Repository of the Max Delbrück Center for Molecular Medicine (MDC)
Soluble epoxide hydrolase is a susceptibility factor
for heart failure in a rat model of human disease
Jan Monti, Judith Fischer, Svetlana Paskas, Matthias Heinig, Herbert Schulz, Claudia Gösele,
Arnd Heuser, Robert Fischer, Cosima Schmidt, Alexander Schirdewan, Volkmar Gross, Oliver
Hummel, Henrike Maatz, Giannino Patone, Kathrin Saar, Martin Vingron, Steven M. Weldon,
Klaus Lindpaintner, Bruce D. Hammock, Klaus Rohde, Rainer Dietz, Stuart A. Cook, Wolf-Hagen
Schunck, Friedrich C. Luft, and Norbert Hubner
Published in final edited form as:
Nature Genetics. 2008 Apr 28 ; 40(5): 529-537 | doi: 10.1038/ng.129
Nature Publishing Group (U.S.A.) ►
MDC Repository | http://edoc.mdc-berlin.de/9531/ 1
Soluble epoxide hydrolase is a susceptibility factor for heart failure in
a rat model of human disease
Jan Monti1,2,9, Judith Fischer1,9, Svetlana Paskas1, Matthias Heinig1,3, Herbert Schulz1, Claudia Gösele1, Arnd
Heuser1,2, Robert Fischer1,2, Cosima Schmidt1, Alexander Schirdewan2, Volkmar Gross1, Oliver Hummel1,
Henrike Maatz1, Giannino Patone1, Kathrin Saar1, Martin Vingron3, Steven M. Weldon4, Klaus Lindpaintner5,
Bruce D. Hammock6, Klaus Rohde1, Rainer Dietz1,2, Stuart A. Cook7, Wolf-Hagen Schunck1, Friedrich C.
Luft1,8, and Norbert Hubner1
1 Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
2 Department of Clinical and Molecular Cardiology, Franz-Volhard Clinic, HELIOS, Charité-Universitätsmedizin Berlin,
Schwanebecker Chaussee 50, 13125 Berlin, Germany
3 Department of Bioinformatics, Max-Planck-Institute for Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany
4 Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877-0368, USA
5 F. Hoffmann-La Roche, Grenzacherstrasse 124, 4070 Basel, Switzerland
6 Departments of Entomology and Nutrition and Cancer Research Center, University of California at Davis, One Shields
Avenue, Davis, California 95616-8584, USA
7 National Heart and Lung Institute, Imperial College, Dovehouse Street, London, SW3 6LY, UK
8 Department of Nephrology/Hypertension, Franz-Volhard Clinic, HELIOS, Charité-Universitätsmedizin Berlin,
Schwanebecker Chaussee 50, 13125 Berlin, Germany
9 These authors contributed equally to this work.
ABSTRACT | We aimed to identify genetic variants associated with heart failure by using a rat model of the human
disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive
heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and
identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2
segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a
more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in
heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload–induced heart failure and cardiac
arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species
role for Ephx2 in this complex disease.
Heart failure is an epidemiologically important disease,
with ≈30% mortality at 1 year after diagnosis [1-3]. Most
affected individuals have impaired systolic function with a
reduced ejection fraction; their prognosis is particularly
poor . Heart failure is thought to result from complex
interactions between genetic susceptibility and lifestyle
and environmental factors . Hypertension is one of the
major risk factors [5,6]. Starting from high blood pressure,
the pathophysiological cardiac remodeling cascade
proceeds to left ventricular hypertrophy (LVH) as a
primarily adaptive process, which then proceeds to left
ventricular dilatation, decreased systolic function and
cardiac arrhythmias in some individuals, whereas others
retain stable systolic function without clinical signs of
heart failure. Elucidation of genetic factors has been
limited by heterogeneity and by gene-environment
interactions, as well as by the confounding presence of
hypertension that serves as the driving force but is not
sufficient as an intermediary phenotype. Familial
predispositions contribute to heart failure in humans .
We used the spontaneously hypertensive heart failure
(SHHF) rat, an inbred, genetically homogenous rat model
that mirrors human hypertension-associated heart failure
. SHHF rats not only develop heart failure late in life
after high blood pressure and left ventricular hypertrophy
have developed , but also show many of the associated
transcriptional and metabolic features of the human
disease [10-12]. The model's genetic propensity is
underscored by the fact that a closely related strain, the
stroke-prone spontaneously hypertensive (SHRSP) rat,
does not develop heart failure despite similarly elevated
We conducted a segregation analysis in F2 hybrids bred
from SHHF and SHRSP, thus removing blood pressure
and LVH as confounding variables. To test the overall
blood pressure– and cardiac hypertrophy–related impact
on the development of heart failure, we generated a
second F2 cross-bred from SHHF and the normotensive
Wistar-Kyoto (WKY) control rat. We integrated linkage
analysis of hemodynamic phenotypes with cardiac
genome-wide expression profiling in the F2 population, a
strategy others and we have successfully used earlier to
elucidate complex traits [13-23]. We identified the gene
encoding soluble epoxide hydrolase, Ephx2, as a heart
failure susceptibility gene. Finally, we validated our
findings using Ephx2 knockout mice and expression
studies in human heart failure.
Determination of cardiac phenotypes
We catheterized the left ventricle to precisely measure
aortic and left ventricular end-diastolic pressures (LVEDP)
and left ventricular end-diastolic volumes (LVEDV), as
well as to construct pressure-volume loops. We found
similar blood pressure and heart weight:body weight
ratios, indicating LVH, in both SHHF and SHRSP
compared to WKY rats. SHHF rats showed substantial left
ventricular dilatation and reduced left ventricular ejection
fraction, consistent with previous observations  (Suppl.
Fig.1). Both SHHF and SHRSP rats had elevated LVEDP,
with higher values in SHHF rats (Suppl. Fig.1).
Blood pressure variance and LVH variance were similar or
Monti J et al.
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smaller in the F2(SHHF × SHRSP) population than among
progenitor strains, showing that blood pressure and LVH
did not segregate in this cross. In contrast, the ejection
fraction showed a large variance, ranging from 14% to
82% with a near normal distribution, consistent with the
independent segregation of multiple alleles. In addition,
we obtained quantitative hemodynamic parameters for
LVEDP, LVEDV and left
(Δpressure/Δtime (mm Hg/s), dP/dtmax) (Suppl. Table 1).
In contrast to F2(SHHF × SHRSP) rats, F2(SHHF × WKY)
rats showed decreased blood pressure and reduced LVH
(Suppl. Table 1).
The F2(SHHF × WKY) data show that the overall lower
average blood pressure was paralleled, on average, by a
higher ejection fraction, decreased LVEDP, lower LVEDV,
improved contractility and decreased B-type natriuretic
peptide (Suppl. Table 1). These observations support our
contention that genetic factors contributing to the complex
heart failure phenotype should be assessed in an
experimental cross that equalizes the influences of high
blood pressures and LVH as permissive heart failure
factors, thus removing them as confounders. The
F2(SHHF × SHRSP) population that we characterized
fulfills both requirements.
Heart failure phenotypes map to rat chromosome 15
We genotyped all F2 rats as described in the Suppl. Note
online. In F2(SHHF × SHRSP), a locus on rat
chromosome 15 centered at marker D15Rat10 showed
statistically highly significant linkage (lod score 4.3) to left
ventricular ejection fraction,
parameter used to characterize the failing heart (Fig.1). At
the same locus, we further demonstrated statistically
significant linkage for cardiac contractility and highly
suggestive linkage to LVEDV. The linkage peak on
chromosome 15 for LVEDV, however, was some 20 cM
away (Fig.1). Nonetheless, the SHHF allele was
invariably associated with cardiac dysfunction. Grouping
F2 rats according to zygosity at D15Rat10 did not show
any genotype association with blood pressure or LVH, as
determined by analysis of variance (data not shown). This
finding indicated that linkage to this locus was
independent of blood pressure. Our data indicated that
this quantitative trait locus (QTL) strongly affected heart
failure in an additive mode, accounting for 15.1% of the
phenotypic variance observed for the ejection fraction. As
we observed clustering of three distinct clinical traits
(ejection fraction, LVEDV and contractility) at D15Rat10
with varying degrees of significance, we speculated that a
composite quantitative trait variable could extract further
genetic information from this locus. Using principal
component analysis, we next derived a composite
phenotype for each F2(SHHF × SHRSP) rat from three
traits (ejection fraction, LVEDV and dP/dtmax) and then
subjected this composite trait to linkage analysis. This
analysis resulted in a statistically more significant linkage
at D15Rat10 (lod 4.8; Fig.1), suggesting a complex but
directional interplay of these phenotypes in the
predisposition of heart failure at this locus, as well as
indicating the limitations of individual, reductionist
parameters as surrogate measurements for a complex
phenotype in general. In contrast to the above, we found
no evidence for linkage of ejection fraction in the
F2(SHHF × WKY) population anywhere in the genome
(Suppl. Note and Suppl. Fig.2 online).
the principal clinical
Transcript profiling in parental strains and F2 populations
Within the region of linkage to heart failure on
chromosome 15, we identified only two transcripts that
were differentially expressed with a false discovery rate
(FDR) of <5% between the parental SHRSP and SHHF
strains and showed a cis-regulated expression QTL
(eQTL) at an FDR <5% in the F2(SHHF × SHRSP)
population, consistent with the close relatedness of SHHF
and SHRSP rats used in this experiment (Suppl. Note). Of
the two cis-regulated transcripts, Ephx2 showed the
strongest genetic evidence for significant allele-specific
regulation in the F2(SHHF × SHRSP) population. The
linkage peak (genome-wide corrected P < 10- 6; FDR <
10-4; Suppl. Fig.3 online) for the Ephx2 eQTL was at
D15Rat10, the same marker that also defined the peak of
the heart failure QTL on chromosome 15 in this cross.
D15Rat10 is the nearest marker to Ephx2 and is located
within 2.7 Mb, based on the rat genome reference
sequence version 3.4. This finding suggested that Ephx2
transcript levels are regulated in cis at the Ephx2 gene
itself and coincide with the peak of linkage to the heart
failure QTL. The second cis-acting eQTL gene in this
region was Mmp14. Even though it was located within the
95% confidence interval encompassing the heart failure
QTL, Mmp14 was located at a distance of more than 10
Mb from the heart failure QTL peak marker D15Rat10.
Strongest eQTL linkage of Mmp14 was observed with
marker D15Rat83 (genome-wide corrected P < 10- 3; FDR
< 0.05). Based on statistical significance and proximity to
the heart failure peak marker, we thus prioritized Ephx2
as candidate for further investigations.
Sequence analysis of Ephx2
We next aimed to elucidate regulatory sequence
variations underlying the observed Ephx2 cis-regulated
eQTL and to screen for coding sequence differences
possibly altering protein function. Sequencing 5,000 bp
upstream of the first exon, we found three SNPs and a 2-
nt deletion in the putative Ephx2 promoter in SHHF as
compared to SHRSP and WKY rats (Fig.2). Genomic
sequencing of the entire coding exon sequence, including
adjacent splice sites, revealed two synonymous and three
nonsynonymous SNPs (Fig.2).
Ephx2 is a heart failure susceptibility gene
We established that Ephx2 was genetically linked to the
peak marker of linkage for heart failure D15Rat10 by
linkage analysis (Suppl. Note). The allelic status at Ephx2
accounted for 64% of the expression differences
observed, with highest expression values associated to
the SHHF genotype (Fig.3a,b).
We measured Ephx2 abundance by protein blot analysis.
Protein amounts in parental rats and F2 rats grouped
according to zygosity at D15Rat10 mirrored the
differences observed at the transcript level (Fig.3c).
Ephx2 encodes the soluble epoxide hydrolase (sEH),
which hydrolyzes cardioprotective epoxyeicosatrienoic
acids (EETs) . To test the functional relevance—that
is, effect on the substrate—of the allele-specific
upregulation of Ephx2, we measured enzymatic hydrolase
activity of Ephx2 in cardiac tissue of the parental and F2
rats grouped by zygosity at the peak marker D15Rat10.
Our results demonstrated that the SHHF allele–specific
upregulation of the transcript and protein also segregated
Monti J et al.
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with an augmented cardiac sEH activity (Fig.3d). Finally,
we directly measured the total amount of 14,15-EETs in
the heart of parental and F2 rats, again grouped according
to genotype. This analysis showed an effect of genotype
on the total amount of cardioprotective EETs, the amount
being significantly reduced when Ephx2 was transcribed
from the SHHF allele (Fig.3e). Taken together, the results
suggest that genetic variation in cis leads to upregulation
of the Ephx2 transcript, elevated protein, increased
hydrolysis of EETs and a decrease of cardioprotective
EETs in the heart mediated by the Epxh2SHHF allele. The
colocalization of this pathophysiological cascade triggered
by the Ephx2 eQTL with the mapping of a heart failure
QTL strongly suggests that Ephx2 contributes to the
observed heart failure QTL.
This interpretation of the data is corroborated by the fact
that, in all instances, the Ephx2SHHF allele was associated
not only with upregulation of the Ephx2 transcript and
protein expression, but also with heart failure in the F2
population. Using the 10% upper and lower values of the
tails of the ejection fraction distribution from F2(SHHF ×
SHRSP) rats, we observed a marked correlation between
Ephx2 transcript levels and ejection fraction (correlation
coefficient r2 = - 0.219, P < 0.01); rats with low ejection
fraction had higher Ephx2 transcript levels, whereas rats
with high ejection fraction had lower transcript levels. No
correlation was detected when the entire dataset was
analyzed (r2 = - 0.048, P not significant).
Ephx2 promoter deletion creates an AP-1 binding site in
To test whether the allelic promoter variants influence
Ephx2 gene expression, we performed luciferase reporter
assays and compared the variant promoters among
SHHF, SHRSP and WKY rats. We found a strong
increase in promoter activity with the SHHF compared to
the SHRSP allele (Fig.4a). The findings are consistent
with the observed cis-regulated eQTL in F2(SHHF ×
SHRSP), in which the SHHF allele was the allele that
showed higher expression (Fig.3a,b).
We also confirmed the effect of allele-specific regulatory
variation using allele-specific real-time PCR of cDNA from
heart tissue of ten F1(SHHF × SHRSP) rats. This
experiment confirmed that the expression of Ephx2 was
regulated in cis and that the Ephx2SHHF allele-specific
transcript levels were significantly elevated compared to
the Ephx2SHRSP allele (P = 8 × 10- 12; Suppl. Fig.4 online).
consensus AP-1 (activator protein 1) transcription factor
binding site in the Ephx2 promoter that exactly covered
the 2-nt deletion in the SHHF strain (Fig.4b). To
investigate whether the mutated AP-1 binding site
affected AP-1 binding
electrophoretic mobility-shift assays; these showed
specific AP-1 binding to the SHHF promoter, whereas AP-
1 binding in SHRSP was abolished (Fig.4b).
computational predictions, we identified a
vitro, we performed
SNPs in the coding region do not alter hydrolase activity
Sequencing detected three nonsynonymous SNPs that
could potentially affect Ephx2 enzymatic activity (Fig.2).
Conceivably, an allele-specific altered protein could
confound the results attributed to allele-dependent
expression differences. We demonstrated by in vitro
expression of recombinant proteins that both SHHF and
SHRSP Ephx2 proteins were equally active, which
excludes a substantial effect of polymorphisms on the
enzyme activity (Suppl. Fig.5 online). Thus, the observed
allele-dependent differences in the hydrolysis of 14,15-
EET (Fig.3d,e) were due to allele-specific expression
differences of Ephx2.
Ephx2 pathway shows genotype-dependent coregulation
Because the transcriptional regulation of pathways related
to Ephx2 function is of major interest, we next focused on
transcripts of the arachidonic acid metabolic pathway that
includes several important enzymatic reactions in the
generation of epoxides (for example, 14,15-EETs) from
arachidonic acid and the enzymes that regulate
arachidonic acid synthesis or beta-oxidation, according to
the Kyoto Encyclopedia of Genes and Genomes. We
ranked the genes from the arachidonic acid metabolic
pathway according to their association with the heart
failure locus on chromosome 15. None of these genes
had a genome-wide significant eQTL at this locus when
correcting for all transcripts tested. Nevertheless, we
observe a concerted differential expression—in trans to
Ephx2—of the top-ranking genes, with individual P values
<0.1. Applying this relaxed filtering resulted in a set of
genes containing six members of the CYP superfamily
and three members of the PLA2 superfamily that will be
referred to as the Ephx2 pathway (Fig.5). To quantify the
small but consistent effect, we computed a score for the
association of gene expression at the level of the whole
pathway with a genetic marker, which was applied as a
genome-scan over all genetic markers used in F2(SHHF ×
SHRSP). We found that the maximum association of the
Ephx2-pathway transcripts occurred at the Ephx2 locus
and that the pathway association score had genome-wide
significance (P = 2.4 × 10- 5).
Ephx2 gene ablation protects from cardiac dysfunction
To corroborate our findings that Ephx2 is involved in the
pathogenesis of hypertension-induced heart failure, we
next studied Ephx2 knockout mice. We hypothesized that
Ephx2 knockout mice could represent an extreme
opposite compared to the detrimental effects associated
with elevated Ephx2 expression observed in F2(SHHF ×
At baseline, Ephx2 null mice and C57BL/6ByJ wild-type
controls had normal blood pressure and normal left
ventricular function (Fig.6a,b) without signs of LVH. To
investigate cardiac function under pressure overload
conditions similar to those we observed in SHHF rats, we
used prolonged (3 weeks) chronic intraperitoneal infusion
of the potent vasoactive peptide angiotensin II (Ang II) at
high dosages. Because the Ephx2 promoter harbors an
Ang II-inducible AP-1 binding site that is also predicted in
mouse, this experimental strategy was expected to
amplify Ephx2-specific pathophysiological responses to
pressure overload in wild-type but not in Ephx2 knockout
Telemetric blood pressure recordings showed a similar
temporal blood pressure increase in both groups (Fig.6a).
As expected from our rat experiments, in which the SHHF
allele was associated with high Epxh2 expression and
reduced ejection fraction, we found a substantially
decreased ejection fraction after 3 weeks of Ang II
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infusion in wild-type mice compared to knockout mice
Cardiac arrhythmias are common features and a principal
cause of death in the failing human heart . We
therefore tested whether Ephx2 gene ablation protected
against Ang II-induced arrhythmias. The ventricular
refractory period was noticeably prolonged in Ephx2
knockout mice compared to wild-type mice (Fig.7a),
supporting a prolonged—that is, beneficial—cardiac
repolarization in Ephx2 knockout mice. Notably, there
were spontaneous supraventricular and premature
ventricular contractions in wild-type mice that were not
observed in Ephx2 knockout mice (Fig.7b). We then
applied programmed electrical stimulations and induced
supraventricular tachycardia and atrial fibrillation, as well
as short episodes of
tachycardia, in all wild-type mice, whereas Ephx2
knockout mice had stable sinus rhythm even under
isoproterenol challenge (Fig.7c). These findings were
corroborated using an independent pressure overload
model using thoracic aortic banding (Suppl. Note and
Suppl. Fig.6 online).
EPHX2 expression is decreased in humans with heart
If an imbalance of EET concentrations is critical to the
pathogenesis of heart failure, we would expect
transcriptional regulation of Ephx2 to maintain high EET
levels to be a general defense mechanism, irrespective of
the trigger eliciting heart failure. To test this hypothesis,
we determined cardiac EPHX2 expression in biopsies
from subjects with ischemic heart failure and from
controls. Cardiac EPHX2 expression was reduced by
61.5% (P = 0.025) in subjects with heart failure,
compared to that in controls (Fig.8). This finding supports
transcriptional downregulation of EPHX2 as a potentially
beneficial adaptive mechanism in human heart failure.
This hypothesis is supported by the fact that wild-type
mice that developed a reduced ejection fraction under two
independent models of pressure overload conditions
(chronic Ang II infusion and aortic banding) showed a
significant downregulation of Ephx2 (Suppl. Note and
Suppl. Fig.7 online).
In humans, hypertension associated with sustained
cardiac hypertrophy represents one of the most common
causes of heart failure
hypertrophy is a primary adaptive response to diminish
wall stress induced by mechanical overload. However, the
resultant LVH is clinically associated with increased
morbidity and mortality . So far, the genetic and
molecular checkpoints gating
remodeling and heart failure are poorly understood
[29,30]. Recent data suggest a substantial contribution of
familial factors that could account for as much as 70%
increased risk for heart failure, after adjustments for other
risk factors including LVH .
We present evidence that genetic variation in Ephx2
facilitates progression from hypertension and cardiac
hypertrophy to heart failure in a rat model of human
hypertension and heart failure. We first generated
experimental F2 intercrosses of SHHF with SHRSP and
WKY as reference strains with different set points
regarding blood pressure and LVH. Hemodynamic
characteristics of parental rat strains were in line with
already established echocardiographic and hemodynamic
data for rats of strains SHHF  and SHR , a close
relative of the SHRSP. We documented marked cardiac
hypertrophy in SHHF rats, together with decreased left
ventricular ejection fraction, indicating the expected
alteration of systolic function. By contrast, SHRSP rats
retain systolic function, as documented by others
[31,32,33,34] and as we have demonstrated here. Thus,
we used SHRSP, a strain that is genetically closely
related to SHHF, as a reference model. However, rats of
our candidate strain, SHHF, clearly demonstrated
decreased ejection fraction and other heart failure
features also common to humans.
Linkage analysis to left ventricular hemodynamic
parameters of cardiac function in experimental SHHF
crosses with WKY and SHRSP revealed significant
segregation of ejection fraction with a QTL on rat
chromosome 15 harboring Ephx2. The fact that significant
linkage of the Ephx2 locus to left ventricular ejection
fraction was detected in the F2(SHHF × SHRSP) cross but
not in the F2(SHHF × WKY) cross underscores the
pathophysiological importance of hypertension and LVH
as permissive factors in deteriorating cardiac function. In
human heart failure, a decreased ejection fraction and
clinical symptoms of heart failure are preceded by
structural and hemodynamic abnormalities, such as
increased LVEDV and reduced contractility . We
identified these phenotypes in F2 populations; both traits
significantly segregated with the Ephx2 locus when both
crosses were analyzed jointly (data not shown),
suggesting a common regulation of hemodynamic
responses to pressure overload by the chromosome 15
Our genome-wide linkage analysis revealed two more
significant QTLs, on chromosome 8 and chromosome 4,
for ejection fraction and associated hemodynamic traits in
the F2(SHHF × SHRSP) population (J.F., J.M., S.P., H.S.,
M.H. et al., unpublished data). Further characterization of
these loci, together with the eQTL data obtained, may
generate additional insights into the genetic factors
contributing to heart failure in this model.
We performed linkage analysis of cardiac gene
transcription as intermediate phenotypes and identified
Ephx2 as a prime candidate gene within the heart failure
chromosome 15 QTL due to its highly significant cis
regulation and its expression pattern across parental
strains. Ephx2 encodes an enzyme responsible for the
conversion of EETs to their corresponding diols , thus
diminishing their biological activity [37-39]. We were aided
by the recent development of pharmacological Ephx2
inhibitors. These experiments showed that EETs elicit a
number of cardioprotective actions including vasodilation,
anti-inflammatory effects, endothelial cell proliferation,
antiaggregatory effects and inhibition of vascular smooth
muscle cell migration . In addition, recent studies have
shown an increased availability of cardioprotective EETs
in Ephx2 knockout mice with associated benefits on left
ventricular function due to reduced infarction size after
ischemic injury . Similar to our own observations,
baseline cardiac anatomy and contractile function in this
study were not different in Ephx2 knockout and wild-type
mice, suggesting that detrimental effects of environmental
stress to the heart in general (ischemia or pressure
Monti J et al.
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overload) can be counteracted by a decreased action of
Ephx2 together with the maintenance of high EET levels.
The Ephx2 locus accounted for almost two-thirds of the
Ephx2 expression variability in a large proportion in the
F2(SHHF × SHRSP) population. However, the association
of the Ephx2 locus with ejection fraction as a far more
complex phenotype was smaller in magnitude. Overall,
the Ephx2 locus accounted for about 15% of the
phenotypic variance for
demonstrated that Ephx2 transcript levels significantly
correlate with the tails of the distribution for ejection
fraction, providing independent evidence that the allelic
modulation of Ephx2 transcript levels plays a causal role
in the development of heart failure. Rats with high
transcript levels had lower ejection fractions, whereas rats
with lower transcript levels had higher ejection fractions. It
is important to note that allelic variants of Ephx2 appear
to impact heart failure as a complex disease through
perturbations of trans-regulated gene networks, and that
transcriptional phenotypes are again intermediate to the
clinically defined disease. We showed that transcriptional
regulation not only of Ephx2 but also of several other
genes in the entire Ephx2 pathway are affected by the
chromosome 15 QTL in our model. We applied pathway
linkage scores to assess the statistical significance of this
genetic effect and found a genome-wide significant
association of expression on a pathway level with the
maximum effect at the Ephx2 locus.
ejection fraction. We
To uncover DNA variants that can account for the
observed alterations of Ephx2 expression and function,
we performed comparative sequence analysis of Ephx2 in
heart failure and reference strains. We detected a
regulatory insertion/deletion polymorphism within the AP-
1 binding site and showed enhanced promoter activity in
SHHF. However, our findings do not preclude that other
regulatory variants contribute to the allele-specific
differences in Ephx2 expression besides this regulatory
insertion/deletion polymorphism in AP-1. Since AP-1
activation has also been demonstrated in human heart
failure , and Ang II serves to increase Ephx2
expression through AP-1 activation , it seems possible
that EPHX2 promoter variants affecting AP-1 binding may
also constitute a contributory mechanism for heart failure
susceptibility in humans. It would be interesting to test
variation at EPHX2 for association with human
hypertensive heart failure.
Ephx2 was established as a primary heart failure
candidate gene by combined evidence from linkage
analyses, global F2 transcription profiling, sequence
analysis and functional data on protein abundance and
Ephx2 enzymatic activity. Additional experimental support
for the specific relevance of Ephx2 in the genesis of
hypertension-induced heart failure was generated by
experiments in mice with targeted disruption of Ephx2. In
keeping with previous studies in two independently-
derived colonies of Ephx2 knockout mice, including the
one used in our experiment , we found no differences
in blood pressure and ejection fraction between knockout
and wild-type mice at baseline. However, after exposing
both strains to pressure-overload conditions, Ephx2
knockout mice showed only about half of the ejection
fraction decline seen in wild-type mice, although blood
pressure increases to high and prolonged dosages of Ang
II were similar in both strains. This situation resembles the
SHHF model, which develops heart failure while its
equally hypertensive cousin, the SHRSP, does not.
Furthermore, hypertensive Ephx2 knockout mice are
protected from cardiac arrhythmias. This is in agreement
with findings using pharmacological inhibition of Ephx2
 and may be explained by previous observations
suggesting that EETs are modulators of ion channel
function in cardiomyocytes (for review, see ref. 25).
We are the first to present evidence that Ephx2 is a heart
failure susceptibility gene. Ephx2 allelic variability leads to
quantitative differences in transcript levels that in turn
quantitatively regulate the metabolism of cardioprotective
epoxyeicosatrienoic acids. The implications of our findings
are important as Ephx2 has already found attention as a
pharmacological target for cardiovascular disease . In
light of elevated Ephx2 expression levels associated with
the development of heart failure in rats, and protection
from heart failure in Ephx2-deficient mice, we interpret
EPHX2 downregulation in human failing hearts as an
adaptive transcriptional process aimed at maintaining high
EET levels. EETs are known to exert cardioprotective
actions . Conversely, individuals with genetically
determined, persistently high cardiac EPHX2 expression,
together with arterial hypertension and LVH, probably
represent a high-risk group for disease progression to
heart failure. The role we have shown for Ephx2 in the
complex initiation of the disease in rats and mice, as well
as its regulation in human heart failure, suggest a
potential avenue for developing new heart failure
Experimental crosses and mice. SHHF/Bbb, WKY/Bbb
and SHRSP/Bbb rats were from our colonies at the Max-
Delbrück Center for Molecular Medicine and were
maintained under strict inbreeding. SHHF breeders
(SHHF/Mcc) were initially obtained from the original
colonies of S.A. McCune (Department of Integrative
Physiology, University of Colorado at Boulder). SHHF
were mated with SHRSP and WKY, respectively, to
generate F1 progenies. F1 rats for each cross were
brother-sister mated, resulting in 166 F2(SHHF × SHRSP)
(87 male and 79 female) and 189 F2(SHHF × WKY) (93
male and 96 female) rats. All rats were investigated at 14
months of age. Ephx2- /- mice were previously described
 and were further backcrossed for nine generations
onto C57BL/6ByJ before
Heterozygote F9 mice were crossed to obtain homozygote
null and wild-type littermates used in this study. All mouse
experiments were performed starting at 16 weeks of age;
only male mice were used. All animal experiments were
conducted according to the guidelines of the American
Physiological Society and with approval of the animal
protection authorities of the state of Berlin (Landesamt Für
Gesundheit und Soziales).
used in our studies.
Hemodynamic measurements. Rats were intubated and
ventilated under isoflurane anesthesia. A 2-French
conductance catheter (SPR 838 Aria, Millar Instruments)
was inserted into the left ventricle through the right carotid
artery. Left ventricular contractility was obtained from the
ventricular pressure curves. Ventricular volumes were
determined as described .
Telemetry. Telemetric blood pressure measurements
were performed as previously described . The data
from the TA11PA-C20 device were sampled every 5 min
for 10 s continuously day and night with a sampling rate of
1,000 Hz. Mice were allowed to recover for three days
Monti J et al.
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before baseline recording started. SBP, DBP and HR
were recorded using the DATAQUEST software.
Echocardiography. Echocardiography was performed
before and after 3 weeks of Ang II treatment as previously
described . Briefly, mice were anesthetized with 2%
isoflurane and kept warm on a heated platform.
Temperature and ECG were continuously monitored.
Cardiac function and morphology were assessed by
echocardiography with a VisualSonics Vevo 770 High-
Resolution Imaging System with the use of a high-
resolution (40 MHz) transducer.
Surface ECG and in vivo electrophysiology studies.
Each mouse was slightly anaesthetized with 1.6%
isoflurane and surface Electrocardiograms (ECG) were
obtained using adhesive electrodes on each limb after 3
weeks of Ang II treatment. ECG standard intervals were
measured in six standard
electrophysiological studies (EPS) with programmed
electrical stimulations (PES) were performed at baseline
and after isoproterenol treatment (intraperitoneal injection
of 0.02mg isoproterenol/g body weight). Briefly, an
octapolar 2-French electrode catheter (CIBer mouse cath,
NuMed Inc.) was placed via the right jugular vein into the
right atrium and ventricle under electrogram guidance.
Electrical stimuli were delivered at twice diastolic
threshold with a pulse duration of 1.0 ms. PES used up to
three extra stimuli after 10 basal stimuli (cycle length
90/100/120 ms). Additionally, short episodes of burst
pacing (10 s) with cycle lengths down to 60 ms were
applied. Only reproducible arrhythmias of three beats or
more were considered. All electrograms were digitally
stored for off-line analysis.
Marker selection and genotyping. SNPs were identified
by the STAR consortium and are publicly available at
http://www.ensembl.org. SNP genotyping in F2 crosses
was carried out using the GoldenGate genotyping
platform (Illumina Inc., San Diego). Microsatellite markers
were genotyped using
automated genotyping on an ABI3730 sequencer. SNP
and microsatellite genotyping results are reported in
Suppl. Table 2 online. Statistical methods for genetic
mapping and linkage analysis are reported in Suppl.
Preparation of labeled cDNA and hybridizations to
microarrays. We pulverized whole frozen hearts and
stored aliquots at - 80 °C for RNA and - 20 °C for protein
experiments. Total RNA was extracted using Trizol
reagent (Invitrogen) and purified using RNeasy Mini kit
(Qiagen) in accordance with the manufacturer's protocol.
Double-stranded cDNA was synthesized from total RNA
using the One-Cycle cDNA Synthesis Kit (Affymetrix) and
cRNA biotinylated from cDNA using the IVT Labeling Kit
(Affymetrix). We hybridized the fragmented cRNA
samples to Affymetrix Rat Genome 230 2.0 arrays in
accordance with the Affymetrix protocol. Statistical
methods for eQTL analysis are reported in Suppl.
Real-time RT-PCR. We reverse-transcribed total RNA
with oligo(dT) primers (Gibco-BRL) and Superscript II
reverse transcriptase (Invitrogen). We designed primers
and probes using Primer Express 2.0 (Applied
Biosystems) and placed them in non-polymorphic regions
of the rat Ephx2 gene (Suppl. Table 3 online). For human
TaqMan probes, Applied
(hs00157403_m1) was used. TaqMan analysis was
performed using an Applied Biosystems 7900HT system.
We normalized expression levels to GAPDH RNA
expression by using the 2- ΔΔCT method.
Analysis of Ephx2 hydrolase activity. Hydrolase
activities were determined by using 10 μg of heart-
extracted proteins (10,000g supernatant) dissolved in 100
mM potassium phosphate buffer, pH 7.2. Proteins were
incubated with 10 μM [1-14C]14,15-EET for 5 min at 37 °C.
Adding ethyl acetate terminated the reaction. After
extraction, products were evaporated under nitrogen and
dissolved in ethanol. Metabolites were resolved by
reverse-phase HPLC (Shimadzu LC 10 Avp). Retention
times for 14,15-EET/-DHET were 23.4 and 12.7 min,
respectively. Methods for protein blot analyses and
production of recombinant proteins are reported in Suppl.
Promoter activity assays. The - 2079 to +41 bp Ephx2
promoter was cloned into a pGL3-Basic vector (Promega)
containing the firefly luciferase gene as a reporter
encompassing either the SHHF or the SHRSP promoters,
and integrity was confirmed after cloning by sequence
analysis. Since SHRSP and WKY share common
haplotypes, we cloned only the promoter variants from
one of the strains (SHRSP) and tested the variants
against the SHHF promoter. For transient transfections,
H9c2 (ATCC) cells were cotransfected with pRL-SV40
plasmid (Promega) as a normalizing control. All
transfections were carried out in triplicates. Firefly and
Renilla luciferase activity was analyzed with the Dual-
Luciferase Reporter Assay
manufacturer's description (Promega).
System according to
Electrophoretic mobility shift assay. Binding of AP-1 to
the Ephx2 promoter in vitro was carried out as previously
described . Briefly, synthetic double-stranded and 3'
biotin-labeled oligonucleotides and recombinant protein
(Promega) were incubated at room temperature for 20
min using the LightShift Chemiluminescent EMSA Kit
(Pierce). The binding reaction contained × 1 binding
buffer, 0.01 μg/μl poly(dI:dC), 0.05% NP-40, 50 mM KCl,
0.5 mM MgCl2 and 0.5 mM DTT. Two-hundred-fold molar
excesses of unlabeled oligonucleotides were used as
competitors. Reaction mixtures were separated with 5%
polyacrylamide gels. Probes were transferred to a
positively charged nylon membrane (Ambion), UV-cross-
linked and detected by stabilized Streptavidin-Horseradish
Peroxidase Conjugate (Pierce).
Quantification of EETs in the heart. Heart tissue was
homogenized using a biopulverizer (Biospec Products).
40 mg aliquots were subjected to alkaline hydrolysis,
followed by a solid phase extraction. Quantification of
EETs in the heart homogenates was done by the
Lipidomix GmbH, Berlin, using HPLC (Agilent 1200) with
tandem mass spectroscopy Triple Quad MS/MS (Agilent
6410). Detection limit for EETs was 0.2 ng per gram of
Human myocardial biopsies for mRNA exxpression
analysis. Full thickness myocardial biopsies were
harvested at the time of cardiac surgery with the approval
of the Hammersmith Hospital Research Ethics Committee.
Heart function was characterized by trans-thoracic
disease but having normal ventricular function were used
as controls. Individuals with heart failure were defined as
an ejection fraction of <45% secondary to ischemic heart
disease. Written informed consent was obtained from all
with coronary artery
Monti J et al.
MDC Repository | http://edoc.mdc-berlin.de/9531/ 7
http://www.ensembl.org and microsatellite markers at
SNPs used are available at
Accession codes. Scanned microarray data have been
submitted to ArrayExpress with accession code E-TABM-
418. Ephx2 DNA variants have been submitted to dbSNP:
ss86236892, ss86236893, ss86236894, ss86236895,
ss86236896, ss86236897, ss86236898, ss86236899 and
We thank A. Müller, H. Kistel, A. Schiche, J. Mothes, J.
Meisel, M. Rothe and M. Taube for technical assistance.
We acknowledge funding to N.H. from the German
Ministry for Science and Education (National Genome
Research Network) and support through STAR and
EURATools (European Commission contract LSHG-CT-
2004-005235 and LSHG-CT-2005-019015); to F.C.L. and
W.-H.S. from the Deutsche Forschungsgemeinschaft; to
B.D.H. from US National Institute of Environmental Health
Sciences grant R37 ES02710; and to S.A.C. from the
British Heart Foundation and the UK Department of
N.H. and J.M. developed the project. N.H. directed the
project. J.F., S.P., C.G., C.S., H.M., G.P., K.S., S.A.C.
and W.-H.S. performed genetic and biochemical analysis.
J.M., A.H., R.F., A.S., V.G., R.D. and F.C.L. performed
and analyzed physiological experiments. M.H., H.S.,
O.H., M.V. and K.R. carried out statistical analysis.
B.D.H., S.M.W., K.L. and S.A.C. contributed materials.
N.H. wrote the paper with J.M., J.F. and F.C.L.
Max-Delbrück Center for Molecular Medicine, Robert-
Rössle-Strasse 10, 13125 Berlin, Germany.
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Figure 1. Colocalization of cardiac phenotype QTL on chromosome 15. Multipoint lod plots in F2(SHHF × SHRSP) for ejection fraction,
dP/dtmax, LVEDV. Lod scores are plotted against genetic map distances in cM. Gray marks on x axis represent marker positions.
Genome-wide corrected significance levels (P = 0.05) were determined by 1,000 permutations. The dashed horizontal line indicates
genome-wide significance for ejection fraction linkage; the thick and thin solid red bar indicates 1- and 2-lod-unit drop confidence
interval, respectively. Significance levels for the other traits reported are slightly different and are not depicted for clarity (genome-wide
significance at lod 3.2 and 3.5 for dP/dtmax and LVEDV, respectively). The composite trait variable constructed from the three traits
notably increased the significance of the findings.
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Figure 2. Polymorphisms in Ephx2 regulatory and coding region. Four polymorphisms were identified in the putative promoter region, of
which a 2-nt insertion in SHRSP and WKY was directly located in an AP-1 binding site (arrow). Five SNPs were detected in Ephx2
exons, three of which led to an amino acid change (marked in parentheses). We found no variation in the 3' UTR. Exons are pictured as
black boxes. In all instances, SHRSP and WKY shared the same allele and haplotype at Ephx2. A variant allele status was observed for
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Figure 3. Ephx2 expression and sEH activity in heart tissue. (a) Ephx2 expression measured using microarrays in parental strains (n =
5 per group) and F2 rats according to their genotype at D15Rat10: SHRSP/SHRSP (SP/SP), n = 29; SHHF/SHRSP (HF/SP), n = 76;
SHHF/SHHF (HF/HF), n = 32. (b) RT-PCR to confirm microarray expression results; parental strains (n = 5 per group) and F2 rats
according to their genotype at D15Rat10 (SHRSP/SHRSP n = 10; SHHF/SHRSP n = 10; SHHF/SHHF n = 10). (c) Representative blot
of sEH protein in F0 and F2 rats. Membranes were stripped and rehybridized with an antibody to α-tubulin as a loading control.
Quantification of the membrane for F2 rats is shown next to blots. (d) sEH hydrolase activity per milligram of heart tissue in parental (n =
5) and F2 rats (n = 10 per genotype). Enzymatic activity was determined by measuring the hydrolysis of 14,15-EET, which is the
preferred substrate of sEH and a prominent metabolite among regioisomeric EET present in rat heart tissue, to 14,15-
dihydroxyeicosatrienoic acids (14,15-DHET). (e) 14,15-EET per gram of heart tissue in parental (n = 5) and F2 rats (n = 10 per
genotype). Data are given as mean ± s.d. (*P < 0.05, **P < 0.01; ***P < 0.001).
Monti J et al.
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Figure 4. Promoter polymorphisms of Ephx2 influence promoter activity. (a) Schematics of reporter constructs used for transfection of
H9c2 cells and, below, luciferase expression of each construct and empty pGL3-Basic vector in H9c2 cells. Each value represents
mean ± s.d. of at least three experiments; ***P < 0.001. RLU, relative light units. (b) Top, sequence alignment of SHHF, SHRSP and
WKY Ephx2 promoter sequences containing the AP-1 binding sequence. Computational analysis predicts that, in SHHF, the AP-1
consensus sequence is completely restored, suggesting that it was intact ancestrally, whereas, in SHRSP and WKY, a 2-bp insertion
disrupts AP-1 binding. Bottom, an electrophoretic mobility shift assay with biotin-labeled probes. Lanes 1 and 4, probes only; lanes 2, 3,
5 and 6, probes and AP-1 recombinant protein; lanes 3 and 6, probes, AP-1 recombinant protein and unlabeled competitor.
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Figure 5. Identification of regulatory trans eQTLs mapping to chromosome 15. A simplified overview of the Ephx2 pathway is depicted.
Each box is headed by an enzyme classification number describing the reaction that is catalyzed. All genes analyzed with the
corresponding enzymatic function are listed in the boxes below. For each gene, a color code indicates the genotype-specific average
expression values according to zygosity—SHHF/SHHF (HF/HF), SHHF/SHRSP (HF/SP) and SHRSP/SHRSP (SP/SP), from left to
right—at the Ephx2 locus. The expression matrix has been centered and scaled to σ= 1 before visualization. Orange, overexpression;
white, baseline; cyan, underexpression. CYP genes are subdivided into two columns, with the gene products of those on the left
producing predominantly EETs, which are converted to dihydroxyeicosatrienoic acids (DHETs) by sEH, and the right producing
predominantly hydroxyeicosatetraenoic acids (HETEs). The Ephx2 pathway has its genome-wide pathway linkage peak at the Ephx2
locus with P < 10- 6.
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Figure 6. Characterization of Ephx2- /- mice by telemetry and echocardiography. (a) Blood pressure measurements (mean arterial
pressure, MAP) in conscious mice (values given as mean ± s.d.; n = 4 per group). (b) Ejection fraction, measured by echocardiography
(mean ± s.d.), in Ephx2- /- (n = 17) and wild-type (n = 15) mice before and after Ang II treatment; **P < 0.01. (c) Examples of M-mode
echocardiography in Ephx2- /- and wild-type mice after Ang II treatment. Blue vertical lines, left ventricular end-systolic diameter; orange
vertical lines, left ventricular end-diastolic diameter.
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Figure 7. Electrophysiological characterization of Ephx2- /- and wild-type mice after Ang II treatment (n = 4 per group). (a,b) Marked
prolongation of ventricular refractoriness (a) and spontaneous premature ventricular contractions (b,*) were seen in all wild-type mice
but could not be observed in any Ephx2 null mice (values given as mean (central line) ± s.d.; wide bar, minimum and maximum values).
Recordings from a single representative mouse from each group are given. The lower trace for ECG recordings in a wild-type mouse
represents a magnification of the boxed area from the upper trace. (c) Representative recordings from in vivo electrophysiological
studies. Programmed stimulation (stim) in the right ventricle of wild-type mice induced non-sustained atrial fibrillation, indicated by the
high frequency signals in the right atrium (RA) and the variability in R–R intervals in the surface ECG (upper panel). Stimulation also
induced ventricular tachycardia in wild-type mice. High rate pacing in the right ventricle (RV) is followed by non-sustained fast ventricular
tachycardia, eventually recovering into normal sinus rhythm (middle panel). Programmed stimulations could not induce arrhythmias in
Ephx2- /- mice (lower panel).
Monti J et al. Download full-text
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Figure 8. EPHX2 mRNA expression is downregulated in individuals with heart failure. Ejection fraction in individuals with heart failure
was severely reduced (n = 6 per group; ejection fraction 29% ± 5% in affected individuals versus >50% in controls), although systolic
and diastolic blood pressures were not different between groups. EPHX2 mRNA expression was determined using quantitative TaqMan
RT-PCR (values given as mean ± s.d.; n = 6 per group); *P = 0.025.