Targeted Deletion of the Extracellular Signal-Regulated

Protein Kinase 5 Attenuates Hypertrophic Response and

Promotes Pressure OverloadInduced Apoptosis in
the Heart
Tomomi E. Kimura,* Jiawei Jin,* Min Zi,* Sukhpal Prehar, Wei Liu, Delvac Oceandy, Jun-ichi Abe,
Ludwig Neyses, Arthur H. Weston, Elizabeth J. Cartwright, Xin Wang
Rationale: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and
intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical
MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating
hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.
Objective: Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac
hypertrophy and hypertrophy-induced apoptosis.
Methods and Results: We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of
the erk5 gene (ERK5cko). In response to hypertrophic stress, ERK5cko mice developed less hypertrophic growth
and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and
Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific
inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte
enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the
inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate
the ERK5-regulated hypertrophic response.
Conclusions: These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and
cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation
of MEF2 activity. (Circ Res. 2010;106:961-970.)
Key Words: cardiac hypertrophy signal transduction genetically modified mice

ardiac hypertrophy is a virtually universal prerequisite

for the development of heart failure. In response to acute
or chronic insults the heart initially develops hypertrophic
growth. However, sustained stress causes chamber dilation,
interstitial fibrosis, and myocyte apoptosis, eventually leading to heart failure or sudden death from arrhythmias.
Numerous signaling pathways including mitogen-activated
protein kinases (MAPKs) are implicated in mediating the
process of cardiac hypertrophy and hypertrophy-induced
cardiomyocyte apoptosis.1 In the heart, all 4 classes of
MAPKs are activated through either mechanical overload or
neurohumoral stimulation.2 However, our understanding of
the critical role of individual MAPK in various aspects of
hypertrophic remodeling remains fragmented and in many
cases controversial.

Extracellular signal-regulated protein kinase (ERK)5 is

an atypical MAPK, also known as big MAPK (BMK)1,
because it is more than twice the size of the other MAPKs,
owing to a very large C-terminal domain.3 It is suggested
that the role of this unique C-terminal tail is to regulate
myocyte enhancer factor (MEF)2 transcriptional activity
and ERK5 subcellular localization.3,4 On exposure to
various stimuli, ERK5 is activated by phosphorylation
downstream of MEK5.5
An important step in deciphering the function of ERK5
is to identify its downstream targets and MEF2A, -C, and
-D are among the best-characterized substrates of ERK5.4,5
Analogous to many MAPKs, ERK5 plays an important role
in cell survival, cell proliferation and differentiation. The
discovery that ERK5 is an important contributor to cell

Original received September 16, 2009; revision received January 4, 2010; accepted January 6, 2010.
From the Faculty of Life Sciences (T.E.K., J.J., W.L., A.H.W., X.W.) and Faculty of Medical and Human Sciences (M.Z., S.P., D.O., L.N., E.J.C.),
University of Manchester, United Kingdom; and Aab Cardiovascular Research Institute (J.-i.A.), University of Rochester School of Medicine and
Dentistry, NY.
*These authors contributed equally to this work.
These authors contributed equally to this work as senior authors.
Correspondence to Dr Xin Wang, Faculty of Life Sciences, University of Manchester, CTF Building, 46 Grafton St, Manchester M13 9NT, United
Kingdom. E-mail xin.wang@manchester.ac.uk
2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org

DOI: 10.1161/CIRCRESAHA.109.209320

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Non-standard Abbreviations and Acronyms

Ad
ANP
BMK
BNP
Col12
Col31
CREB
CTGF
ERK5
FS
HIF
HW/TL
ISO
JNK
MAPK
MEF
MEK5
Myh6
Myh7
NRCM
PKB
siRNA
TAC

adenovirus
atrial natriuretic peptide
big mitogen-activated protein kinase
brain natriuretic peptide
procollagen type I, 2
procollagen type III, 3
Ca2/cAMP response element-binding protein
connective tissue growth factor
extracellular signal-regulated protein kinase 5
fractional shortening
hypoxia inducible factor
heart weight/tibia length
isoproterenol
c-Jun N-terminal kinase
mitogen-activated protein kinase
myocyte enhancer factor
mitogen-activated protein kinase kinase 5

myosin heavy polypeptide

myosin heavy polypeptide
neonatal rat cardiomyocyte
protein kinase B
small interfering RNA
transverse aortic constriction

survival has increased interest in this signaling pathway. In

sympathetic neurons, ERK5 is required to mediate the
survival response to nerve growth factor by suppressing
the expression of Bad and Bim.6 The analysis of mice with
an endothelial-specific deletion of ERK5 provides physiological evidence that ERK5 is essential for the survival of
endothelial cells via the activation of MEF2.7 Moreover,
recent studies have demonstrated that ERK5 activation
prevents cardiomyocyte apoptosis, likely through the inhibition of a feedback loop of phosphodiesterase 3A/inducible cAMP early repressor.8 Apart from its role in cardiomyocytes survival, ERK5 has also been implicated in
regulating cardiac hypertrophy.9,10
ERK5 can be activated in the heart by pressure overload
and by hypertrophy-stimulating factors including leukemia
inhibitory factor, adrenergic agonists, oxidative, and osmotic stresses in cultured cardiomyocytes.11,12 However,
previous studies of transgenic mice with cardiac-specific
overexpression of activated MEK5 provided conflicting
results. Decompensated eccentric hypertrophy with no
cardiomyocyte apoptosis was observed in MEK5 transgenic mice, which is in stark contrast to the normal cardiac
function and morphology found in MEK5 transgenic
mice.12,13 On hypertrophic stress, MEK5 transgenic mice
develop hypertrophic growth to a similar degree to controls but with reduced apoptosis and better cardiac function.8 Regarding the capacity of activating ERK5, we have
demonstrated that MEK5 is an active enzyme, whereas
MEK5 is a stronger activator because of its higher

affinity for ERK5.14 In contrast, Cameron et al proposed

that MEK5 is a dominant negative variant that can
suppress ERK5 signaling.15 Moreover, expression of
MEK5 has been suggested to be restricted to brain and
liver, whereas MEK5 is ubiquitously expressed.16 Given
these contradictions emerging from MEK5 studies, it is
necessary to investigate the function of ERK5 in the heart
using cardiomyocyte-specific ERK5 deletion mice
(ERK5cko), a physiologically relevant model compared to
the overexpressing transgenic lines. Hayashi et al have
previously generated and described a cardiomyocytespecific ERK5 deletion mouse model.7 In concordance
with their observation, we also found ERK5cko mice
(generated in our laboratory) were viable, fertile, and
appeared normal, suggesting the specific ablation of erk5
in cardiomyocytes alone does not affect cardiac development. To characterize the role of ERK5 in cardiac hypertrophy we subjected ERK5cko mice to hypertrophic stimuli,
the mutant mice developed less hypertrophic growth and
fibrosis compared to controls (ERK5f/f), also increased
apoptosis with upregulated expression of p53 and Bad
were observed in the mutant heart. Consistently, we found
that silencing ERK5 expression or specific inhibition of its
kinase activity using a novel compound BIX02189 in
neonatal rat cardiomyocytes (NRCMs) reduced MEF2
transcriptional activity and blunted the hypertrophic response. Moreover, the inhibition of MEF2 activity in
NRCMs using a non-DNA binding mutant form of MEF2C
was found to diminish the ERK5-regulated hypertrophic
response. These results clearly reveal an important function of ERK5 in stress-induced cardiac hypertrophic remodeling and cardiomyocyte survival.

Methods
Cardiac hypertrophy was induced by transverse aortic constriction
(TAC) or chronic infusion of isoproterenol (ISO) (Sigma-Aldrich) at
10 mg/kg per day for 7 days in 8- to 10-week-old male ERK5f/f and
ERK5cko mice as previously described.17
See the expanded Methods section in the Online Data Supplement
(available at http://circres.ahajournals.org) for the following: generation of ERK5cko mice, echocardiography, histological analysis,
immunoblot analysis, small interfering (si)RNA transfection, adenoviral infection, apoptosis assays, quantitative PCR, luciferase reporter assay, and [3H]leucine incorporation.

Results
Characterization of ERK5cko Mice

Using myosin heavy chain promoter driven-Cre transgenic

mice, we generated ERK5cko mice. Normal levels of ERK5
mRNA were present in brain and liver, whereas ERK5
mRNA was significantly reduced (approximately 86%) in the
mutant ventricle (Online Figure I, A). Immunoblot analysis of
ventricular extracts from ERK5cko mice at 8 weeks old
showed more than 80% deletion of ERK5 protein (Online
Figure I, B). It was evident that ablation of ERK5 was
specific to the heart because protein levels of ERK5 were
equivalent in brain, liver and skeletal muscle from both
ERK5cko and ERK5f/f mice (Online Figure I, B). The absence

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Figure 1. Less response to 1 week

TAC induced-hypertrophy in ERK5cko
mice. A, Hematoxylin/eosin staining of
heart cross-sections (scale bar: 20 m).
B, HW/TL ratios of ERK5f/f and ERK5cko
mice were calculated following TAC or
sham operation, n7 to 9 per group. C,
The mean cross-sectional areas of cardiomyocytes from two genotypes were
measured, n5. D, Massons trichromestaining of cross-sections shows less
interstitial fibrosis in ERK5cko hearts.
Arrows indicate areas of fibrosis. Quantification of the relative area of fibrosis is
expressed as percentage of the fibrosis
area in the microscope views, n7. Data
are presented as meansSEM.

of ERK5 in the heart did not cause any compensatory changes

in the protein levels of other MAPKs, such as ERK1/2, c-Jun
N-terminal kinase (JNK), p38, and MEK5 (Online Figure I,
C). Furthermore, cardiac structure and contractile function
were examined by histological analysis and echocardiography, respectively, and no differences were found between the
two genotypes (data not shown).

ERK5cko Mice Displayed Less Cardiac

Hypertrophic Remodeling on Pressure Overload
To determine whether ERK5 is required for pressure
overload-induced cardiac hypertrophic remodeling, ERK5f/f
and ERK5cko mice were subjected to pressure overload
stimulation by fallowing 1 week of TAC, ERK5f/f mice
showed a 34% increase in heart weight/tibia length (HW/TL).
In contrast, ERK5cko mice showed only a 23% increase in
HW/TL (Figure 1A and 1B). Consistent with these results,
greater cross-sectional area of cardiomyocytes was observed
in ERK5f/f-TAC mice (280.056.27 m2), in comparison to
256.518.03 m2 in ERK5cko-TAC mice (Figure 1C). Pressure overloadinduced hypertrophy is often accompanied by
interstitial fibrosis. Based on Massons trichrome staining,
less ventricular fibrosis was seen in ERK5cko-TAC hearts
(Figure 1D). Furthermore, cardiac structure and function
were assessed by echocardiography. In response to TAC,
ERK5cko mice showed a significant reduction in end-diastolic
left ventricular posterior wall thickness compared with the
control group. Meanwhile, we also found in the mutant hearts

a trend toward a decrease in fractional shortening percentage

(FS%) and a markedly slowed aorta maximum velocity,
which is a sign of contractile dysfunction (Table). Fetal gene
activation is a hallmark of hypertrophic remodeling. As
shown in Figure 2, upregulation of the transcripts of the
hypertrophic gene markers atrial natriuretic peptide (ANP),
brain natriuretic peptide (BNP), skeletal -actin (Acta1), and
-myosin heavy polypeptide (Myh7) was remarkably blunted
in the mutant hearts following TAC; we also found the
Table. Echocardiographic Assessment of ERK5f/f and ERK5cko
Mice After One Week of TAC
Sham
f/f

ERK5

TAC
CKO

ERK5

f/f

ERK5

ERK5CKO

dPW (mm)

0.750.03

0.750.06

0.990.05

0.840.03*

dIVS (mm)

0.880.01

0.850.04

1.130.08

1.150.06

LVEDD (mm)

4.020.04

3.880.13

3.780.18

3.990.17

LVESD (mm)

2.660.04

2.690.09

2.550.17

2.950.24

FS (%)

34.10.5

30.42.1

32.81.9

27.12.9

Ao Vmax (cm/sec)

63.45.3

62.72.7

65.74.3

56.32.1*

Ratio of E/A wave

1.150.09

1.260.17

1.430.21

1.440.13

Ao Vmax, aorta maximum velocity; dPW, end-diastolic left ventricular

posterior wall thickness; dIVS, end-diastolic interventricular septal wall thickness; FS, fraction shortening; LVEDD, diastolic left ventricular internal dimension; LVESD, systolic left ventricular internal dimension. Data are presented as
meansSEM (n7 to 9 per group). *P0.05, ERK5cko-TAC mice vs ERK5f/fTAC mice.

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Figure 2. Quantitative realtime PCR analyses of gene

markers associated with hypertrophy and fibrosis after 1
week of TAC. The data are
derived from three independent experiments performed in
triplicate and are normalized to
the GAPDH content (n3 to 5
per group). Data are presented
as meansSEM.

mRNA level of myosin heavy polypeptide (Myh6) was

compromised in the mutant hearts (Figure 2). Consistent with
Massons trichrome staining, the fibrosis marker genes connective tissue growth factor (Ctgf), procollagen type I, 2
(Col12) and procollagen type III, 3 (Col31) were significantly downregulated in ERK5cko-TAC mice compared with
the controls (Figure 2). Finally, we investigated the activation of
ERK5 and an array of hypertrophic regulators such as ERK1/2,
protein kinase (PK)B, p38, and JNK after TAC. ERK5 was
activated by TAC in ERK5f/f hearts but not in ERK5cko hearts,
meanwhile there was no difference observed in the activation of
other hypertrophic regulators between the two genotypes although all were activated following TAC (Figure 3). These
results suggest that the blunted hypertrophic response in the
mutant mice is likely attributable to the absence of ERK5 in the
heart.

TAC (Figure 4A through 4C). In accordance with this

finding, ERK5cko-TAC mice showed augmented protein
levels of Bad and p53, whereas the expression level of
Bcl-2 was comparable in both genotypes (Figure 4D). A
recent study demonstrated a protective role of ERK5 in
sympathetic neurons through the phosphorylation of Ca2/
cAMP response element-binding protein (CREB), thereby
suppressing the transcription of Bad.6 However, we did not
observe any change in the protein level and phosphorylation status of CREB in the mutant hearts (Figure 4D).
Furthermore, in the two sham groups, no difference was
detected in expression levels of these apoptosis-associated
proteins (Online Figure II). Together, these data suggest
that ERK5 is required for cardiomyocyte survival in
response to pressure overload by suppressing the
mitochondrion-dependent apoptotic pathway.

ERK5cko Cardiomyocytes Were More Vulnerable

Under Hypertrophic Stress

Prolonged Pressure Overload Caused Cardiac

Dysfunction in ERK5cko Mice

Sustained hypertrophic stress causes cardiomyocyte apoptosis which contributes to the transition of pathological
hypertrophy to heart failure. To determine the effect of
ERK5 on cardiomyocyte viability under hypertrophic
stress TUNEL staining and caspase 3 activity assays were
performed to detect cardiomyocyte apoptosis. We observed increased TUNEL-positive nuclei along with enhanced caspase 3 activity in the mutant hearts following

To further examine whether the lack of ERK5 in cardiomyocytes predisposes mice to heart failure following long-term
pressure overload stimulation, ERK5f/f and ERK5cko mice
were subjected to 5 weeks of TAC. Likewise, 5 weeks of
TAC also caused compromised HW/TL ratio, smaller myocyte cross-sectional areas, and less fibrosis in ERK5cko mice
compared with the controls (Figure 5A through 5C). Analysis
of gene expression profile showed that mRNA levels of ANP,

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Interstitial fibrosis was also more noticeable in the control

hearts (Online Figure IV). Furthermore, the transcript levels
of ANP and BNP were found to be remarkably elevated in the
ERK5f/f hearts, compared with the mutant hearts (Online
Figure IV). These results indicate that ERK5 regulates
cardiac hypertrophic remodeling not only by pressure overload but also by chronic -adrenergic stimulation.

Knockdown of ERK5 or Inhibition of Its

Activation Suppressed MEF2 Transcriptional
Activity and Promoted Apoptosis in NRCMs

Figure 3. Analysis of hypertrophic regulators in ERK5ckoTAC hearts. Protein extracts from ERK5f/f and ERK5cko hearts
subjected to TAC or sham operation were examined by immunoblotting for total ERK5, ERK1/2, PKB, p38, and JNK expression, as well as their phosphorylation levels using specific antibodies. The results suggest that blunted hypertrophic response
in the mutant mice is specifically attributable to the absence of
ERK5 in the heart.

BNP, Myh7, Acta1, Ctgf, Col12, and Col31 were significantly lower in ERK5cko-TAC hearts (Online Figure III).
Furthermore, a much more pronounced apoptosis was detected in ERK5cko cardiomyocytes (Figure 5D). Interestingly,
we observed detrimental changes in ERK5cko cardiac function after 5 weeks of TAC, which was evident by significant
decreases in FS% and aorta maximum velocity (Figure 5E).
Collectively, these data demonstrate the functional importance of ERK5 in protecting the heart from failure following
chronic pressure overload stimulation.

ERK5cko Mice Showed an Attenuated Cardiac

Hypertrophic Response to Chronic
-Adrenergic Stimulation
We next investigated whether ERK5 is required for cardiac
hypertrophic remodeling in response to chronic -adrenergic
stimulation. After 7 days administration of ISO, ERK5cko
mice developed blunted hypertrophy reflected by a 32%
increase in HW/TL, compared with the controls (48%).
Consistently, ERK5cko mice showed a reduction in crosssectional area (254.353.37 m2), compared with ERK5f/f
mice whose cardiomyocyte cross-sectional area was
306.75.4 m2 after the ISO-treatment (Online Figure IV).

MEF2 transcription factor is a key regulator of pathological

cardiac hypertrophy.18 Led by the result that the transcript
levels of Ctgf, Myh6, and Acta1 (known to be downstream
targets of MEF218 20) were compromised in ERK5cko-TAC
hearts we further examined whether MEF2 transcriptional
activity was mediated by ERK5 in cardiomyocytes in response to hypertrophic stimuli. Using the MEK5/ERK5selective inhibitor (BIX02189, 10 mol/L), ERK5 phosphorylation was specifically inhibited which led to attenuated
MEF2 activity in NRCMs following ISO treatment (Figure
6A and 6B). To corroborate this result, NRCMs were transfected with the reporter plasmid pG5E1bLuc together with a
construct encoding Gal4, Gal4-MEF2A, or Gal4-MEF2D. As
expected, reduced MEF2 transcriptional activity was detected
in BIX02189-treated NRCMs after ISO stimulation (Online
Figure V). In parallel, using an siRNA method we specifically suppressed endogenous ERK5 expression by 83% in
NRCMs, a similar inhibitory effect on MEF2 transcriptional
activity was also found (Figure 6C and 6D; Online Figure V).
Furthermore, siERK5-treated NRCMs showed an impaired
hypertrophic response which was evident by decreased
mRNA levels of ANP and Acta1 and diminished protein
synthesis (Figure 7A and 7B). To further critically examine
the effect of MEF2 on ERK5-mediated hypertrophic response, an adenovirus containing a non-DNA binding mutant
form of MEF2C (Ad-MEF2C-R3T) was used to inhibit
MEF2 activity.8,21 After ISO stimulation, we observed an
increased rate of protein synthesis and elevated transcript
level of ANP in Ad-ERK5-infected NRCMs; however, such
hypertrophic increases were appreciably blunted by the coinfection of Ad-MEF2C-R3T (Figure 7C through 7E). Combined, these data strongly suggest that MEF2 acts downstream of ERK5 to promote a hypertrophic response. Lastly,
we evaluated the effect of ERK5 knockdown or the inhibition
of its kinase activation on apoptosis in NRCMs. Consistent
with apoptosis results obtained from ERK5cko-TAC mice,
increased apoptosis was detected in siERK5-treated or
BIX02189-treated NRCMs on exposure to the proapoptotic
agent sorbitol, which confirms the protective role of ERK5 in
cardiomyocytes (Online Figure VI).

Discussion
The major finding of this study is that ERK5 plays an
important role in promoting hypertrophic remodeling and
cardiomyocyte survival. In the absence of ERK5, hypertrophic growth, interstitial fibrosis, and fetal gene reactivation

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Figure 4. ERK5 is required for cardiomyocyte survival in response to

hypertrophic stress. A, Increased apoptosis in ERK5cko ventricular myocardium after 1 week of TAC was detected
by TUNEL assay (scale bar: 50 m). Triple staining was performed: TUNEL
(green), Hoechst (blue), -actinin (red).
Arrows point to TUNEL-positive nuclei.
B, The bar graph summarizes the percentage of TUNEL-positive nuclei in
ERK5cko hearts compared with that in
control hearts (n7 to 11 per group). C,
Caspase 3 activity was measured by
caspase activity assay after TAC (n7).
D, Immunoblot analyses of protein levels
of Bad, Bcl-2, p53, and CREB and phosphorylation of CREB by specific antibodies. Tubulin expression is the protein
loading control. The ratios of Bad/Bcl-2
and p53/tubulin are represented by the
bar graphs (n4 per group). Data are
meansSEM.

were profoundly compromised; meanwhile, increased cardiomyocyte death was seen following hypertrophic stimuli.
Further studies in NRCMs treated with either siERK5 or a
novel ERK5 kinase inhibitor (BIX02189) have provided clear
evidence that MEF2 is a critical component downstream of
the ERK5 signaling pathway in regulating hypertrophic
response.

Role of ERK5 in Hypertrophic Remodeling

ERK5 is implicated in the induction of cardiac hypertrophy
and heart failure in humans10,22; however, our understanding
of its involvement in various aspects of cardiac remodeling is
limited and, in some cases, controversial. For example,
previous studies using transgenic mice with cardiac-specific
overexpression of activated MEK5 provided conflicting results,8,12,13 which can be partially explained by the fact that
different isoforms (, form) of MEK5 were used to
generate the transgenic lines. An additional explanation is the
transgenic approach, as phenotypes observed from transgenic

mouse models are largely associated with the copy number of

overexpressed genes. With respect to the fact that ERK5 may
exert actions independent of its kinase activity,23 it was hoped
that the analysis of ERK5cko mice would generate more direct
information on the specific contribution of ERK5 in the heart.
In the present study, phenotypes observed from ERK5cko
mice were reminiscent of observations in MEF2D-null
mice19; they also demonstrated blunted hypertrophic growth
and fibrosis, together with the downregulation of mRNA
levels of MEF2-regulated genes. In line with these observations, enhanced hypertrophy was seen in mice with cardiacspecific overexpression of MEF2A after TAC.20 Interestingly, ERK5 and p38 MAPK are able to phosphorylate and
activate MEF2A and MEF2C, whereas MEF2D is a specific
substrate of ERK5.3,5 Of note, the major MEF2 isoforms in
the adult heart are MEF2A and MEF2D, which participate in
mediating different aspects of pathological cardiac signaling.19,20 Recent studies in conventional and cardiomyocytespecific p38 knockout mouse models suggest that p38 does
not promote cardiac hypertrophy and may, in fact, prevent

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Figure 5. Prolonged pressure overload

caused diminished hypertrophic response
and cardiac dysfunction in ERK5cko mice. A,
HW/TL ratios of ERK5f/f and ERK5cko mice following 5 weeks TAC or sham operation. B,
Quantification of the mean cross-sectional
areas of cardiomyocytes from two genotypes.
C, Quantification of interstitial fibrosis induced
by TAC. D, TUNEL-positive nuclei in ERK5cko
hearts were significantly more than that in the
controls after 5 weeks of TAC. E, Echocardiography assessment (FS%, aorta maximum
velocity [Ao Vmax]) demonstrated impaired cardiac function in ERK5cko mice. Data are presented as meansSEM (n7 to 9 per group).

it.24,25 Considering the above evidence, MEF2 is most likely

to perform as a hypertrophic player downstream of ERK5,
rather than p38. Furthermore, responding to ISO stimulation,
a decrease was found in MEF2 transcriptional activity in

NRCMs in which ERK5 was exclusively knocked down or its

kinase activation was specifically inhibited. In addition to
this, reduced protein synthesis and downregulated mRNA
levels of ANP and Acta1 were detected in siERK5-treated

Figure 6. ERK5 is required for mediating

MEF2 activity in hypertrophic cardiomyocytes. A, Immunoblot analyses of expression and phosphorylation levels of ERK5,
ERK1/2, p38, and JNK in BIX02189 treatedNRCMs. B, NRCMs were infected with
Ad-MEF2-Luc for 24 hours before the incubation of BIX02189. After 2 hours of incubation of the inhibitor, the MEF2 transcriptional activity was measured by the
luciferase reporter assay system following
ISO stimulation. C, NRCMs were transfected with ERK5 siRNA or negative control
siRNA. At 72 hour after the transfection, cell
lysates were prepared for analysis of ERK5
protein level by immunoblotting. MEK5 and
ERK1/2 protein levels were also examined
to determine the specificity of ERK5 knockdown. D, The MEF2 transcriptional activity
was measured in siRNA-treated NRCMs
following ISO treatment. Data are
meansSEM (n6 per group).

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Figure 7. The critical role of

MEF2 in ERK5-mediated hypertrophy. A, Quantitative
real-time PCR analyses of
hypertrophic gene markers in
siRNA treated-NRCMs after
ISO stimulation. The data are
derived from three independent experiments performed in
triplicate and are normalized to
the GAPDH content (n3). B,
[3H]Leucine incorporation was
measured in siRNA-treated
NRCMs after ISO treatment
(n6). C, Immunoblot analyses
of ERK5, MEF2C and GFP
expression in adenoviral
infected NRCMs. D, NRCMs
were infected with Ad-MEF2CR3T, Ad-ERK5, or Ad-ERK5
together with Ad-MEF2C-R3T.
Transcript level of ANP was
measured after ISO treatment.
E, [3H] Leucine incorporation
was measured in NRCMs
infected with indicated adenoviruses before and after ISO
stimulation (n6). Data are
meansSEM.

NRCMs following ISO treatment. Moreover, the finding that

the inhibition of MEF2 activity was able to attenuate the
ERK5-regulated hypertrophic response strengthens the notion
that MEF2 acts downstream of ERK5 in regulating
hypertrophy.
CTGF is a key mediator of tissue fibrosis, which is
predominantly expressed in fibroblasts; however, during
cardiac remodeling it is also secreted by cardiomyocytes.26,27
Several lines of evidence indicate the important role of MEF2
in regulating CTGF expression.19,20,28 In the present study,
the decreased Ctgf mRNA level along with less interstitial
fibrosis were seen in ERK5cko-TAC hearts, thus it is plausible
to propose that absence of ERK5 in cardiomyocytes is likely
responsible for such phenotypes via downregulated MEF2
activity. Taken together, these data provide unequivocal
evidence that ERK5 is responsible for controlling MEF2
activity in different aspects of hypertrophic remodeling.
In parallel, we also investigated the activation of a number
of hypertrophic regulators following TAC stress, including
ERK1/2,29 p38,24 JNK17 and PKB.30 However, we did not
observe any difference in their activities between the two
genotypes; this underpins the notion that ERK5 is an important character in mediating hypertrophic remodeling. It is
worth noting that residual hypertrophic growth seen in

ERK5cko-TAC mice is likely attributable to the activation of

ERK1/2 and PKB, which are known as promoters of
hypertrophy.29,30

ERK5 Is Required for Cardiomyocyte Survival

Apart from the altered hypertrophic response, we also
detected an increase in apoptotic cardiomyocytes with
enhanced Bad and p53 protein levels in TAC treatedmutant hearts, indicating that ERK5 prevents hypertrophy
induced-cardiomyocyte loss. The ability of ERK5 to protect endothelial cells from shear stress via inhibition of
Bad activation was established by Pi et al.31 Furthermore,
evidence has been provided that ERK5 is required to
mediate neuronal survival by suppressing Bad expression
via a mechanism dependent on CREB transcriptional
activity.6 Consistent with this study, we also found upregulated Bad expression in ERK5cko-TAC cardiomyocytes,
although we did not observe any change in the protein
level and phosphorylation status of CREB. CREB is a
prosurvival transcription factor which either acts as an
activator for increasing Bcl-2 expression or as a repressor
of Bad expression. 6,32 Interestingly, cardiomyocytespecific inactivation of CREB did not cause any increase in
apoptosis at basal level32; nevertheless, further studies are

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Kimura et al
required to address the role of CREB in protecting cardiomyocytes following stress. Prolonged pressure overload is
known to cause an accumulation of p53, which is a pivotal
mediator of apoptosis.33,34 It has been demonstrated that
p53 is able to induce a number of Bcl-2 family genes,
including Bad.34,35 Thus, the upregulated p53 expression
found in ERK5cko-TAC hearts is proposed to be responsible for the increased level of Bad. It is also worth noting
that impaired contractility seen in the mutant hearts after
TAC is very likely attributed to the increased apoptosis.
Although the actual rate of apoptosis is low, it is, however,
reasonable to believe that this level of apoptosis is sufficient to cause cardiac dysfunction.36 As discussed, it is
suggested that ERK5 is necessary for protecting cardiomyocyte against hypertrophic stress and for determining
cardiac function during hypertrophic remodeling.

Novel Pharmacological Inhibitors of the

MEK5/ERK5 Pathway
ERK5 is one of the least-studied MAPK subfamilies, in
part, because of the lack of biological/pharmacological
tools, including specific inhibitors. Recently, the MEK5/
ERK5-selective inhibitors BIX02188 and BIX02189 have
been identified. BIX02189 is demonstrated to have greater
potency in inhibiting ERK5 activity than BIX02188.37,38 In
the present study, by applying BIX02189 to NRCMs, we
detected that ERK5 phosphorylation was specifically
blocked without affecting the activation of other MAPKs;
MEF2 transcriptional activity was also impaired. Thus, for
the first time, our results provide important information
regarding the effect of BIX02189 in cardiomyocytes. It has
been demonstrated that ERK5 is required for tumor growth
because of its essential role in the development of tumor
vasculature.39 Selectively targeting the ERK5 pathway by
pharmacological compounds is thought to be a promising
approach for treating cancers. Although the availability of
selective MEK5/ERK5 inhibitors may shed light in developing such a therapeutic strategy, it must nevertheless be
considered that ERK5 has a role in many other tissues.
Further investigation of these MEK5/ERK5 inhibitors in
vivo under basal and various stress conditions is certainly
required.
In conclusion, the present study provides convincing evidence that clarifies the in vivo role of ERK5 in the heart.
ERK5 regulates hypertrophic remodeling and protects cardiomyocytes from hypertrophic stress mostly likely by regulating the MEF2 transcription activity. The recognition of the
functional importance of ERK5 in the heart provides invaluable information regarding the possible beneficial or adverse
effects of new therapeutic strategies.

Acknowledgments
We thank Dr Roger Snow (Department of Medical Chemistry,
Boehringer Ingelheim Pharmaceuticals Inc) for kindly providing us
with the ERK5 inhibitor BIX02189.

Sources of Funding
This work was supported by British Heart Foundation grant PG/07/
055/23144 (to X.W., E.J.C., and A.H.W.).

Erk5 Deletion Blunts Cardiac Hypertrophy

969

Disclosures
None.

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Novelty and Significance

What Is Known?

ERK5 (an atypical mitogen-activated protein kinase) is implicated in

the induction of heart failure in humans.
ERK5 can be activated in the hearts of animal models by pressure
overload and in cultured cardiomyocytes by various hypertrophystimulating factors.

What New Information Does This Article Contribute?

The present study elucidates the in vivo role and molecular basis
whereby ERK5 regulates cardiac remodeling and protects cardiomyocytes from hypertrophic stress.

Heart failure (HF) is a debilitating condition that remains a major

cause of mortality worldwide. Understanding the molecular determinants of hypertrophic remodeling, a virtually universal prerequisite of the development of HF, is a key step in elucidating the

pathogenesis of HF. Extracellular signal-regulated protein kinase

(ERK)5 has been implicated in the induction of HF. However, its role
in various aspects of hypertrophic remodeling has thus far not been
elucidated. Using a range of approaches including a cardiomyocyte-specific ERK5 knockout mouse model (ERK5cko), and an
MEK5/ERK5-selective inhibitor (BIX02189), we found that ERK5cko
mice developed less hypertrophic growth but increased apoptosis
along with cardiac dysfunction in response to pressure overload,
suggesting the functional importance of ERK5 in protecting against
HF. Moreover, we have identified the transcription factor myocyte
enhancer factor 2 as a downstream effector of ERK5 in regulating
this hypertrophic response. Together, the present study demonstrates for the first time the in vivo role of ERK5 in regulating
hypertrophic growth and prevention of HF. Overall, the recognition
of the functional importance of ERK5 in the heart provides
invaluable information regarding the possible beneficial or adverse
effects of new therapeutic strategies to combat human HF.

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Targeted Deletion of the Extracellular Signal-Regulated Protein Kinase 5 Attenuates

Hypertrophic Response and Promotes Pressure OverloadInduced Apoptosis in the Heart
Tomomi E. Kimura, Jiawei Jin, Min Zi, Sukhpal Prehar, Wei Liu, Delvac Oceandy, Jun-ichi
Abe, Ludwig Neyses, Arthur H. Weston, Elizabeth J. Cartwright and Xin Wang
Circ Res. 2010;106:961-970; originally published online January 14, 2010;
doi: 10.1161/CIRCRESAHA.109.209320
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571

The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/106/5/961

Data Supplement (unedited) at:

http://circres.ahajournals.org/content/suppl/2010/01/14/CIRCRESAHA.109.209320.DC1.html

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CIRCRESAHA/2009/209320/R1

Supplement Material
Materials and Methods
Generation of ERK5 Cardiac Knockout Mice
The erk5-flox mice (referred to as ERK5f/f) were previously generated1. ERK5f/f mice were mated
with mice expressing Cre under myosin heavy chain (MHC) promoter to generate cardiacspecific ERK5 knockout mice (referred to as ERK5cko). The MHC-Cre line (kindly provided by
Dr. MD Schneider, National Heart and Lung Institute, UK) is a well established model that
provides efficient Cre recombinase activity in the myocardium. We did not observe any
abnormality in cardiac morphology and function in the MHC-Cre line up to six months of age.
All mice used in this study were maintained in a pathogen-free facility at the University of
Manchester. The animal studies were performed in accordance with the UK Home Office and
institutional guidelines.
Echocardiography
For cardiac morphological and functional analysis, M-mode and Doppler echocardiographic
recordings were performed using an Acuson Sequoia C256 system (Siemens) following a
protocol described previously2. Parameters of intraventricular septal thickness (IVS), left
ventricular posterior wall thickness (LVPW), left ventricular end-diastolic dimension (LVEDD),
left ventricular end-systolic dimension (LVESD) and fractional shortening (FS %), aorta
maximum velocity (Ao Vmax) and ratio of the E/A-wave were obtained.
Histology
Freshly dissected heart tissue was fixed in 4% paraformaldehyde, dehydrated and embedded in
paraffin. 5-7m thick sections were cut and stained with hematoxylin & eosin or Massons
trichrome method as described2. To calculate the mean cross-sectional area approximately 150
randomly selected cardiomyocytes were measured. 45 randomly chosen frames from Massons
trichrome stained sections were quantified to assess the degree of myocardial fibrosis using
Image J software.
Preparation of Lysates and Immunoblot Analysis
Proteins were homogenised and extracted from tissues in Triton lysis buffer (20 mM Tris-HCl,
pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM -glycerophosphate, 10%
glycerol, 1 mM orthovanadate, 1 mM phenylsulphfonyl fluoride, 10 g/ml leupeptin, 10 g/ml
aprotinin). Protein extracts (30 g) were subjected to Western blot analysis with antibodies
against JNK, p38 MAPK, p53 (Santa Cruz); ERK1/2, PKB, Bcl-2, CREB, phospho-PKB (Ser
473), phospho-ERK1/2, phospho-JNK, phospho-p38 MAPK, phospho-CREB (Ser 133), MEF2C
(Cell Signalling); Bad (BD Transduction); ERK5 (Upstate); phospho-ERK5, GFP (Invitrogen)
and tubulin (Sigma). Immunecomplexes were detected by enhanced chemiluminescence with
anti-mouse, anti-rabbit, or anti-goat immunoglobulin G coupled to horseradish peroxidise as the
secondary antibody (Amersham-Pharmacia).
Characterization of Effect of BIX02189 on Neonatal Rat Cardiomyocytes
Primary cultures of neonatal rat cardiomyocytes (NRCMs) were prepared as previously
described2. NRCMs were incubated with 10M BIX02189 (Boehringer Ingelheim) for 2h prior to
the addition of isoproterenol (10M, 30min). Cell extracts were lysed for immunoblotting to
determine the effect of BIX02189 on inhibition of the activation of ERK5.
siRNA Transfection
NRCMs were transfected with siRNA (100nM) using Lipofectamine LTX and Plus reagents
according to the manufacturers instructions (Invitrogen). Rat ERK5 siRNA (5AAAGGGTGCGAGCCTATAT-3) was purchased from Dharmacon, siRNA negative control (Si
Neg) was obtained from Eurogenetec. To assess the specific effect of ERK5 siRNA on silencing
ERK5 expression, the protein levels of MEK5 and ERK1/2 were detected by immunoblot
analysis 72h post-transfection.

CIRCRESAHA/2009/209320/R1
Adenoviral Infection
NRCMs were infected with either Ad-MEF2C-R3T (Seven Hills Bioreagents), or Ad-ERK5
(kindly provided by Jun-ichi Abe, Rochester University), or Ad-MEF2C-R3T combined with AdERK5 at 50 MOI in serum-free medium for 24h, followed by additional 24h incubation of ISO,
afterwards cells were then subjected to quantitative real-time PCR analyses or [3H] leucine
incorporation assay. Ad-GFP was used for a control experiment.
Apoptosis Assays
TUNEL assay to detect apoptosis was performed on paraffin-embedded heart sections using the
in situ Cell Death Detection kit (Roche). Triple staining with Hoechst, anti--actinin antibody
(Sigma), and TUNEL was performed to confirm apoptotic morphology in cardiac nuclei. An
average of total 10,000 myocyte cells from random fields was analyzed. The data are obtained
from three independent experiments. For caspase activity assay, proteins were homogenised and
extracted from heart tissues in the lysis buffer (25mM HEPES pH 7.5, 10mM KCl, 1.5mM
MgCl2, 1mM EDTA, 1mM EGTA, 1mM benzamidine, 1mM dithiothreitol [DTT], 1mM
phenylsulphfonyl fluoride, 1% Triton X100). Protein extracts (50 g) were incubated with
200M DEVD-AMC caspase-3 specific fluorogenic substrate (Alexis Biochemicals) for 1h in the
caspase reaction buffer (20mM HEPES, 100mM NaCl, 10mM DTT, 0.1% CHAPS, 10% w/v
sucrose). Cleavage of the substrate was measured by spectrofluorometer at 380 nm excitation and
460 nm emission wavelengths. To evaluate the effect of ERK5 knockdown or the inhibition of its
kinase activation on apoptosis, BIX02189-treated or siERK5-treat NRCMs were stimulated with
sorbitol (Sigma) at 300mM to induce apoptosis3, 4. After 4h sorbitol stimulation, cell lysates were
prepared for measuring caspase 3 activity.
Quantitative Real-Time PCR
Tissues or NRCMs were extracted to prepare total RNA using Trizol reagent, followed by the
synthesis of cDNA. Real-time quantitative PCRs were performed using the SYBR-green I Core
Kit (Eurogentec). The primers used for ANP, BNP, Myh6, Myh7, Acta1, Ctgf, Col12, Col31
and GAPDH were obtained from Qiagen. PCR products were detected in the ABI-PRISM 7700
sequence detection system (Applied Biosystems), and the results were analyzed using the 2-CT
method5. The level of expression of mRNA was normalized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA.
Luciferase Reporter Assay
To measure MEF2 luciferase activity after blocking ERK5 activation, NRCMs were infected with
recombinant adenovirus encoding the MEF2-luciferase reporter gene (AdMEF2-Luc, Seven Hills
Bioreagents) at 25 MOI in serum-free medium for 24h, prior to the incubation of BIX02189.
After 2h incubation with the inhibitor, the cells were then treated with 10M isoproterenol for an
additional 18h in the presence of BIX02189. To measure MEF2 luciferase activity in the absence
of ERK5 expression, 48h post-transfection of siRNA, NRCMs were infected with AdMEF2-Luc
for 24h followed by isoproterenol stimulation. After various treatments, aliquots of NRCM
lysates were assayed for the MEF2 luciferase activity using the luciferase assay kit (Promega). To
further assess whether MEF2 transcriptional activity is regulated by either ERK5 knockdown or
the inhibition of ERK5 kinase activation, NRCMs were transiently transfected with the reporter
plasmid pG5E1bLuc together with a construct encoding the fusion proteins Gal4-MEF2A, or
Gal4-MEF2D3, prior to 2h incubation with BIX02189. After BIX02189 incubation, MEF2
transcriptional activity was measured at 24h post ISO stimulation by the dual-luciferase reporter
assay system (Promega). Gal4 was used for a control experiment. A pRL-TK plasmid encoding
Renilla luciferase was employed to monitor transfection efficiency. In parallel, siRNA-treated
NRCMs were transfected with various plasmid vectors as described above using Lipofectamine
LTX and Plus reagents (Invitrogen). Following 24h ISO stimulation, MEF2 transcriptional
activity was measured by the dual-luciferase reporter assay system (Promega).

CIRCRESAHA/2009/209320/R1
[3H] Leucine Incorporation
NRCMs were transfected with siRNA (100nM) for 48h, or infected with various adenoviruses
(Ad-GFP, Ad-MEF2C-R3T, Ad-ERK5, or Ad-ERK5 plus Ad-MEF2C-R3T) at 50 MOI for 24h
following the isoproterenol treatment (10M, 24h). The cells were then incubated in the same
medium with 1.0Ci/ml [3H] Leucine for an additional 24h. After washing in ice-cold PBS, the
cells were precipitated with 10% trichloroacetic acid for 30 minutes at 4C. The precipitates were
then solubilized in 0.4 N NaOH for 1h and neutralized with 1N HCl. Radioactivity was measured
in a liquid scintillation counter. Each well was normalized to the total DNA content measured at
260nm.
Data Analysis
Data are expressed as mean SEM and analyzed using one-way or two-way ANOVA followed
by Bonferonnis post-test where appropriate. Comparisons between two groups were performed
using Students t-test. P-values <0.05 are considered statistically significant.
Expanded Discussion
The Loss of ERK5 in Cardiomyocytes Does Not Have a Primary Influence on HypertrophyInduced Angiogenesis
Enhanced angiogenesis under pressure overload is a critical feature of adaptive hypertrophic
growth. Sustained stress eventually causes deregulation of microvascularization and insufficient
oxygenation which contribute to the pathogenesis of heart failure6. ERK5 is reported to be a
negative regulator of angiogenesis7, 8. During embryonic development, deficiency of ERK5
results in upregulated hypoxia inducible factor 1 (HIF-1) activity and increased expression of
the vascular endothelial growth factor (VEGF) which impedes the angiogenesis process and
vascular maturation8. Interestingly, a recent study reported that cardiomyocyte-specific deletion
of HIF-1 caused significantly less microvessels and impaired cardiac function after TAC6.
Taking these findings into account, we assessed capillary density in TAC-treated ERK5cko hearts.
No difference in the capillary-to-cardiomyocyte ratio was found in the two genotypes (data not
shown); suggesting the lack of ERK5 in cardiomyocytes does not have a primary influence on
capillary density. However, whether ERK5 expressed in nonmyocytes is involved in regulating
capillary density in the process of hypertrophic remodeling needs further study.
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Cartwright EJ, Wang X. Cardiac-Specific Deletion of Mkk4 Reveals Its Role in
Pathological Hypertrophic Remodeling, but Not in Physiological Cardiac Growth. Circ
Res. 2009;104:905-914.
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Kayama Y, Harada M, Shimizu I, Asahar T, Hamada H, Tomita S, Molkentin JD, Zou Y,
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overload. Nature. 2007;446:444-448.

CIRCRESAHA/2009/209320/R1
7. Pi X, Garin G, Xie L, Zheng Q, Wei H, Abe J, Yan C, Berk BC. BMK1/ERK5 is a novel
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CIRCRESAHA/2009/209320/R1
A

P<0.001

Supplement Fig. I

n. s.

n. s.

ERK5f/f ERK5CKO
Brain

ERK5f/f ERK5CKO
Liver

ERK5/GAPDH mRNA
expression (% maximum)

100
80
60
40
14%

20
0

ERK5f/f ERK5CKO
LV

ERK5
ERK5f/f

ERK5CKO

P<0.001

LV

ERK5/tubulin ratio
(% maximum)

100

Brain

Liver

80
60
40
19%

20
0

SM

ERK5f/f

ERK5CKO

Tubulin

LV
ERK5f/f

ERK5CKO

ERK1/2

JNK

p38

MEK5

Supplement Figure I. Characterization of cardiac-specific deletion of ERK5. (A) Quantitative RT-PCR

analysis of the mRNA levels of ERK5 in the left ventricle (LV), brain and liver, demonstrating a 86%
decrease in ERK5 mRNA in LV. (B) Western blot analyses to determine specificity of the deletion of ERK5
in the left ventricle compared to protein extracts from various tissues, tubulin expression served as the protein
loading control. The ratio of ERK5 expression to tubulin is shown in the bar graph. Data are presented as
mean SEM, n=3 for each group. (C) Immunoblot analyses show similar expression levels of ERK1/2, JNK,
p38 MAPK and MEK5 in the two genotypes. n.s.: no difference found between two groups.
5

CIRCRESAHA/2009/209320/R1
Supplement Fig. II

Bad/Bcl-2 ratio
(Arbitrary unit)

2.0

ERK5f/f

1.5

n. s.

1.0
0.5

ERK5CKO
0
sham

Bad

ERK5f/f

ERK5CKO

2.0
P/T CREB ratio
(Arbitrary unit)

Bcl-2
p53

P-CREB

CREB

1.5

n. s.

1.0
0.5

0
sham

ERK5f/f

ERK5CKO

Tubulin

P53/tubulin ratio
(Arbitrary unit)

2.0
n. s.

1.5
1.0
0.5

0
sham

ERK5f/f

ERK5CKO

Supplement Figure II. Immunoblot analyses of protein levels of Bad, Bcl-2, p53, CREB, and
phosphorylation level of CREB in the two sham groups. Tubulin expression is the protein loading
control. The ratios of Bad/Bcl-2, p53/tubulin and P/T CREB are represented by the bar graphs, n=4
per group. Data are presented as mean SEM, n.s.: no difference found between two groups

CIRCRESAHA/2009/209320/R1
Supplement Fig. III

8
6
4
2

mRNA (arbitrary units)

mRNA (arbitrary units)

4
3
2
1

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

sham
5w-TAC

5
4

P<0.05

3
2
1
ERK5f/f ERK5CKO ERK5f/f ERK5CKO
sham
5w-TAC

Col12
6
P<0.05

4
3
2
1

Col31
6
P<0.05

mRNA (arbitrary units)

mRNA (arbitrary units)

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

sham
5w-TAC

Ctgf
6

Myh7
6

P<0.05

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

sham
5w-TAC

Acta1
6

P<0.05
8

P<0.05

4
3
2
1
0

0
ERK5f/f ERK5CKO ERK5f/f ERK5CKO
sham
5w-TAC

mRNA (arbitrary units)

BNP
10

P<0.05

mRNA (arbitrary units)

mRNA (arbitrary units)

ANP
10

5
4
3
2
1
0

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

sham
5w-TAC

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

sham
5w-TAC

Supplement Figure III. Quantitative real-time PCR analyses of gene markers associated with hypertrophy and
fibrosis after 5 weeks of TAC. The data are derived from three independent experiments performed in
triplicate and are normalized to the GAPDH content, n= 5 per group. Data are presented as mean SEM.

CIRCRESAHA/2009/209320/R1
Supplement Fig. IV
A
vehicle

ISO
ERK5CKO

48% P<0.001

ERK5f/f

32% P<0.001

P<0.05
Myocyte cross-sectional
area (m2)

10
HW/TL (mg/mm)

ERK5CKO

8
6
4
2
0

6
P<0.001
P<0.05

400
300
200
100
0

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

vehicle
ISO

P<0.001

500

Interstitial myocardial
fibrosis (%)

ERK5f/f

P<0.05

0
ERK5f/f ERK5CKO ERK5f/f ERK5CKO
vehicle
ISO

ERK5f/f

ERK5CKO
ISO

E
BNP
8

P<0.001
P<0.01

mRNA (arbitrary units)

mRNA (arbitrary units)

ANP
8

P<0.01

4
2

P<0.001
6

4
2

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

vehicle
ISO

P<0.01
P<0.01

ERK5f/f ERK5CKO ERK5f/f ERK5CKO

vehicle
ISO

Supplement Figure IV. The loss of ERK5 attenuated isoproterenol induced-cardiac hypertrophy. (A) ERK5cko
mice showed blunted cardiac hypertrophy following isoproterenol infusion (scale bar: 20m). (B) HW/TL ratios
of ERK5f/f and ERK5cko mice were evaluated after 7-day isoproterenol treatment, n=4 to 9 per group. (C) Mean
cross-sectional areas of cardiomyocytes in ERK5f/f and ERK5cko mice were calculated, n=4. (D) Ventricular
interstitial fibrosis was minimal in ERK5cko hearts detected by Massons trichrome-staining of cross-sections.
Quantification of the relative fibrotic area is expressed as percentage of the area of fibrosis in the microscope
views, n=4. (E) Quantitative real-time PCR analyses of hypertrophic gene markers, ANP and BNP. The data are
derived from three independent experiments performed in triplicate and are normalized to the GAPDH content,
n=3. Data are presented as mean SEM.
8

CIRCRESAHA/2009/209320/R1
Supplement Fig. V
A

*#

Vehicle
BIX02189
*#

Luciferase activity
(arbitrary units)

1
0
ISO

+
Gal4

+
+
Gal4-MEF2A

+
+
Gal4-MEF2D

B
5

Control siRNA
ERK5 siRNA

*#

Luciferase activity
(arbitrary units)

4
*#
3

1
0
ISO

+
Gal4

+
+
Gal4-MEF2A

+
+
Gal4-MEF2D

Supplement Figure V. The ERK5 knockdown or the inhibition of its kinase activation suppressed MEF2
transcriptional activity in NRCMs. (A) NRCMs were transfected with the reporter plasmid pG5E1bLuc together
with constructs encoding Gal4-MEF2A, or Gal4-MEF2D, followed by BIX02189 treatment. After 2h inhibitor
treatment, MEF2 transcriptional activity was measured by the dual-luciferase reporter assay system before and
after ISO stimulation. A control experiment was performed with pG5E1bLuc and Gal4. As expected, both Gal4MEF2A and Gal4-MEF2D activities were reduced in BIX02189-treated NRCMs, indicating diminished MEF2
transcriptional activity. (B) siRNA-treated NRCMs were co-transfected with various plasmid vectors as
indicated. MEF2 transcriptional activity was measured by the dual-luciferase reporter assay system following
ISO stimulation. Firefly luciferase activity was normalized to that of Renilla luciferase. The data are derived
from three independent experiments performed in duplicate, n=6. Data are presented as mean SEM. *P<0.05
versus vehicle alone (A) or control siRNA alone (B); #P<0.05 versus BIX02189 treated + ISO (A) or ERK5
siRNA treated + ISO (B).

CIRCRESAHA/2009/209320/R1
Supplement Fig. VI

A
Caspase 3 activity (fold)

2.5
P<0.01

2.0
1.5
1.0
0.5

0
Sorbitol
BIX02189

+
+

P<0.01

2.5
Caspase 3 activity (fold)

+
-

2.0
1.5
1.0
0.5

0
siRNA control

ERK5

vehicle

control

ERK5

Sorbitol

Supplement Figure VI. The ERK5 knockdown or the inhibition of its kinase activation sensitizes NRCMs to
apoptosis. BIX02189-treated NRCMs (A); or siERK5-treated NRCMs (B) were stimulated with sorbitol for
4h. Following stimulation, caspase 3 activity was measured by caspase activity assay, n=6. Data are presented
as mean SEM.

10