MEK1-induced physiological hypertrophy inhibits chronic post-myocardial infarction remodeling in mice

Article (PDF Available)inJournal of Cellular Biochemistry 114(1) · January 2013with26 Reads
DOI: 10.1002/jcb.24299 · Source: PubMed
AIMS: Although activation of MEK-ERK signaling is known to be cardioprotective during acute reperfusion injury, the effect of MEK activation on chronic changes in ventricular structure and function during the more complex process of remodeling after myocardial infarction (MI) with or without reperfusion remains uncertain. METHODS/RESULTS: Four weeks after permanent coronary ligation, LV fractional shorting, preload recruitable stroke work and end-systolic elastance were all preserved in transgenic mice with CM-specific upregulation of the MEK1-ERK1/2 signaling pathway (MEK1Tg) compared to wildtype (WT) controls (5.8% decline vs. 17.3%, P < 0.01; 603 ± 98 mmHg vs. 335 ± 98, P < 0.05; 6.14 ± 0.57 mmHg/µL vs. 3.92 ± 0.60, P< 0.05, respectively). Despite similar initial infarct sizes, post-MI remodeling was significantly reduced in MEK1Tg, demonstrated by reductions in chronic infarct size (28.5 ± 3.1% vs. 47.8 ± 3.2%), myocardial fibrosis (3.98 ± 0.74% vs. 9.27 ± 1.97%) and apoptosis (0.66 ± 0.11% vs. 1.60 ± 0.34%). Higher phosphorylation (i.e. activation) of pro-survival transcription factor STAT3, higher expression of anti-apoptotic protein Bcl2, and higher phosphorylation (i.e. inactivation) of pro-apoptotic BAD were observed in the post-MI remote myocardium of MEK1Tg. MMP2 activity was higher in MEK1Tg, while expression of TIMP3 and MMP9 activity were lower in transgenic mice. CONCLUSION: Beyond any immediate cardioprotective effect, therapeutic activation of MEK1-ERK1/2 signaling during the chronic post-MI period may preserve LV function by increasing the expression of pro-survival factors and by suppressing factors, such as the balance between matrix modulating proteins, that promote pathological remodeling in the remote myocardium. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.
MEK1-Induced Physiological Hypertrophy Inhibits Chronic
Post-Myocardial Infarction Remodeling in Mice
Che-Chung Yeh, Deepak Malhotra, Yi-Lin Yang, Yanchun Xu, Yanying Fan,
Hongzhe Li, and Michael J. Mann
Division of Cardiothoracic Surgery, University of California, San Francisco and VA Medical Center,
San Francisco, California
Although activation of MEK-ERK signaling is known to be cardioprotective during acute reperfusion injury, the effect of MEK activation on
chronic changes in ventricular structure and function during the more complex process of remodeling after myocardial infarction (MI) with or
without reperfusion remains uncertain. Four weeks after permanent coronary ligation, LV fractional shorting, preload recruitable stroke work,
and end-systolic elastance were all preserved in transgenic mice with CM-specific upregulation of the MEK1-ERK1/2 signaling pathway
(MEK1 Tg) compared to wildtype (WT) controls (5.8% decline vs. 17.3%, P < 0.01; 603 98 mmHg vs. 335 98, P < 0.05; 6.14 0.57 mmHg/ml
vs. 3.92 0.60, P < 0.05, respectively). Despite similar initial infarct sizes, post-MI remodeling was significantly reduced in MEK1 Tg,
demonstrated by reductions in chronic infarct size (28.5 3.1% vs. 47.8 3.2%), myocardial fibrosis (3.98 0.74% vs. 9.27 1.97%) and
apoptosis (0.66 0.11% vs. 1.60 0.34%). Higher phosphorylation (i.e., activation) of pro-survival transcription factor STAT3, higher
expression of anti-apoptotic protein Bcl2, and higher phosphorylation (i.e., inactivation) of pro-apoptotic BAD were observed in the post-MI
remote myocardium of MEK1 Tg. MMP2 activity was higher in MEK1 Tg, while expression of TIMP3 and MMP9 activity were lower in
transgenic mice. Beyond any immediate cardioprotective effect, therapeutic activation of MEK1-ERK1/2 signaling during the chronic post-MI
period may preserve LV function by increasing the expression of pro-survival factors and by suppressing factors, such as the balance between
matrix modulating proteins, that promote pathological remodeling in the remote myocardium. J. Cell. Biochem. 114: 47–55, 2013.
ß 2012 Wiley Periodicals, Inc.
he majority of morbidity and mortality associated with post-
MI cardiomyopathy and heart failure results from chronic
remodeling of the surviving myocardium after MI. Despite a
growing body of knowledge regarding the changes in intracellular
signaling that accompany post-MI remodeling [Feuerstein and Young,
2000; Woodcock et al., 2008; Dobaczewski and Frangogiannis, 2009;
Hori and Nishida, 2009; Zamilpa and Lindsey, 2010], links between
these empiric observations and the etiology of very complex and
poorly understood chronic changes in cardiac myocyte (CM) biology
that lead to macroscopic changes in ventricular structure and
function over time in the post-MI heart remain elusive.
Correlative studies in human pathologic specimens have consis-
tently indicated an upregulation in multiple signaling pathways
during various stages of cardiac remodeling leading to heart failure
[Ungerer et al., 1993; Abbate et al., 2002; Baba et al., 2003]. Although
acute interventions in CM signaling are currently being contemplated
as early responses to ischemic events, such molecular interventions
would not be available to large numbers of patients who present much
later in the course of their post-MI cardiomyopathy with chronic
and evolving changes. Pathways that are believed to instigate a decline
in myocardial function have been assumed to contribute to a
pathologic process in chronic remodeling, whereas those believed to
have an ameliorative effect, on the other hand, are assumed to
represent a natural compensatory mechanism. It is imperative,
however, to confirm these assumptions and to better understand the
roles of specific signaling pathways in long-term ventricular
remodeling in order to effectively design novel interventions that
will be relevant to the treatment of millions of post-MI patients.
It has been postulated that either pharmacologic or genetic
manipulation of various signaling pathways might succeed in
Journal of Cellular
Journal of Cellular Biochemistry 114:47–55 (2013)
Disclosure statement: None.
Grant sponsor: National Institutes of Health; Grant numbers: K08HL079239, R01HL083118.
*Correspondence to: Michael J. Mann, Division of Cardiothoracic Surgery, 500 Parnassus Avenue, Suite W420, San
Francisco, CA. E-mail:
Manuscript Received: 7 May 2012; Manuscript Accepted: 16 July 2012
Accepted manuscript online in Wiley Online Library ( 20 July 2012
DOI 10.1002/jcb.24299 ß 2012 Wiley Periodicals, Inc.
reducing the progressive apoptotic loss of CMs and thereby lessen
ventricular wall thinning and fibrosis during post-MI remodeling,
the leading cause of human heart failure [Dorn, 2009]. We have
shown recently, however, that a cell type-specific elucidation of
molecular regulation in the diseased myocardium may be essential
for successful application of this type of advanced targeted therapy,
as non-specific induction of anti-apoptotic signaling in cardiac
non-myocytes might actually enhance fibrosis rather than impede
myocardial wall thinning [Yeh et al., 2010].
Multiple arms of the mitogen activated protein (MAP) kinase
pathway have been shown to be upregulated during the develop-
ment of post-MI remodeling. Data from our laboratory and others’
have suggested that the balance between so-called stress activated
kinases p38 and JNK and the putative anti-apoptotic kinases ERK1/2
may be disrupted during this pathologic progression, resulting in
an increase in CM dysfunction and apoptosis [Zhang et al., 2003;
Qin et al., 2005; Ren et al., 2005; Yeh et al., 2010]. Supraphysiologic
upregulation of ERK1/2 signaling, achieved via CM-specific
overexpression of a constitutively active form of the ERK activator
kinase MEK1 (aMEK1), has been shown to prevent the acute
functional loss of left ventricle through the reduction of the size of
infarct 24 h after ischemia-reperfusion (I/R) [Bueno et al., 2000].
These observations regarding alterations of short-term bursts of pro-
and anti-apoptotic signaling that contribute heavily to the survival
of CMs after the acute insult of ischemia and reperfusion, however,
have little bearing on long-term and complex changes in CM
signaling during chronic post-MI remodeling. Given the greater
complexity of pathologic stresses and the more prolonged time
course of this pervasive pathologic entity, we undertook focused
gain-of-function studies utilizing a transgenic mouse model of
low-moderate CM-specific aMEK1 overexpression to establish the
potential role of enhanced ERK1/2 signaling in ameliorating both
the structural degeneration of the remote myocardium as well as
progressive ventricular dysfunction during the evolution of post-MI
Eight to ten week old aMEK-1 transgenic (MEK1 Tg) mice (kindly
provided by Dr. Jeffrey Molkentin) and their wild-type (WT)
littermates were anesthetized with isoflurane prior to intubation
with a 24 gauge catheter. Inhalation anesthesia was then instituted
with 1.5% isoflurane using a rodent ventilator (Harvard) at
115 breath cycles/min. A left lateral thoracotomy incision was
placed at the level of the fourth interspace and a 7.0 polypropylene
suture was used to ligate the left anterior descending artery (LAD) at
approximately 1/3 the distance from the base to the apex of the
heart. The left chest was then closed and the animal was recovered
after extubation in a light-warmed incubator. Animals were
subsequently sacrificed at 4 weeks after infarction; at sacrifice,
some hearts were fixed and sectioned for histologic analysis, while
others were snap frozen for protein extraction. All procedures
conformed with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996), and were approved by the
Institutional Animal Care and Use Committee of the San Francisco
Veterans Affairs Medical Center.
At the time of harvest of specimens for protein extraction, the left
ventricle was dissected from the remainder of the heart. The infarct
area was identified visually and dissected from the remainder of the
myocardium. In addition, myocardial tissue from the free wall and
septum clearly distant from the area of infarction was collected as
remote myocardium. Any attempt to accurately isolate the irregular
border zone from these small mouse hearts was felt to introduce a
prohibitive sampling error. Specimens were snap frozen in liquid
nitrogen. Myocardial tissues were homogenized in a lysis buffer
containing 0.13 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na
5 mM NaF, 20 mM HEPES, and Protease inhibitor cocktail tablet
(Roche Diagnostics, Indianapolis, IN) and centrifuged at 35,000 rpm
for 30 min. Supernatants were separated by gel electrophoresis,
blotted onto a PVDF membrane (Invitrogen), and detected by
chemiluminescence kit (Pierce) after incubation with primary
antibody and corresponding horseradish peroxidase-labeled sec-
ondary antibody according to the manufacturer’s instructions. To
determine MMP activity, tissue lysates were applied to 10%
Zymogram Gelatin Gel (Invitrogen) and electrophoresis was done
according to the manufacturer’s manual. After electrophoresis, the
gels were washed twice in incubation buffer (50 mM Tris–HCl, 5 mM
, 150 mM NaCl, and 0.05% NaN
) for 20 min each at room
temperature before incubation in fresh buffer overnight at 378C. The
gels were stained in 2% Coomassie Brilliant Blue G/25% methanol/
10% acetic acid for 2 h and then destained for 1 h in 2% methanol/
4% acetic acid. Gel images were captured by AlphaImager. Band
densities were analyzed by Image J Software (Bethesda, MA).
Transthoracic echocardiography was performed in conscious mice
using an Acuson Sequoia 512 machine and a 13-MHz probe. A two-
dimensional short-axis view of the left ventricle was obtained at the
level of the papillary muscles, that is, in myocardium remote to the
infarction, and two-dimensional M-mode tracings were also recorded.
LV fractional shortening was calculated as (LVDd–LVDs)/LVDd 100,
where LVDd ¼ LV diastolic dimension and LVDs ¼ LV systolic
dimension [Yeh et al., 2010]. In vivo hemodynamic measurements
were done in the anesthetized mice by inserting a 1.4 F Millar
pressure–volume (PV) catheter into the LV camber via the right
carotid artery [Pacher et al., 2008] and hemodynamic parameters
were recorded and analyzed by Millar PV system MPVS-400 and
PVAN program (Millar Instruments Inc.).
Infarct area 24 h after LAD ligation was determined by triphenylte-
trazolium chloride (TTC; Sigma-Aldrich) staining. Briefly, after
perfusion with Evan’s blue dye, each heart was removed and sliced
horizontally into 2 mm slices. The slices were incubated in 1% TTC in
phosphate buffered saline for 5 min at 378C. The infarct ratio was
determined by dividing the infarct area (white) by the total area-at-
risk (white and red). Five-micron sections of pressure-fixed and
paraffin-embedded hearts were stained with Gomori trichrome and
Sirius Red to assess LV infarct size and fibrosis in the remote
myocardium. Myocyte size was evaluated via staining with Texas
Red-conjugated wheat germ agglutinin (WGA, Invitrogen). All
acquired images were analyzed and compared using the Image J
Software (Bethesda, MA). Sections were also subjected to TUNEL
staining (ApopTag Peroxidase In Situ Apoptosis Detection Kit,
Chemicon, Temecula, CA), and apoptotic cells and total cells,
identified via hematoxylin nuclear counterstaining, were counted
in three sections/heart hematoxylin–eosin staining was used to
distinguish the morphology of cells containing apoptotic nuclei on
adjacent sections. The level of apoptosis is expressed as apoptotic
cells as a percentage of total nuclei.
Values are reported as mean SEM. Comparisons among groups
were made using ANOVA, followed by Neuman–Keuls post-hoc
testing. P-values lesser than 0.05 were considered statistically
significant, with Bonferroni correction where appropriate.
Although the cardiac-specific upregulation of MEK-ERK signaling
has been linked to stable, physiologic hypertrophy of the myocardium,
the level of constitutive aMEK1 expression has been shown to
impact the resulting phenotype. Whereas a high level of expression
is associated both with an increase in myocardial mass and a
demonstrable improvement in functional parameters such as
fractional shortening, lower expression levels have been found to
have a much more moderate increase in myocardial wall thickness
and overall muscle mass [Bueno et al., 2000]. Given our interest in
the impact of MEK-ERK signaling on chronic, post-MI remodeling,
we hoped to minimize the confounding effects of pre-MI
hypertrophy and of MEK-ERK signaling during the evolution of
acute infarction that are inevitably associated with a transgenic
model of constitutive CM-specific expression. We therefore worked
with a model of low to moderate constitutive aMEK1 expression that
minimized the difference in pre-MI cardiac structure and function
between our experimental Tg animals and their littermate, WT
Low to moderate CM-specific aMEK1 expression resulted in a
21% increase in HW/BW ratio, compared to the 11% and 37%
increases observed previously in low and intermediate CM aMEK1
overexpression, respectively [Bueno et al., 2000]. Constitutively
high aMEK1 expression leads to a 23% and 45% reduction in
baseline LV end diastolic and end systolic dimensions, respectively,
while our MEK1 Tg mice did not display any reduction in these
baseline (i.e., pre-infarct) parameters of LV structure. Furthermore,
whereas high levels of aMEK1 expression lead to a measurable 33%
increase in both fractional shortening and LV dP/dt
[Bueno et al.,
2000], our aMEK1 transgenics did not display any difference in
baseline cardiac contractile function compared to WT littermate
controls (Table I).
Persistent expression of a constitutively active form of MEK1 in CM
was associated not only with an increase in ERK 1/2 phosphoryla-
tion (i.e., activation), but also with an interesting increase in total
ERK 1/2 protein expression. Although a similar increase in ERK
protein expression was observed previously in the MEK1 Tg [Bueno
et al., 2000], the mechanism of MEK1 regulation of ERK 1/2
expression has yet to be elucidated. Upregulation of ERK1/2 protein
expression after MI, however, was substantially higher in the
myocardium of non-transgenic mice (Fig. 1) compared to aMEK1
transgenic littermates. In contrast, the amount of phosphorylated
ERK 1/2 (P-ERK) was significantly higher in MEK1 Tg compared to
littermate WT controls in stressed cardiac cells after MI (Fig. 1).
Expression of MAP kinase phosphatases (MKPs) 1 and 3 did not
differ between the myocardial tissues of MEK1 Tg and WT controls
after MI, suggesting that this difference in ERK phosphorylation
was not mediated by an alteration in MKP expression. Levels of
stress activated kinase p38 and JNK expression and phosphorylation
were not significantly different in MEK1 Tg compared to WT
TABLE I. Functional Parameters of the Left Ventricles 4 Weeks After LAD Ligation
MEK1 Tg (n ¼ 13) WT (n ¼ 14) MEK1 Tg (n ¼ 45) WT (n ¼ 45)
FS (%) 52 152 149 1
43 1
LVEDV (ml) 39 436 3
42 3
54 4
LVESV (ml) 9 18 1
11 1
17 2
SW (mmHg ml) NA NA 732 141
371 91
dPdt max (mmHg/s) NA NA 5341 628 4257 438
Ees (mmHg/ml) NA NA 6.14 0.57
3.92 0.6
PRSW (mmHg) NA NA 603 98
335 40
bMHC/aMHC</bold NA NA 1.20 0.08
1.37 0.09
ANP/18S NA NA 1.29 0.13
1.54 0.11
HW/BW (%) 0.58 0.02
0.48 0.01
0.59 0.01
0.51 0.01
P < 0.05;
P < 0.01;
Tg versus WT;
sham versus MI; for in vivo hemodynamic measurements, n ¼ 9 in MEK1 Tg and n ¼ 7 in WT; for real-time PCR analysis of the
expression of MHCs and ANP, n ¼ 13 in MEK1 Tg and n ¼ 4 in WT. FS, fract ional shortening; LVEDV, left ventricle end-diastolic volume; LVESV, left ventricle end-
systolic volume; SW, stroke work; Ees, end-systolic elastance; PRSW, preload recruitable stroke work.
It has previously been documented that post-MI remodeling of the
remote myocardium is associated with a significant increase in
myocardial apoptosis [Qin et al., 2005; Yeh et al., 2010]. The large
increase in P-ERK in MEK1 Tg hearts compared to littermate WT
controls was associated with a statistically significant 59%
reduction in myocardial apoptosis (apoptotic rate of 0.66 0.11%
in MEK1 Tg vs. 1.60 0.34% in littermate WT controls, P < 0.05), as
measured by TUNEL staining (Fig. 2A). Furthermore, it has been
shown that constitutive MEK1-ERK1/2 signaling in the uninfarcted
hearts of these transgenic mice results in a stable hypertrophy of
CMs that does not degenerate into a dilated cardiomyopathy. Even
after MI, surviving CMs in the remote myocardium of MEK1 Tg mice
remained significantly larger than the CM of littermate WT controls
(Fig. 2B).
The loss of CMs to a slow but relentless increase in apoptosis is
believed to contribute to chronic extension of infarction in the LV
wall, and to weakening and fibrosis of the post-MI remote
myocardium. Reperfusion injury involves a significant degree of
CM apoptosis that contributes to acute infarct size. Even though
overexpression of anti-apoptotic aMEK1 has been shown to protect
the myocardium from acute reperfusion injury and reduce acute
infarct size in the area-at-risk, we found that aMEK1 overexpression
did not affect the initial infarct size 24 h after permanent ligation of
the LAD in which necrosis, rather than apoptosis, predominantes
and in which there is little reperfusion injury (Fig. 2C, acute
infarction of 57.5 4.7% and 54.0 4.6% of the area-at-risk in
MEK1 Tg and WT controls, respectively, P ¼ 0.96). However, at
4 weeks post-MI the percentage of infarct in the LV wall was
significantly lower in MEK1 Tg mice than in their littermate WT
controls (Fig. 2D). Fibrosis was also found to be substantially
reduced in the remote myocardium of post-MI MEK1 Tg mice
compared to their littermate controls (Fig. 2E).
The role of matrix metalloproteinases (MMPs) and tissue inhibitors
of metalloproteinases (TIMPs) in pathologic remodeling remains
Fig. 1. Expression of MAPK related proteins in the remote myocardium 4 weeks after MI. A: Representative Western blots of protein expression and phosphorylation of
MAPK-related proteins. B–E: Quantification of phosphorylated ERK1/2 (P-ERK1/2), total ERK1/2, phosphorylated MEK1/2 (P-MEK1/2), and total MEK1/2 expression levels
(n ¼ 14, MEK1 Tg; n ¼ 7, WT;
< 0.05).
somewhat controversial, but specific MMPs and TIMPs have been
suggested by some to moderate the accumulation of collagen during
myocardial fibrosis [Baker et al., 1998; Hayashidani et al., 2003;
Matsusaka et al., 2005; Mias et al., 2009; Givvimani et al., 2010].
Although the protein levels of MMP2, known to play an important
role in the myocardium and associated in some studies with
protection from fibrotic remodeling, were similar in infarcted hearts
from both MEK1 Tg and WT hearts, the activity of this enzyme was
significantly higher in the post-MI remote myocardium of the
transgenic animals (Fig. 3). Similarly higher MMP2 activity was also
observed in non-infarcted MEK1 Tg hearts than in non-transgenic
control hearts, suggesting a possible role for MMP2 in the resistance
Fig. 2. Histological changes in MEK1 Tg and WT remote myocardium 4 weeks after MI. A: Apoptosis by TUNEL staining (400). B: Myocyte surface revealed by WGA staining
(100), C: Infarct size in the risk area measured by TTC staining (10). D: Percentage of LV infarction measured via trichrome staining (20). E: Fibrosis as measured by collagen
staining (100). n ¼ 5–10,
< 0.05,
< 0.01, NS, no significant difference.
that has been demonstrated in these hearts to deterioration from
stable to pathologic hypertrophy. MMP9 and TIMP3 have been
associated with pathologic remodeling in some studies [Baker et al.,
1998; Givvimani et al., 2010]; interestingly, both higher MMP9
activity (in some animals) and significantly higher expression of
TIMP3 (in all animals tested) were observed in the remote
myocardium of non-transgenic infarcted hearts, but not in the
infarcted hearts of MEK1 Tg mice (Fig. 3).
Echocardiography of MEK1 Tg animals after MI reflected a
preservation of global LV structure, with no significant increase
in either diastolic or systolic LV dimensions compared to non-
infarcted controls (Table I). In contrast, the LV chambers of post-MI
WT hearts were dilated, with 50% ( P < 0.01) and 113% ( P < 0.01)
increases in LV diastolic and systolic dimensions, respectively
(Table I).
The preservation of microscopic and macroscopic structure in the
remote myocardium of post-MI MEK1 Tg mice also coincided with
a significant preservation of LV function compared to WT controls.
A 17.3% decline in fractional shortening (regional function) was
observed at a point remote from the LV infarct in the myocardium of
WT controls. In contrast, this decline was only 6% in hearts with
CM-specific aMEK1 overexpression during the post-MI period
(Table I). This functional benefit from MEK1-ERK1/2 gain-of-
function was confirmed with invasive measurements of stroke work
(SW), end-systolic elastance (Ees), and preload recruitable stroke
work (PRSW), confirming a significant improvement in ventricular
contractility after MI compared to non-transgenic littermates
(Table I).
The phosphorylation (i.e., activation) of various known downstream
mediators of MEK1-ERK1/2 signaling was assessed in an attempt
to better understand the therapeutic mechanism of aMEK1 over-
expression in reducing post-MI remodeling of the remote
myocardium. A significant increase was observed in the phosphor-
ylation of pro-survival transcription factor STAT3 (Fig. 4). The
increase in absolute levels of P-ERK1/2 in MEK1 Tg hearts was also
associated with an increase in protein levels of anti-apoptotic Bcl-2
and in phosphorylation (i.e., inactivation) of pro-apoptotic BAD,
while expression of anti-apoptotic Bcl-xL and pro-apoptotic protein
BAX remained the same (Fig. 5).
Although previous studies have begun to address the causal and
potentially therapeutic roles of changes in MEK-ERK signaling in
the setting of acute ischemia and reperfusion, the objective of these
studies was an elucidation of the impact of myocardial MEK-ERK
activation on chronic post-MI remodeling, possibly the largest
Fig. 3. Expression of MMP-related proteins in the remote myocardium 4 weeks after MI. A: Representative Western blots of protein expression of MMPs and TIMPs. B:
Quantification of MMP2 activity in the remote myocardium after MI (n ¼ 5;
< 0.05). C: Representative zymography of MMP2 and MMP9 activity in the remote myocardiu m
of MEK1 Tg and WT mice with or without MI.
single cause of congestive heart failure in the United States. While
these previous studies have demonstrated a role for changes in
MEK-ERK signaling in ameliorating the apoptosis and extent of
infarction related to very rapid changes instigated by acute stresses
in CM, they do not speak to the possible role of long-term alteration
in the balance of MAPK pathways in the much more complex
and insidious process of chronic ventricular fibrosis and remodeling.
We worked with a transgenic mouse model in which low to moderate
levels of constitutive aMEK1 expression from birth led only to a
moderate increase in baseline HW/BW ratio and individual CM surface
size, but no other demonstrable differences in other parameters of
myocardial structure or function prior to a standardized permanent
coronary ligation. The prior influence of low–moderate over-
expression of MEK-ERK signaling in this model before MI may
therefore have had only a minimal confounding effect on the
chronic remodeling observed after MI, allowing at least tentative
conclusions to be drawn from the effect of post-MI MEK-ERK
overexpression on this important pathologic process.
In this study, a transgenic model of stable, long-term CM-specific
upregulation of MEK1-ERK1/2 signaling did, in fact, suggest that
modulation of the balance among MAP kinase pathways can play a
role in the context of chronic LV remodeling. Overexpression of
aMEK1 resulted in a reduction in myocardial apoptosis, documented
via TUNEL staining, and was associated with increased levels of
anti-apoptotic Bcl-2 protein and of phosphorylation (inactivation)
of pro-apoptotic BAD. The chronic protection against CM apoptosis
afforded by long-term P-ERK1/2 upregulation may also have been
mediated, at least in part, by an observed increase in phosphoryla-
tion (activation) of the cardioprotective transcription factor STAT3
[Obana et al., 2010].
Fig. 4. Expression of STATs in the remote myocardium 4 weeks after MI. A:
Representative Western blots of protein expression and/or phosphorylation
(P-) of STAT3 and STAT5. B: Quantification of phospho-STAT3 (P-STAT3)
expression and phosphorylation level in the post-MI remote myocardium,
n 6,
< 0.05.
Fig. 5. Regulation of apoptosis-related genes in the 4 weeks post-MI remote
myocardium. A: Representative Western blots of protein expression and/or
phosphorylation (P-) of anti-apoptotic and pro-apoptotic factors. B–C:
Quantification of Bcl-2 and phosphorylated BAD/BAD level in the post-MI
remote myocardium, n 6,
< 0.05,
< 0.01.
Chronic inhibition of apoptosis in the surviving post-MI
myocardium was correspondingly associated both with a substan-
tially lower degree of infarct extension and with an inhibition
of post-MI fibrosis in the remote myocardium. Although the
expression and activity of MMP2 has been linked to the rupture of
infarcted myocardium in acute MI models, the role of MMP2 in
the uninfarcted remote myocardium during chronic remodeling
remains to be fully elucidated [Hayashidani et al., 2003]. In fact,
MMP2, the activity of which was increased in the remote post-MI
myocardium of MEK1 Tg mice, has been shown to suppress
inflammation and preserve cardiac function in a model of TNF-
induced cardiomyopathy, and may also promote adaptive remodel-
ing via the removal of excess collagen from the region remote to a
chronic infarct [Matsusaka et al., 2005; Mias et al., 2009]. In fact,
nonspecific inhibition of MMP activity has been found to enhance
pathological hypertrophy in the mouse aortic constriction model
[Vinet et al., 2008]. Another recent study suggested that MMP2 may
promote angiogenesis during adaptive hypertrophy [Givvimani
et al., 2010]. On the other hand, increased expression of TIMP3 has
been shown to induce apoptosis, and both TIMP3 and MMP9 have
been found to contribute to decompensatory hypertrophy [Baker
et al., 1998; Givvimani et al., 2010]. Both of these potentially
pathologic modulators of myocardial matrix were found to be
elevated in specimens from WT but not MEK1 Tg mice after MI. It has
been reported that aMEK1 can induce both MMP2 and MMP9
protein translation and increase their activities [Lemieux et al.,
2009], but only an increase in MMP2 activity was observed in MEK1
Tg after MI. This observation of increased MMP2 and a possible
change in the balance among different MMPs and TIMPs in MEK1 Tg
mice may contribute to a favorable environment for maintaining
compensatory hypertrophy rather than pathologic hypertrophy in
the post-MI myocardium.
Extensive in vitro studies of cardiac myocyte biology have
documented a well established role for MEK1 in instigating a
downstream cardioprotective cascade involving ERK1/2 phopshor-
ylation, both through pharmacologic and genetic MEK1 inhibition
[Zhu et al., 1999; Yue et al., 2000; Huang et al., 2007], and through
MEK1 gain of function [Bueno et al., 2000]. In addition,
augmentation of ER1/2 phosphorylation through down regulation
of MKP3 has also been associated with enhanced cell survival and
adaptive cardiac hypertrophy [Maillet et al., 2008]. These and other
studies, however, have demonstrated that changes in MEK1-ERK1/2
signaling develop rapidly (on the order of minutes) in the context
of acute hypoxia [Seko et al., 1996]; rapid changes in ERK1/2
phosphorylation have similarly been observed in ex vivo and in vivo
studies of I/R injury [Knight and Buxton, 1996; Ballard-Croft et al.,
2006]. Cardiac upregulation of P-ERK1/2 in MEK1 Tg mice has also
been found to reduce infarct size after I/R [Bueno et al., 2000].
Whereas it is understandable that rapid changes in MEK1-ERK1/2
signaling influence the balance of pro-apoptotic and survival
signaling within cardiac cells in this acute setting of CM death
immediately after I/R, very little is known about the functional
significance of chronic changes in this signaling pathway as it
relates to the insidious loss of CM during longer-term post-MI
remodeling of the remote, uninfarcted myocardium. It is this chronic
remodeling of the post-MI heart, however, that instigates the
transition from compensated function after MI to cardiomyopathy
in the majority of post-MI heart failure patients. The results of this
study are among the first to directly suggest via gain-of-function
that a causal relationship exists between P-ERK1/2 levels and the
progression of myocardial apoptosis, fibrosis, and dysfunction
during this chronic remodeling process. Given the enormous
complexity of the stresses and of the biological responses within the
myocardium during this chronic process, this kind of cell-type
specific molecular elucidation is essential for confirming hypotheses
regarding the contributory roles of various signaling moieties both
to pathogenesis and to potential therapeutic molecular intervention.
Significant limitations remain, however, regarding the conclu-
sions that can be drawn from these first-generation gain-of-
function experiments. Although our model of low–moderate aMEK1
expression did minimize the impact of constitutive MEK1-ERK1/2
upregulation on pre-MI structure and function, the potentially
confounding variable of modest, pre-existing cardiac hypertrophy
in MEK1 Tg hearts at the time of acute infarction in this model could
not be entirely eliminated. It cannot therefore be determined from
this model what role, if any, was played by this modest pre-existing
difference in myocardial structure on the outcome of post-MI
remodeling. Measurement of acute infarct size in this model of
permanent coronary ligation, in which apoptosis likely plays a
smaller role relative to necrosis than in models of I/R, did not reveal
a strong influence of anti-apoptotic ERK signaling on immediate
infarct size. The larger infarcts seen at 4 weeks therefore likely
reflect the contribution of this anti-apoptotic pathway in limiting
more chronic infarct expansion.
Despite their limitations, the current studies have established, for
the first time, an important verification of the contributory role that
chronic changes in MEK1-ERK1/2 signaling can have on the
complex, slow processes of CM loss and of regional/global changes
in ventricular structure and function during pathologic cardiac
remodeling. They also provide a necessary proof-of-concept with
regard to the efficacy of molecular intervention in post-MI
cardiomyopathy. Whereas previous studies have begun to explore
therapeutic roles for manipulation of MAP kinase and other
signaling pathways in the setting of acute ischemia and I/R [Bueno
et al., 2000; Ren et al., 2005; Engel et al., 2006; Li et al., 2006], a
greater impact on human heart failure may eventually be achievable
through novel strategies that address the large pool of patients who
live with and eventually succumb to chronic, post-MI remodeling of
the remote myocardium.
The authors express their appreciation for the gift of the transgenic
mouse model (aMEK1 Tg) provided by Dr. Jeffrey Molkentin and for
the technical assistance provided by Dr. Richard Tu. This work was
supported by National Institutes of Health grants K08HL079239 and
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    • "The phosphorylation of CREB and IKK stimulates the transcription of pro-survival factors (Yuan and Yankner 2000). Phosphorylation of STAT3 at Ser727 by pERK and the consequent higher expression of anti-apoptotic protein Bcl2 were investigated before (Ng et al. 2006; Yeh et al. 2013; Zhou and Too 2011). Additionally, 0.5 mM acrylamide treatment induced expression of Bcl2 and DNA ligase III (a DNA repair protein) in human astrocytoma cells (Chen et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Acrylamide is a neurological and reproductive toxicant in humans and laboratory animals; however, the neuron developmental toxicity of acrylamide remains unclear. The aims of this study are to investigate the cytotoxicity and neurite outgrowth inhibition of acrylamide in nerve growth factor (NGF)- or fibroblast growth factor 1 (FGF1)-mediated neural development of PC12 cells. MTS assay showed that acrylamide treatment suppresses NGF- or FGF1-induced PC12 cell proliferation in a time- and dose-dependent manner. Quantification of neurite outgrowth demonstrated that 0.5 mM acrylamide treatment resulted in significant decrease in differentiation of NGF- or FGF1-stimulated PC12 cells. This decrease is accompanied with the reduced expression of growth-associated protein-43, a neuronal marker. Moreover, relative levels of pERK, pAKT, pSTAT3 and pCREB were increased within 5-10 min when PC12 cells were treated with NGF or FGF1. Acrylamide (0.5 mM) decreases the NGF-induced activation of AKT-CREB but not ERK-STAT3 within 20 min. Similarly, acrylamide (0.5 mM) decreases the FGF1-induced activation of AKT-CREB within 20 min. In contrast to the NGF treatment, the ERK-STAT3 activation that was induced by FGF1 was slightly reduced by 0.5 mM acrylamide. We further showed that PI3K inhibitor (LY294002), but not MEK inhibitor (U0126), could synergize with acrylamide (0.5 mM) to reduce the cell viability and neurite outgrowth in NGF- or FGF1-stimulated PC12 cells. Moreover, acrylamide (0.5 mM) increased reactive oxygen species (ROS) activities in NGF- or FGF1-stimulated PC12 cells. This increase was reversed by Trolox (an ROS scavenging agent) co-treatment. Together, our findings reveal that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K-AKT-CREB signaling, along with the production of ROS.
    Full-text · Article · Dec 2013
    • "In heart diseases, TIMP-3 may contribute to the regulation of myocardial remodeling, it deficiency disrupts matrix homeostasis and causes spontaneous left ventricular dilation, cardiomyocyte hypertrophy and contractile dysfunction 7. Our published paper proposed that the interaction between MMP-2, MMP-9 and TIMP-3 may contribute to atrial ECM remodeling of atrial fibrillation 19. In addition, many studies also appear that the association of MMP-2, MMP-9 and TIMP-3 in heart diseases 31, heart remodeling 32, 33 and cardiocytes function properly 34, and supported that MMP-2, MMP-9 and TIMP-3 may play important role in VSD. "
    [Show abstract] [Hide abstract] ABSTRACT: Ventricular septal defect (VSD) is the most common form of congenital heart diseases. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in causal cardiac tissue remodeling. We studied the changes of circulating MMP-2 and MMP-9 activities in the patients with VSD severity and closure. There were 96 children with perimembranous VSD enrolled in this study. We assigned the patients into three groups according to the ratio of VSD diameter/diameter of aortic root (Ao). They were classified as below: Trivial (VSD/Ao ratio ≤ 0.2), Small (0.2 < VSD/Ao ≤ 0.3) and Median (0.3 < VSD/Ao) group. Plasma MMP-2 and MMP-9 activities were assayed by gelatin zymography. There was a significant higher MMP-2 activity in the VSD (Trivial, Small and Median) groups compared with that in Control group. The plasma MMP-9 activity showed a similar trend as the findings in MMP-2 activity. After one year follow-up, a significant difference in the MMP-9 activity was found between VSD spontaneous closure and non-closure groups. In conclusion, a positive trend between the severity of VSD and activities of MMP-2 and MMP-9 was found. Our data imply that MMP-2 and MMP-9 activities may play a role in the pathogenesis of VSD.
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    • "Specifically, differences in the activation distribution and in the heart and body surface geometry can be due to underlying pathophysiology. This hypothesis is indirectly supported by the experimental studies, which identified signaling pathways, affecting some, but not all features of remodeling in different disease models [16,40]. Further theoretical and clinical studies are needed to clarify this intriguing discrepancy. "
    [Show abstract] [Hide abstract] ABSTRACT: Post-myocardial infarction (MI) structural remodeling is characterized by left ventricular dilatation, fibrosis, and hypertrophy of the non-infarcted myocardium. The goal of our study was to quantify post-MI electrical remodeling by measuring the sum absolute QRST integral (SAI QRST). We hypothesized that adverse electrical remodeling predicts outcomes in MADIT II study participants. Baseline orthogonal ECGs of 750 MADIT II study participants (448 [59.7%] ICD arm) were analyzed. SAI QRST was measured as the arithmetic sum of absolute QRST integrals over all three orthogonal ECG leads. The primary endpoint was defined as sudden cardiac death (SCD) or sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) with appropriate ICD therapies. All-cause mortality served as a secondary endpoint. Adverse electrical remodeling in post-MI patients was characterized by wide QRS, increased magnitudes of spatial QRS and T vectors, J-point deviation, and QTc prolongation. In multivariable Cox regression analysis after adjustment for age, QRS duration, atrial fibrillation, New York Heart Association heart failure class and blood urea nitrogen, SAI QRST predicted SCD/VT/VF (HR 1.33 per 100 mV*ms (95%CI 1.11-1.59); P = 0.002), and all-cause death (HR 1.27 per 100 mV*ms (95%CI 1.03-1.55), P = 0.022) in both arms. No interaction with therapy arm and bundle branch block (BBB) status was found. In MADIT II patients, increased SAI QRST is associated with increased risk of sustained VT/VF with appropriate ICD therapies and all-cause death in both ICD and in conventional medical therapy arms, and in patients with and without BBB. Further studies of SAI QRST are warranted.
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