human congenital disease aortic coarctation, which is characterized by severe
stenosis or obliteration of the distal aortic
arch around the insertion of the ductus
arteriosus (Weinstein et al., 1995). Most
importantly, grl was the first zebrafish
mutant to be successfully used in an in vivo
small-molecule screen. In this screen, a
class of compounds was identified that
suppress the coarctation phenotype by
upregulation of vascular endothelial
growth factor (VEGF) (Peterson et al.,
2004). This approach offers tremendous
opportunities for future drug development
to treat human heart disease.
Cardiac muscle cells in humans are postmitotic and cannot be regenerated adequately if lost through events such as
myocardial infarction. A growing field in
cardiology focuses on the regeneration of
functional cardiac myocytes from cardiac
stem cells through transdifferentiation, or
by inducing cardiac muscle cells to re-enter
the cell cycle. Interestingly, zebrafish hearts
are able to regenerate after injuries (Poss et
al., 2002). The zebrafish mutant island beat
(isl), which harbors a mutation in the alpha1
C L-type calcium channel subunit (CLTCC) leading to a silent ventricle as a result
of impaired calcium signaling, fails to add
an adequate number of cells to the cardiac
ventricle, causing the ventricles to be
hypoplastic (Rottbauer et al., 2001). Another
pathway controlling cardiac growth was
discovered with the help of the zebrafish
mutant liebeskummer (lik). Reptin, a transcriptional co-repressor of β-catenindependent transcription, is activated in lik
mutants leading to augmentation of cardiac
myocyte numbers, so-called cardiac hyperplasia. Reptin signaling is antagonized by
the transcriptional cofactor pontin, which
regulates β-catenin signaling in cardiomyocytes (Rottbauer et al., 2002). Aside from
the coarctation phenotype described above,
grl mutants have also been shown to
develop hyperplastic hearts. Gridlock, a
basic helix-loop-helix transcription factor,
forms a complex with the cardiac tran19
Disease Models & Mechanisms DMM
PRIMER
Zebrafish as a model for CVD
Fig. 2. Zebrafish functional characterization. (A) Schematic illustration of a zebrafish heart. (B) Onedimensional section [(see red bar in (A)] through the cardiac ventricle (ordinate), based on a video
recording of a zebrafish heart at 72 hpf plotted over time (abscissa). The resulting image is similar to an
M-mode echocardiogram and allows accurate determination of ventricular fractional shortening.
(C) Ventricular fractional shortening (FS) of wild-type zebrafish embryos and heart failure mutants at the
indicated time points. (D) Electrocardiogram (ECG) of an adult wild-type zebrafish. The P wave, resulting
from atrial excitation, is followed by the QRS complex, which represents ventricular depolarization, and
the T wave, representing ventricular repolarization.
scription factor Gata5 and represses Gata5
transcriptional activity leading to inhibition
of myocardial proliferation (Jia et al., 2007).
By contrast, the hearts of the zebrafish
mutants heart of glass (heg), santa (san) and
valentine (val) do not show concentric
growth because they fail to add additional
myocardial cell layers, even though the total
cell number is not altered. As a result, these
mutants have hypocontractile, monolayered, giant cardiac ventricles (Mably et al.,
2003; Mably et al., 2006). Further dissection
of these pathways could allow us to reverse
the final exit of cardiac myocytes from the
cell cycle and help us to engineer functional
neo-myocardium.
Cardiomyopathies
Human cardiomyopathies are diseases that
primarily affect the myocardium. The two
most prevalent forms are dilated cardiomyothapy (DCM) and hypertrophic cardiomyopathy (HCM). Dilated cardiomyopathy is a condition that, in humans, is
characterized by decreasing cardiac pump
function, leading to congestive heart failure.
HCM is defined as hypertrophy of the
myocardium in the absence of any other diseases that cause myocardial hypertrophy,
20
such as high blood pressure or storage diseases. Whereas HCM is caused exclusively
by genetic mutations, DCM is considered
to be multifactorial with a strong genetic
influence. Several zebrafish mutants have
been identified that display heart phenotypes resembling the features of the human
disease.
In the zebrafish mutant dead beat (ded),
cardiac contractility initially resembles that
of wild-type fish, but then decreases from
48-60 hpf. The gene responsible for this
phenotype is the zebrafish homologue of
phopholipase C gamma 1 (PLCG1). PLCG1
is known to associate with the VEGFreceptor Flt-1 in endothelial cells. Interestingly, using the zebrafish mutant ded,
this pathway is now known to be crucial
for cardiac pump function in cardiac
myocytes in a cell-autonomous fashion.
Conservation of this pathway throughout
evolution into mammals was confirmed
using cultured rat cardiac myocytes (Rottbauer et al., 2005).
One mechanism allowing for the development of DCM is defective cardiac stretch
sensing (Knoll et al., 2002). The zebrafish
mutant main squeeze (msq) is characterized
by a loss of cardiac contractility from 60-
72 hpf. Positional cloning identified a missense mutation in the homologue of the
integrin-linked kinase (ilk) gene, leading to
loss of kinase activity and failure to bind to
the ILK-associated protein β-parvin/affixin.
Loss of contractility in msq mutant
zebrafish is preceded by a loss of atrial
natriuretic factor (anf) expression, a gene
that is known to be expressed in a stretchdependent manner. This led to the hypothesis that ILK might be part of a potential
cardiac stretch sensor that recognizes an
increase in preload by augmented stretching of the heart, and then transmits these
signals to the contractile apparatus in order
to increase contractility (Bendig et al.,
2006). Indeed, based on the msq findings,
ILK mutations have been identified in
human patients suffering from DCM,
demonstrating the usefulness of the unbiased, zebrafish forward genetics approach
for the identification of truly novel candidate genes for human disease (Knoll et al.,
2007; Dahme et al., 2008).
Several zebrafish mutants have been
identified that never establish normal ventricular contractility and, when analyzed
ultrastructurally, do not form cardiac sarcomeres. pickwick (pik) heart failure
mutants harbor a mutation in a cardiacspecific exon of the gene encoding the
giant filament protein titin. Mutations in
the cardiac-specific exon of titin are also
found in human patients suffering from
DCM (Gerull et al., 2002; Xu et al., 2002).
The mutant tell tale heart (tel), which has
a similar phenotype to the pik mutant, also
fails to assemble functional cardiac sarcomeres and has been shown to be caused
by a mutation in the cardiac regulatory
myosin light chain gene mlc2 (Rottbauer
et al., 2006). Mutations in the human gene
encoding ventricular MLC-2 (MYL2) are
found in patients suffering from HCM, a
condition were cardiac myocytes are
enlarged leading to thickening of the cardiac ventricular walls. Indeed, HCM is
considered to be mainly a disease of inefficient sarcomerogenesis, because most
mutations found in human HCM patients
affect genes encoding sarcomeric proteins.
silent heart (sil) is another example of a
mutant that displays ventricular acontractility owing to impaired myofibrillogenesis; sil harbors a mutation in the cardiac
troponin T gene (Sehnert et al., 2002). Cardiac Troponin T (TNNT2) mutations are
also a major cause of HCM in humans.
dmm.biologists.org
PRIMER
Zebrafish as a model for CVD
Disease Models & Mechanisms DMM
Arrhythmias
Arrhythmias are defined as diseases with an
abnormal heart rhythm. A major limitation
for arrhythmia research, in vivo, is the difference in the molecular underpinnings of
the cardiac action potential between
humans and classic model organisms, such
as mice. Mice have a heart rate of 300-600
beats per minute (bpm), whereas human
hearts have a rate of 60-100 bpm. As a consequence, much of the heart cycle, especially repolarization – meaning the reestablishment of resting conditions, is
accomplished by a completely different set
of ion channels in humans compared with
in mice. Interestingly, zebrafish have a heart
rate of 80-160 bpm and repolarization is
achieved by similar mechanisms as in
humans. As zebrafish embryos are transparent, arrhythmias can be detected by simple observation of the heartbeat. For example, atrioventricular block, where only every
second or third atrial contraction is conducted to the ventricle or where atrioventricular conduction is completely blocked,
can be observed in the embryos. Another
common arrhythmia phenotype, and the
most common arrhythmia in humans, is
atrial fibrillation, which is characterized by
uncoordinated contraction of atrial cardiac
myocytes.
Two zebrafish mutants have been isolated that harbor mutations in the main
repolarizing ion channel gene kcnh2 (erg).
A mutant with complete loss of kcnh2 function displays complete atrioventricular
block and ventricular asystole (Arnaout et
al., 2007). This mutant has been proposed
as a model for human long QT syndrome,
a repolarization disorder associated with
sudden cardiac death. By contrast, reggae
(reg) mutants have a kcnh2 gain-of-function
mutation leading to accelerated repolarization and, thus, can be considered a model
for human short QT syndrome (Hassel et
al., 2008). Interestingly, reg mutants present
with paroxysmal atrial fibrillation, a condition that has been found in human short QT
syndrome (Schimpf et al., 2008). Slow mo
(smo) is a mutant that is characterized by
bradycardia (a slow heart rate). The underlying mechanism is severe reduction in the
cardiac pacemaker ‘funny current’, If (Baker
et al., 1997; Warren et al., 2001). Interestingly, an If-channel blocker (ivabradin),
which leads to a reduction of the If-current,
has been introduced recently into clinical
practice to treat conditions where tachyDisease Models & Mechanisms
Advantages of the
zebrafish as a model for
cardiovascular disease
• Zebrafish have a closed
cardiovascular system and a cardiac
cycle that is highly reminiscent of
human physiology
• Zebrafish embryos develop externally
and are transparent, enabling the
analysis of cardiac morphology and
dynamics
• Zebrafish embryos develop rapidly
and receive a sufficient supply of
oxygen and nutrients by diffusion,
thus even severe cardiac defects
remain sublethal throughout
embryogenesis and early larval
stages
• Zebrafish have very tractable genetics
and their genome has been
sequenced
cardia (a fast heart rate) is undesirable.
Interestingly, unlike most zebrafish
mutants, reg and smo mutants can survive
into adulthood allowing additional experimental approaches to be carried out in
these zebrafish, such as ECG recordings,
isolation of cardiomyocytes and determination of action potentials – all of which are
more easily achieved in adult zebrafish than
in embryonal zebrafish. Another zebrafish
arrhythmia mutant is tremblor (tre), which
harbors a mutation in the gene that encodes
the cardiac-specific sodium-calciumexchanger (NCX) and leads to defective calcium extrusion during diastole and a subsequent intracellular calcium overload and
atrial fibrillation (Ebert et al., 2005; Langenbacher et al., 2005). Zebrafish island
beat (isl) mutants have defective L-type calcium currents which, aside from the
reduced number of ventricular cardiac
myocytes, also leads to atrial fibrillation
(Rottbauer et al., 2001). Zebrafish mutants
with atrial fibrillation have mutations in various genes that are involved in ion transport
and, therefore, recapitulate the complexity
of human disease, where mutations in multiple genes are known to contribute to atrial
fibrillation (Tsai et al., 2008).
Conclusion
Several hundred cardiac mutants have been
identified through various zebrafish muta-
genesis screens. The saturation mutagenesis approach, which proposes that every
gene of the zebrafish genome should be
altered for analysis, suggests that the genetic
basis of cardiovascular function could be
completely dissected in a simplified system.
As discussed here, many of the zebrafish
gene programs are conserved between
species and the essential genes are also candidates for human CVD. Eventually, cardiac
genes that have been recognized in zebrafish
will be analyzed, either by direct sequencing or by single nucleotide polymorphism
(SNP)-based whole genome analysis. Small
molecule screens and similar techniques
should provide a detailed molecular understanding of the underlying pathophysiology
of human CVD and help in the development
of novel therapies. So, let’s keep fishing for
novel cardiovascular disease genes!
COMPETING INTERESTS
The authors declare no competing financial interests.
REFERENCES
Amsterdam, A. and Hopkins, N. (2004). Retroviralmediated insertional mutagenesis in zebrafish.
Methods Cell Biol. 77, 3-20.
Arnaout, R., Ferrer, T., Huisken, J., Spitzer, K., Stainier,
D. Y., Tristani-Firouzi, M. and Chi, N. C. (2007).
Zebrafish model for human long QT syndrome. Proc.
Natl. Acad. Sci. USA 104, 11316-11321.
Baker, K., Warren, K. S., Yellen, G. and Fishman, M. C.
(1997). Defective “pacemaker” current (Ih) in a
zebrafish mutant with a slow heart rate. Proc. Natl.
Acad. Sci. USA 94, 4554-4559.
Bendig, G., Grimmler, M., Huttner, I. G., Wessels, G.,
Dahme, T., Just, S., Trano, N., Katus, H. A., Fishman,
M. C. and Rottbauer, W. (2006). Integrin-linked
kinase, a novel component of the cardiac mechanical
stretch sensor, controls contractility in the zebrafish
heart. Genes Dev. 20, 2361-2372.
Chi, N. C., Shaw, R. M., De Val, S., Kang, G., Jan, L. Y.,
Black, B. L. and Stainier, D. Y. (2008). Foxn4 directly
regulates tbx2b expression and atrioventricular canal
formation. Genes Dev. 22, 734-739.
Dahme, T., Weichenhan, D., Grimmler, M., Meder, B.,
Hassel, D., Just, S., Hess, A., Hartkopf A. D., Vogel,
B., V., Katus, H. A. et al. (2008). Mutationen des
integrin-linked kinase/β-parvin - proteinkomplexes
führen durch gestörte kardiomyozytäre
dehnungswahrnehmung zu dilatativer
kardiomyopathie. Clin. Res. Cardiol. 97 Suppl. 1, P225.
Doyon, Y., McCammon, J. M., Miller, J. C., Faraji, F.,
Ngo, C., Katibah, G. E., Amora, R., Hocking, T. D.,
Zhang, L., Rebar, E. J. et al. (2008). Heritable targeted
gene disruption in zebrafish using designed zincfinger nucleases. Nat. Biotechnol. 26, 702-708.
Ebert, A. M., Hume, G. L., Warren, K. S., Cook, N. P.,
Burns, C. G., Mohideen, M. A., Siegal, G., Yelon, D.,
Fishman, M. C. and Garrity, D. M. (2005). Calcium
extrusion is critical for cardiac morphogenesis and
rhythm in embryonic zebrafish hearts. Proc. Natl. Acad.
Sci. USA 102, 17705-17710.
Garrity, D. M., Childs, S. and Fishman, M. C. (2002).
The heartstrings mutation in zebrafish causes
heart/fin Tbx5 deficiency syndrome. Development
129, 4635-4645.
21
Disease Models & Mechanisms DMM
PRIMER
Gerull, B., Gramlich, M., Atherton, J., McNabb, M.,
Trombitas, K., Sasse-Klaassen, S., Seidman, J. G.,
Seidman, C., Granzier, H., Labeit, S. et al. (2002).
Mutations of TTN, encoding the giant muscle filament
titin, cause familial dilated cardiomyopathy. Nat.
Genet. 30, 201-204.
Hassel, D., Scholz, E. P., Trano, N., Friedrich, O., Just,
S., Meder, B., Weiss, D. L., Zitron, E., Marquart, S.,
Vogel, B. et al. (2008). Deficient zebrafish ether-a-gogo-related gene channel gating causes short-QT
syndrome in zebrafish reggae mutants. Circulation
117, 866-875.
Jia, H., King, I. N., Chopra, S. S., Wan, H., Ni, T. T.,
Jiang, C. C., Guan, X., Wells, S., Srivastava, D.,
Zhong, T. P. (2007). Vertebrate heart growth is
regulated by functional antagonism between Gridlock
and Gata5. Proc. Natl. Acad. Sci. USA 104, 14008-14013.
Knoll, R., Hoshijima, M., Hoffman, H. M., Person, V.,
Lorenzen-Schmidt, I., Bang, M. L., Hayashi, T.,
Shiga, N., Yasukawa, H., Schaper, W. et al. (2002).
The cardiac mechanical stretch sensor machinery
involves a Z disc complex that is defective in a subset
of human dilated cardiomyopathy. Cell 111, 943-955.
Knoll, R., Postel, R., Wang, J., Kratzner, R., Hennecke,
G., Vacaru, A. M., Vakeel, P., Schubert, C., Murthy,
K., Rana, B. K. et al. (2007). Laminin-alpha4 and
integrin-linked kinase mutations cause human
cardiomyopathy via simultaneous defects in
cardiomyocytes and endothelial cells. Circulation 116,
515-525.
Langenbacher, A. D., Dong, Y., Shu, X., Choi, J., Nicoll,
D. A., Goldhaber, J. I., Philipson, K. D. and Chen, J.
N. (2005). Mutation in sodium-calcium exchanger 1
(NCX1) causes cardiac fibrillation in zebrafish. Proc.
Natl. Acad. Sci. USA 102, 17699-17704.
Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T.
and Murray, C. J. (2006). Global and regional burden
22
Zebrafish as a model for CVD
of disease and risk factors, 2001, systematic analysis of
population health data. Lancet 367, 1747-1757.
Mably, J. D., Mohideen, M. A., Burns, C. G., Chen, J. N.
and Fishman, M. C. (2003). heart of glass regulates
the concentric growth of the heart in zebrafish. Curr.
Biol. 13, 2138-2147.
Mably, J. D., Chuang, L. P., Serluca, F. C., Mohideen,
M. A., Chen, J. N. and Fishman, M. C. (2006). santa
and valentine pattern concentric growth of cardiac
myocardium in the zebrafish. Development 133, 31393146.
Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D. and
Wolfe, S. A. (2008). Targeted gene inactivation in
zebrafish using engineered zinc-finger nucleases. Nat.
Biotechnol. 26, 695-701.
Mori, A. D. and Bruneau, B. G. (2004). TBX5 mutations
and congenital heart disease: Holt-Oram syndrome
revealed. Curr. Opin. Cardiol. 19, 211-215.
Peterson, R. T., Shaw, S. Y., Peterson, T. A., Milan, D. J.,
Zhong, T. P., Schreiber, S. L., MacRae, C. A. and
Fishman, M. C. (2004). Chemical suppression of a
genetic mutation in a zebrafish model of aortic
coarctation. Nat. Biotechnol. 22, 595-599.
Poss, K. D., Wilson, L. G. and Keating, M. T. (2002).
Heart regeneration in zebrafish. Science 298, 21882190.
Rottbauer, W., Baker, K., Wo, Z. G., Mohideen, M. A.,
Cantiello, H. F. and Fishman, M. C. (2001). Growth
and function of the embryonic heart depend upon
the cardiac-specific L-type calcium channel alpha1
subunit. Dev. Cell 1, 265-275.
Rottbauer, W., Saurin, A. J., Lickert, H., Shen, X.,
Burns, C. G., Wo, Z. G., Kemler, R., Kingston, R., Wu,
C. and Fishman, M. (2002). Reptin and pontin
antagonistically regulate heart growth in zebrafish
embryos. Cell 111, 661-672.
Rottbauer, W., Just, S., Wessels, G., Trano, N., Most, P.,
Katus, H. A. and Fishman, M. C. (2005). VEGF-
PLCgamma1 pathway controls cardiac contractility in
the embryonic heart. Genes Dev. 19, 1624-1634.
Rottbauer, W., Wessels, G., Dahme, T., Just, S., Trano,
N., Hassel, D., Burns, C. G., Katus, H. A. and
Fishman, M. C. (2006). Cardiac myosin light chain-2: a
novel essential component of thick-myofilament
assembly and contractility of the heart. Circ. Res. 99,
323-331.
Schimpf, R., Borggrefe, M. and Wolpert, C. (2008).
Clinical and molecular genetics of the short QT
syndrome. Curr. Opin. Cardiol. 23, 192-198.
Sehnert, A. J., Huq, A., Weinstein, B. M., Walker, C.,
Fishman, M. and Stainier, D. Y. (2002). Cardiac
troponin T is essential in sarcomere assembly and
cardiac contractility. Nat. Genet. 31, 106-110.
Sood, R., English, M. A., Jones, M., Mullikin, J., Wang,
D. M., Anderson, M., Wu, D., Chandrasekharappa,
S. C., Yu, J., Zhang, J. et al. (2006). Methods for
reverse genetic screening in zebrafish by
resequencing and TILLING. Methods 39, 220-227.
Tsai, C. T., Lai, L. P., Hwang, J. J., Lin, J. L. and Chiang,
F. T. (2008). Molecular genetics of atrial fibrillation. J.
Am. Coll. Cardiol. 52, 241-250.
Warren, K. S., Baker, K. and Fishman, M. C. (2001). The
slow mo mutation reduces pacemaker current and
heart rate in adult zebrafish. Am. J. Physiol. Heart Circ.
Physiol. 281, H1711-H1719.
Weinstein, B. M., Stemple, D. L., Driever, W. and
Fishman, M. C. (1995). Gridlock, a localized heritable
vascular patterning defect in the zebrafish. Nat. Med.
1, 1143-1147.
Xu, X., Meiler, S. E., Zhong, T. P., Mohideen, M.,
Crossley, D. A., Burggren, W. W. and Fishman, M. C.
(2002). Cardiomyopathy in zebrafish due to mutation
in an alternatively spliced exon of titin. Nat. Genet. 30,
205-209.
dmm.biologists.org