Atrial ERK1/2 activation in the embryo leads to incomplete Septal closure: a novel mouse model of atrial Septal defect
Yeh et al. Journal of Biomedical Science
Atrial ERK1/2 activation in the embryo leads to incomplete Septal closure: a novel mouse model of atrial Septal defect
Che-Chung Yeh 0 2
Yanying Fan 0 2
Yi-Lin Yang 2
Michael J. Mann 1 2
0 Equal contributors
1 Division of Cardiothoracic Surgery , 500 Parnassus Avenue, Suite W420, San Francisco, CA 94143 , USA
2 Cardiothoracic Translational Research Laboratory, University of California, San Francisco , San Francisco, CA , USA
Background: MEK1 mutation and activated MAPK signaling has been found in patients with RASopathies and abnormal cardiac development. Previous studies have suggested that regulation of fetal MAPK signaling is essential for normal cardiac development. We investigated the effect of active MEK1 overexpression on fetal atrial septal development. Methods and results: An inducible double transgenic (DTg) mouse model was developed in which cardiacspecific fetal expression of a constitutively active form of human MEK1 (aMEK1) was induced primarily in the atrium via the withdrawal of doxycycline from the drinking water of pregnant mice. Atrial septal defect (ASD) was found in 51% (23/45) of DTg mice. Fifty-two percent (12/23) of ASD mice died before weaning, and surviving ASD mice exhibited hypertrophic hearts with enlarged right atria and decreased fractional shorting (40 ± 2% vs. 48 ± 0%, p < 0. 05). The model mimicked human ASD in several key clinical features: severe ASD was associated with growth impairment; ASD-specific mortality was highest within the early postnatal period; despite an even distribution of ASD among the sexes, early mortality was significantly higher in males. The expression of aMEK1 and increased phosphorylation of ERK1/2 was documented via Western blot in DTg fetal hearts, with the largest increases seen in atrial tissue. In an alternative transgenic aMEK1 model with elevated atrial MKP3 expression and corresponding suppression of increases in ERK1/2 phosphorylation, animals did not develop ASD. Conclusion: This new model of ASD suggests that enhanced atrial MEK1-ERK1/2 signaling during fetal development disrupts normal atrial septation, possibly regulated by the balance of ERK1/2 phosphorylation.
Atrial septal defect; RASopathies; Mitogen-activated protein kinase; MAP kinase phosphatase; Mitogenactivated protein kinase pathway
Atrial septal defects (ASD) are among the most common
types of congenital heart disease (CHD), with an
estimated incidence of 56–100 per 100,000 live births [
While mutations in GATA4, MYH6, NKX2–5 and TBX5
genes have been linked to the abnormal septation of
atrial chambers, most patients with ASD are diagnosed
without known etiologic causes [
]. However, the risk
of a secundum defect is increased in siblings of ASD
patients, suggesting that an inherited molecular
mechanism may play a role in abnormal atrial septation.
The function of MEK1 signaling in the adult heart has
been associated with “physiologic” hypertrophy [
Overexpression of active MEK1 induces hypertrophic
changes in adult left ventricular (LV) structure that
increase cardiac function and that do not degenerate into
]. Increased MEK1 activity also
protects hearts under stress conditions such as
ischemiareperfusion and myocardial infarction [
]. In contrast
to this positive influence of MEK1 activity in adult
hearts, recent advances in the study of RASopathies have
demonstrated a potentially detrimental role for
Ras-RafMEK-ERK signaling during embryonic/fetal heart
development . RASopathies occur as a cluster of
syndromes with germline mutations in genes
participating in the Ras-Raf-MEK-ERK kinase signaling pathway.
Independent of mutations and other syndromes, CHD
including ASD - are common in RASopathies patients
and are a major source of morbidity and even mortality
]. Although mutations in human MEK1 genes have
been identified in RASopathies, and the early application
of a MEK1 inhibitor in animal models has been able to
ameliorate associated defects [
], there is no direct
evidence indicating how MEK1 may contribute to CHD.
We observed a very high incidence of ASD in double
transgenic (DTg) mice in which expression of a
constitutively active isoform of human MEK1 (aMEK1) regulated
by an α-myosin heavy chain (αMHC) promoter was
induced during fetal development. Although the αMHC
promoter is generally used as a means to induce
myocardial-specific gene expression in adult hearts, this
promoter is also active in the embryonic/fetal heart with
higher activity in the atrial region [
]. No previous
model of relatively atrial-specific overexpression of
aMEK1 in the developing fetus has been reported, nor
are there reproducible, specific model.s of congenital
ASD that closely mimic the human clinical scenario.
Hemagglutinin (HA)-tagged, constitutively active human
MEK1 (aMEK1) cDNA, kindly provided by Dr. Natalie
Ahn, was subcloned into the pTet-Splice vector
(Invitrogen Corp.) for most of the experimentation reported, and
also into the pTREtight vector (Clontech, Mountain View,
CA) for validation of the transgenic model. The aMEK1
expression DNA cassettes were excised from both the
pTet-Splice vector and the pTREtight vector, and each
was used for pronuclear injection to generate founders
carrying tetracycline responsive element regulated
expression of aMEK1 gene (TAMEK). Genotyping was done by
PCR using Primers
5′-GAGCTGGGGGCTGGCAATGG3′ and 5′-CCTTGGCCTGGGTGGGGTCT-3′. Positive
founders derived from each of the vectors were bred with
C57BL/6 mice to obtained stable TAMEK transgenic (Tg)
lines. The cardiac specific expression of
tetracyclineresponsive transcriptional activator (tTA) αMHC-tTA Tg
mice (MH) were obtained from the Jackson Laboratory
and were crossed with TAMEK mice to generate TAMEK/
MH DTg mice. The tTA transgene was detected by PCR
5′-CGCTGTGGGGCATTTTACTTTAG3′ and 5′-CATGTCCAGATCGAAATCGTC-3′. DTg
mice and breeding pairs were treated with Doxycycline
(DOX, 0.5 mg/ml) in drinking water for suppression of
transgene expression. DOX was withdrawn from the
drinking water to induce fetal aMEK1 transgene
expression. Wild type and /or single Tg littermates were used as
controls for DTg mice. The MEK1 transgenic mouse, in
which aMEK1 is driven by the αMHC promoter without
DOX regulation, was kindly provided by Dr. Jeffrey
]. Time mating was validated by checking the
vaginal plugs in the morning and marking as 0.5 day post
copulation (dpc). All animals were maintained at a 12 h
light–dark cycle with ad libitum access to food and water.
All procedures conformed to the Guide for the Care and
Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85–23, revised
1996), and were approved by the Institutional Animal
Care and Use Committee of the University of California,
San Francisco. Please see Additional file 1 S1 for
additional details on Methods.
Anatomy and histology analysis
Mouse hearts were arrested in diastole with i.p. injection
of 1 ml of 1 M KCl in saline, formalin-fixed and embedded
in paraffin. To assess the morphology of the atrial septum,
the right atrial appendage was removed, and the
anatomical structure of septal wall was assessed under dissection
microscopy. To document ASD and possible ventricular
septal defects, serial continuous sections of paraffin
embedded hearts were stained with Gomori’s trichrome
]. Stained sections were reviewed for occurrence
of septal defect and photographic images were acquired.
Western blotting assay
Fetal hearts were collected from fetuses at 10.5 to 14.5
dpc; some fetal hearts were then divided into atrial and
ventricular portions. Each tissue sample was
homogenized in lysis buffer containing 150 mM NaCl, 50 mM
Tris-HCl, 1 mM Na3VO4, 5 mM NaF, 1% NP40, and
protease inhibitor cocktail tablet (Roche Diagnostics,
Indianapolis, IN). Samples containing equal amounts of
protein were separated by NuPAGE Novex Bis-Tris Gels
(Invitrogen, Carlsbad, CA) and transferred to PVDF
membranes (Invitrogen). Blots were first probed with
antibodies against phospho-ERK1/2 (P-ERK1/2, rabbit
monoclonal, 1:2000, #4376, Cell Signaling, Danvers,
MA,) or total ERK1/2(rabbit polyclonal, 1:4000, #9102,
Cell Signaling), total MEK1/2 (rabbit monoclonal,
1:4000, #9126, Cell Signaling), MKP1(rabbit polyclonal,
1:1000, SC-1102, Santa Cruz Biotech, Santa Cruz, CA),
MKP3(mouse monoclonal, 1:1000, SC-1000374, Santa
Cruz Biotech), tTA (mouse monoclonal anti-TetR,
1:2000, #631131, Clontech, Mountain View, CA),
GAPDH (rabbit polyclonal, sc-25,778, Santa Crus
Biotech) or HA (mouse monoclonal, 1:2000, #2367, Cell
Signaling), and then with appropriate horseradish
peroxidase-conjugated secondary antibodies (goat
antirabbit IgG-HRP, 074–1056 and goat anti-mouse
IgGHRP, 074–1806, KPL, Gaithersburg, MD). SuperSignal
West Femto Maximum Sensitivity substrate (Thermo
Fisher Scientific Inc., Waltham, MA) and Immobilon
Western HRP Substrate (EMD Millipore, Billerica, MA)
were used for the chemiluminescent visualization of
proteins. Exposed films were then subjected to density
analysis using Image J software (NIH).
Transthoracic echocardiography was performed in
conscious 8-week old female mice using an Acuson Sequoia
512 machine and a 13-MHz probe [
]. A two
dimensional short-axis view of the left ventricle was obtained
at the level of the papillary muscles. Two-dimensional
M-mode tracings were also recorded. LV fractional
shortening (FS) and ejection fraction (EF) were
calculated using the following equation: FS (%) = 100 × (LV
end of diastolic dimension - LV end of systolic
dimension) /LV end of diastolic dimension; EF (%) = 100 × (end
of diastolic volume - end of systolic volume)/end of
Results are expressed as mean ± SEM. Mean values
were compared by the unpaired 2-tailed Student’s t
test or ANOVA. Mortality rates were compared by
Fisher’s exact test. P-values less than 0.05 were
considered statistically significant. Although we examine
many potential effects, we report nominal p-values,
without adjustment for multiple testing. Such
adjustment would be focused on avoidance of one or more
results with p < 0.05 in the case where all differences
are truly zero [
], which is an extremely
unrealistic hypothesis about the state of nature in our
situation. We therefore rely on scientific judgment rather
than formal adjustment methods to indicate where
caution is warranted despite findings with p < 0.05. In
addition, adjustment would require that each result
detract from the others, but there are clear
biological relationships among the issues that we
examine, and these permit coherent sets of findings
to reinforce each other rather than detract from one
Doxycyline induced aMEK1 expression and activity in
TAMEK/MH double transgenic mice (DTg)
Even though αMHC expression is traditionally
associated with the adult heart, previous studies have
demonstrated the expression of this MHC isoform and
the activity of its promoter in fetal hearts [
]. In the
absence of DOX in the drinking water of pregnant mice,
expression of the HA-tagged human aMEK1 transgene,
dependent on tTA expression driven by the αMHC
promoter, was observed in DTg fetuses. Its expression was
subsequently suppressed in DTg fetuses via introduction
of DOX into the drinking water of pregnant mice
(Fig. 1A). Although expression of the aMEK1 transgene
was detectable via identification of an otherwise absent
HA tag, the level of expression achievable in the fetal
heart as driven by a combination of the relatively low
activities of the αMHC promoter and the tTA promoter in
the fetal heart was not strong enough to yield a
measurable increase in total MEK1 protein. As has been
observed previously, however [
], the constitutive activity
even of this relatively smaller amount of aMEK1 protein
compared to the total pool of non-phosphorylated and
non-activated endogenous MEK1 resulted in a
measurable increase in downstream ERK1/2 activation (i.e.,
phosphorylation, Fig. 1A-C).
During fetal development, aMEK1 transgene
expression was identified in 14.5 dpc hearts, but aMEK1
expression was absent in the fetus’ brain tissue (Fig. 1B).
Furthermore, there was a differential expression pattern
of aMEK1 transgene even within the developing fetal
hearts. Expression of the HA-tagged aMEK1 gene, as
well as increased phosphorylation of ERK1/2, was
significantly higher in atrial tissue than in ventricular tissue
from 14.5 dpc hearts of DTg fetuses (Fig. 1C).
Atrial septal defect in DTg in the absence of DOX (and in the presence of upregulated ERK1/2 phosphorylation)
Hearts from a large number of DTg mice (23 out of 45)
that had not been exposed to DOX in utero exhibited
enlarged right atria compared to the right atria from
their non-DTg littermates (Fig. 2A). Further anatomical
analyses of 8 week-old hearts with hypertrophied right
atria revealed the large, unfused foramen ovale
connecting both atrial chambers, resembling secundum atrial
septal defects (Fig. 2B, Additional file 2 S2). In contrast,
only small vestigial foramena ovale were observed in
WT hearts (Fig. 2B). The ASDs in DTg hearts were
confirmed on whole heart histologic sections taken at
14 days of age, although no other ventricular or
atrioventricular abnormalities were observed in DTg
hearts, with or without in utero exposure to DOX
Premature mortality and compromised cardiac function in DTg
In the absence of DOX exposure, crossbreeding of
heterozygous TAMEK1 and MH mice yielded pups with
four different transgenic genotypes in a classic
Mendelian distribution (Fig. 2C). By postnatal day 21, however,
only 73% (33/45) of the DTg pups had survived, while
there was no mortality among the other three genotypes
(Fig. 3A). Although the incidence of ASD was similar in
male and female DTg in the early postnatal period,
mortality was observed in 11/14 (79%) of male ASD-DTg’s at
21 days, compared to only 1/9 (11%) of female
ASDDTg’s at that time point (P = 0.003, Fig. 3B).
Even though DTg pups as a whole had similar initial
birth bodyweights compared to their non-DTg
littermates, postnatal growth of DTg pups subsequently
diagnosed with ASD, but not DTg pups without ASD, was
delayed at day 14 (6.1 ± 0.4 g for ASD-DTg vs. either 8.2
± 0.2 g for non-DTg or 8.2 ± 0.5 g non-ASD DTg, p <
0.01, Fig. 2C & 3C). Among the 14-day old DTg pups
with ASD, the average bodyweight of mice that survived
at wean (21 days) was marginally higher than those mice
that did not subsequently survive (7.1 ± 0.4 g vs 6.1 ±
0.4 g, P = 0.07), suggesting a link between severity of the
ASD and early postnatal growth. This early growth
impairment may therefore serve as a diagnostic marker for
severe ASD in this mouse model.
At 8 weeks of age, the body weight difference between
surviving female ASD mice and other
genotype/phenotype female mice had disappeared (we included only
female groups in our statistical analysis due to the
insufficient number of surviving male ASD-DTg mice,
Table 1), reflecting better overall compensation for ASD
in surviving ASD mice. All single transgenic and WT
mice survived past the time of wean. Echocardiography
was performed on 8-week old female mice from the four
different genotypes. Compared to the non-ASD groups,
the ASD-affected female DTg mice, despite equivalent
body weights, had significantly decreased LV function as
reflected both in lower fractional shortening (40 ± 2% vs
48 ± 0%, p < 0.05) and reduced ejection fraction (67 ± 2%
vs. 75 ± 0%, p < 0.05; Table 1, Additional file 3 S3).
Differential phosphorylation of ERK1/2 in the developing atria of two different aMEK1 transgenic models correlates to differential development of ASD
In addition to the DTg model of inducible,
cardiacspecific aMEK1 expression described here, our
laboratory has also studied an alternative transgenic model,
the MEK1 Tg mouse, in which cardiac-specific
expression of aMEK1 is constitutive under the αMHC
promoter and is not affected by DOX. In contrast to the
inducible TAMEK/MH DTg model, the constitutively
expressing MEK1 Tg mouse (a generous gift from the
Jeffrey Molkentin laboratory) did not develop ASD or
other congenital cardiac defects. We evaluated fetal
transgene expression in the MEK1 Tg model, and did
observe aMEK1 expression in 14.5 dpc fetal hearts as
reflected by an increase in total MEK1 protein (Fig. 4).
As in the TAMEK/MH DTg model, the 14.5 dpc
expression of aMEK1 in the constitutively expressing
transgenic was also higher in the atria than in the ventricles.
However, the pattern of atrial phosphorylation of ERK1/
2 was markedly different in the two transgenic models.
Although aMEK1 expression in adult MEK1 Tg hearts is
associated with increased ERK1/2 expression and
], neither atrial nor ventricular
phosphorylation of ERK1/2 was significantly increased in heart
tissues from constitutively expressing fetal 14.5 dpc MEK1
Tg mice compared to their 14.5 dpc WT siblings (Fig. 4).
This difference in downstream ERK1/2 activation despite
similar fetal atrial aMEK1 expression in the MEK1 Tg and
our novel TAMEK/MH DTg mice correlated with the
different phenotypes observed with regard to congenital
ASD development, as ASD was only observed in DTg
mice in which atrial ERK1/2 phosphorylation was greater
than that of both ventricular tissue from the same hearts
and heart tissues from non-DTg littermates,.
In order to evaluate the possible roles of integration site
and the use of different transgene expression vectors in the
different molecular and clinical phenotypes observed in
TAMEK/MH DTg and MEK1 Tg mice, we developed
a second inducible model using the pTREtight vector.
We observed a similar incidence and clinical impact of
ASD in this second DTg line (Additional file 4, S4).
We also observed another interesting difference
between the inducible TAMEK/MH DTg and the
constitutively expressing MEK1 mouse with regard to MEK/ERK
regulation. Expression of the negative regulator of ERK
phosphorylation/activation, MAP kinase phosphotase 3
Fig. 3 a Mortality in ASD mice. Fifty-one percent early postnatal
mortality in ASD-affected DTg (12 out of 23 by 3 weeks of age),
compared to 100% 3-week survival in the non-DTg and non-ASD
DTg groups. b Gender difference in mortality. Male ASD mice (n = 14)
had higher mortality than female ASD mice (n = 9, 79% vs 11%;
P = 0.003). c Early mortality is associated with poor weight gain.
There is no difference in average bodyweights among ASD DTg mice
and other groups at birth. However, the average body weight at
14 days after birth is significantly lower in the ASD DTg mice. ASD DTg
non-survivor (NS) group n = 4; ASD DTg - survivor (S) group, n = 6;
non-ASD DTg, n = 9; non-DTg group, n = 40. **P < 0.01, *P < 0.05
] was significantly higher in the atria and
ventricles of 14.5 dpc TAMEK/MH DTg mice than in
cardiac tissues from their WT siblings (Fig. 4, Additional
file 4 S4, P < 0.05). However, a similarly high level of
MKP3 expression was observed in both (constitutively
expressing) MEK1 Tg mice and in their respective WT
siblings (Fig. 4). In contrast, the expression of MKP1,
which also regulates p38 and JNK phosphorylation [
was similar among atrial and ventricular tissues from all
four groups of mice (Fig. 4).
22 ± 0
64 ± 2*
37 ± 2*
The severity of human ASD differs drastically among
affected individuals, ranging from life-threatening to
symptomless. Although ASD is one of the most
common congenital heart defects, this clinical variability in
phenotype makes identification of factors that interrupt
normal atrial septal development even more difficult.
There are several genetic mouse models that exhibit
defects in atrial septation. Among the most often reported
are models of functionally impaired or heterozygous null
NKX2–5 and GATA4, based largely on early associations
of mutations in these genes for these transcription
factors with human ASD and other congenital defects
2, 3, 21–23
]. These models of transcription factor
disruption, however, have proven to be extremely
complex, as their mechanisms involve disruption not
only of the target transcription factor but also
impaired expression of other related transcription factors
such as TBX5 and MEF2c [
]. As a result, changes
in numerous downstream effectors regulated by these
multiple transcription regulators are at play.
Confounding the mechanistic analyses of these models is
their frequent association with extracardiac
abnormalities, and the fact that they are often embryonic lethal
and demonstrate compound septal defects involving
both atria and ventricles.
In contrast, the model described here of isolated
congenital ASD offers a relatively focused mechanistic
pathway for future study; it is directed to a
wellcharacterized signaling pathway rather than a complex
of pleiotropic upstream transcription factors. In addition
to the mechanistic complexity of previously reported
genetic models of ASD, the perinatal characteristics of
those models do not closely mimic the clinical findings
in human ASD patients. Our novel model of isolated
ASD, however, resembles human ASD in the following
important ways: severe ASD is associated with growth
impairment; ASD-specific mortality is highest within the
early postnatal period; although the incidence of ASD is
evenly distributed among the sexes, this early mortality
is significantly higher in males ([
]; Table 2).
Our current mouse model likely reflects an important
role of Ras-MEK-MAPK signaling in the development of
RASopathy syndromes, including atrial septal defects. In
fact, we have observed for the first time that
upregulation of MEK-ERK signaling in an atrial-specific manner,
resulting in higher atrial ERK1/2 phosphorylation, is
associated with abnormal atrial septation. Furthermore, by
comparing two aMEK1 transgenic mouse models, we
substantiated the potential role for site-specific ERK1/2
activation in the development of ASD.
Since MAPK signaling pathways can be regulated by
various stimuli, such as growth factors, hormones,
exogenous chemicals and even mechanical stresses [
our findings also suggest that genetic-independent,
aberrant activation of ERK1/2 during pregnancy may lead to
the sporadic occurrence of ASD.
The mechanism by which upregulated ERK1/2
activation is absent in the fetal atria of the constitutively
expressing MEK1 mouse, despite fetal cardiac aMEK1
expression driven by the αMHC promoter, remains
unclear. One explanation for differences in phenotype
between the two transgenic models studied may be related
to differences in integration site and the use of different
transgene expression vectors in their generation.
However, we observed a similar incidence and clinical impact
of ASD in a second DTg line derived via a distinct
integration using an alternative vector.
It is also possible that the difference in genetic
backgrounds between the double transgenic (C57Bl/6-FVBN
mix in both lines studied) and single transgenic (pure
FVBN) mouse models may modulate the downstream
effects of aMEK1 transgene expression. The importance
of genetic background as a possible regulator of
congenital defects has been demonstrated previously in the
Nkx2–5 transgenic mouse [
]. Heterozygous Nkx2–5
knockout mice in the inbred strain background C57Bl/6
frequently developed atrial and ventricular septal defects,
whereas the incidences of these defects were
substantially reduced in the Nkx2–5(+/−) progeny outcrossed to
the strains FVB/N or A/J [
]. Since the pure
FVBNderived single Tg MEK1 and its WT siblings both had
higher fetal cardiac MKP3 expression than the WT
siblings of the C57Bl/6-FVBN-derived DTg or control
C57Bl/6 mice, it is possible that the expression level of
MKP3 is related to this difference in genetic
backgrounds. Higher pre-existing MKP3 levels in the fetal
atria of MEK1 single Tg hearts may have suppressed
activation of ERK1/2, thereby resulting in resistance to
ASD despite aMEK1 transgene expression (Fig. 5). The
higher levels of MKP3 observed in the DOX-withdrawn
DTg may, in turn, reflect a feedback loop that is not as
effective as high constitutive MKP3 expression in
controlling ERK1/2 activation. In fact, previous studies have
indicated that MKP3-mediated suppression of ERK
activation is required for proper development of the
kidneys, neural plate and limb bud mesenchyme [
similar mechanism may be at play to explain why lower
levels of αMHC-driven aMEK1 expression in fetal
MEK1 Tg hearts did not exhibit the increased ERK1/2
phosphorylation seen in adult MEK1 Tg hearts with
significantly higher αMHC-driven aMEK1 expression.
Studies in MKP3-null mouse lines derived from mice
with different genetic backgrounds have also
demonstrated variable influences on development even with
similar increases in ERK phosphorylation [
results reported here do suggest that the fine tuning of
ERK1/2 signaling via MKP3 expression levels at specific
locations within the developing heart may be essential
for the proper organ and tissue development .
Developmental defects in the brain have also been
observed in models of upstream ERK activation via Braf
Q241R or Neurofibromatosis type 1 gene
overexpression. Treatment with MEK inhibitors was found to
correct developmental defects in these experimental
]. While ERKs are known to be
preferentially activated both in the regions of the primitive
brain and in the heart during early development, our
new evidence underscores the importance of carefully
regulated MAPK signaling during fetal development.
Any dysregulation or over-activation of this pathway that
leads to an increase in ERK1/2 phosphorylation may
impact the normal development of specific organ
structure or function.
Our data indicate that the regulation of MAPK signaling
is essential for normal development of the atrial septum.
Specifically, atrial upregulation of activated ERK1/2 was
associated with failure of normal septal closure. The
TAMEK1/MH DTg mouse is the first ASD-specific
model that closely mimics the clinical manifestations of
human congenital ASD, and therefore may serve as a
novel tool to elucidate both the development of human
fetal septal defects as well as possible therapeutic
interventions. Furthermore, our data also begin to suggest
that modifiers of MAPK signaling in fetal hearts, such as
MKP3 and other internal/environmental factors, should
be further evaluated to better understand the complex,
multifactorial development of ASD and other congenital
Additional file 1: S1 Methods. (DOCX 34 kb)
Additional file 2: S2 Figure. Comparison of progeny of 4 genotypes at
8 weeks of age showed atrial septal defect in DTg (+/+). (PPTX 1404 kb)
Additional file 3: S3 Figure. Survived ASD affected DTg (+/+, from line #25)
and its non- DTg siblings at 8 weeks old. (PPTX 206 kb)
Additional file 4: S4 Figure Anatomical dissection revealed ASD in two
independent DTg lines at 8 weeks old. A. line #25, derived from expression
sequence using pTet-Splice vector; B. line #8, derived from expression
sequence using pTREtight vector. C. MEK1/2, P-ERK1/2, ERK1/2, MKP1, and
MKP3 protein levels as detected by Western blot in atrial (A) and ventricular
(V) tissue from 14.5 dpc DTg (+/+) and WT (−/−) littermates from line #8
(C57BI/6-FVBN mixed background) and MKP1, and MKP3 from control pure
C57BI/6 mice. D. Survival among DTg mice from line #8 with (n = 11) and
without (n = 12) ASD, and among non-DTg littermates (n = 66). (PDF 1225 kb)
Additional file 5: S5. Original blots in fig. 1. (PPTX 1641 kb)
Additional file 6: S6 Table Echocardiographic measurements of LV
function in the 8 weeks old DTg mice (ASD and non-ASD). (DOCX 14 kb)
Additional file 7: S7. Original blots in fig. 4. (PPTX 1028 kb)
A: Atria; aMEK1: Active form of human MEK1; ASD: Atrial septal defects;
CHD: Congenital heart disease; DOX: Doxycycline; Dpc: Day post copulation;
DTg: Double transgenic (DTg); EF: Ejection fraction; ERK1/2:
Mitogenactivated protein kinase ½; FS: Fractional shortening; HA: Hemagglutinin;
i.p.: Intraperitoneal; LV: Left ventricular; MEK1: Mitogen-activated protein
kinase kinase 1; MH: αMHC-tTA transgenic mouse; MKP: MAP kinase
phosphatase; P-ERK1/2: Phospho-ERK1/2; TAMEK: Transgenic mouse with
tetracycline transactivator regulated aMEK1 gene; TetR: Tetracycline repressor
protein; Tg: Transgenic mouse; tTA: Tracycline-responsive transcriptional
activator; V: Ventricle; α-MHC: α-myosin heavy chain
The authors appreciate the generous gifts of the HA-tagged human active
MEK1 cDNA and the transgenic mouse model (MEK1 Tg) provided by
Dr. Natalie G. Ahn and Dr. Jeffrey Molkentin.
This work was supported by National Institutes of Health; Grant numbers:
K08HL079239 and R01HL083118.
Availability of data and materials
All datasets, on which the conclusions of the manuscript rely on, are
presented in the paper.
CY and MJM conceived the study, CY was responsible for the design of the
research; CY, YF and YY performed the experiments; CY analyzed the data
and interpreted the results of the experiments; CY and YF prepared the
figures and the manuscript; CY and MJM revised the manuscript. All authors
read and approved the final manuscript.
All procedures conformed to the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication
No. 85–23, revised 1996), and were approved by the Institutional Animal
Care and Use Committee of the University of California, San Francisco.
Consent for publication
The authors declare that they have no competing interests.
Our study doesn’t include any data about people or human tissue samples.
All animal experiments in this research were reviewed and approved by the
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