MG132, a proteasome inhibitor, attenuates pressure-overload-induced cardiac hypertrophy in rats by modulation of mitogen-activated protein kinase signals.

Abstract

Proteasome inhibitors are involved in cell cycle control, growth and inflammatory signaling, and transcriptional regulation of mitotic cells. A recent study has suggested that specific proteasome inhibitor MG132 may suppress cardiomyocyte hypertrophy in vitro. However, the underlying molecular mechanisms are not clear. In this study, we investigated the effects of long-term MG132 treatment on cardiac hypertrophy and the related molecular mechanisms in vivo. MG132 (0.1 mg/kg/day) was intraperitoneally injected to rats with abdominal aortic banding (AAB) for 8 weeks. Results showed that treatment with MG132 significantly attenuated left ventricular (LV) myocyte area, LV weight/body weight, and lung weight/body weight ratios, decreased LV diastolic diameter and wall thickness, and increased fractional shortening in AAB rats. AAB induced the phosphorylation of ERK1/2, JNK1, and p38 in cardiac myocytes. The elevated phosphorylation levels of ERK1/2 and JNK1 in AAB rats were significantly reversed by MG132 treatment. In conclusion, our results suggested that long-term treatment with MG132 attenuates pressureoverload-induced cardiac hypertrophy and improves cardiac function in AAB rats through regulation of ERK1/2 and JNK1 signaling pathways.

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Original Article
MG132, a proteasome inhibitor, attenuates pressure-overload-induced cardiac
hypertrophy in rats by modulation of mitogen-activated protein kinase signals
Baolin Chen1,2, Yuedong Ma1,3, Rongsen Meng1, Zhaojun Xiong1, Chengxi Zhang1, Guangqin Chen1, Aixia Zhang1,
and Yugang Dong1,3*
1Department of Cardiology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China
2Department of Cardiology, The People’s Hospital of Guizhou Province, Guiyang 550002, China
3Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou 510080, China
*Correspondence address. Tel: þ86-20-87332200-8149; Fax: þ86-20-87332200-8151; E-mail: yg.dong@yahoo.com
Proteasome inhibitors are involved in cell cycle control,
growth and inflammatory signaling, and transcriptional
regulation of mitotic cells. A recent study has suggested
that specific proteasome inhibitor MG132 may suppress
cardiomyocyte hypertrophy in vitro. However, the under-
lying molecular mechanisms are not clear. In this study, we
investigated the effects of long-term MG132 treatment on
cardiac hypertrophy and the related molecular mechanisms
in vivo. MG132 (0.1 mg/kg/day) was intraperitoneally
injected to rats with abdominal aortic banding (AAB) for 8
weeks. Results showed that treatment with MG132 signifi-
cantly attenuated left ventricular (LV) myocyte area, LV
weight/body weight, and lung weight/body weight ratios,
decreased LV diastolic diameter and wall thickness, and
increased fractional shortening in AAB rats. AAB induced
the phosphorylation of ERK1/2, JNK1, and p38 in cardiac
myocytes. The elevated phosphorylation levels of ERK1/2
and JNK1 in AAB rats were significantly reversed by
MG132 treatment. In conclusion, our results suggested
that long-term treatment with MG132 attenuates pressure-
overload-inducedcardiachypertrophy
cardiac function in AAB rats through regulation of ERK1/2
and JNK1 signaling pathways.
and improves
Keywords
transduction; abdominal aortic banding
MG132; cardiac hypertrophy; MAPK; signal
Received: November 15, 2009Accepted: January 11, 2010
Introduction
Cardiac hypertrophy is a key compensatory mechanism in
response to pressure or volume overload that involves
alterations in the regulation of signaling transduction path-
ways and transcription factors [1]. Hypertrophic signals are
further integrated within the cardiomyocytes and result in
enhanced protein synthesis, altered cell cycle regulation,
and hypertrophic gene expression [2]. Thus, cardiac hyper-
trophy appears to be a specialized form of cellular growth
that requires mechanisms normally involved in proliferation
control and cell cycle regulation [2]. The ubiquitin–protea-
some system is the major pathway for intracellular protein
degradation in mitotic cells [3], controlling the level of
many key proteins involved in cell cycle control, cellular
mass, growth signaling, and transcriptional regulation [4–
6]. Previous evidences indicated an association of protea-
some dysfunction with the pathogenesis of heart disease,
such as ischemia–reperfusion injury [7], doxorubicin cardi-
otoxicity [8], heart failure [9], and hypertrophic cardiomyo-
pathy [10,11]. It has been reported that pharmacological
inhibition of the proteasome was associated with alterations
in protein expression profile of cardiomyocyte, and modu-
lated cardiovascular disease progression [12,13]. MG132, a
specific proteasome inhibitor, has been shown to suppress
proliferation and inflammation [14],and possess proapopto-
tic [15,16] and antineoplastic [17,18]activities.
Recently, another report has shown that MG132 inhibits
isoproterenol-induced hypertrophy in cultured cardiomyo-
cytes [4]. However, there has been no similar study about
the effects of MG132 on hypertrophic cardiomyocytes
in vivo. Thus, the aim of the present study was to investigate
whether long-term treatment with MG132 could attenuate
left ventricular (LV) hypertrophy induced by pressure over-
load in rats and to elucidate the underlying mechanisms.
Materials and Methods
Animal models of cardiac hypertrophy
Male Sprague–Dawley rats (8 weeks old, 180 – 230 g)
were used toestablishpressure-overload
described previously [19]. All animals were separated into
four groups (10 rats per group): (i) vehicle-treated sham
model as
Acta Biochim Biophys Sin (2010): 253–258 | ª The Author 2010. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmq012.
Advance Access Publication 2 March 2010
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group; (ii) MG132-treated sham group; (iii) vehicle-treated
abdominalaortic banding
MG132-treated AAB group. Under intraperitoneal pento-
barbital (50 mg/kg) anesthesia, AAB was created using a
5-0 suture tied twice around the abdominal aorta in which
a 21-gauge needle was inserted. The needle was then
retracted yielding a 70 – 80% constriction with an outer
aortic diameter of ?0.8 mm. In the sham surgery rats, the
same surgery was performed as described above except the
aorta was constricted. At Day 3 after the surgery,
MG132-treated rats were intraperitoneally injected with
0.1 mg/kg/dayof MG132
C26H41N3O5; ALEXIS, San Diego, USA) for 8 weeks. All
control animals were injected with a corresponding volume
of vehicle only (0.1% DMSO). Animals and all surgeries
involved in the experiments were approved by the Animal
Care and Use Committee of Sun Yat-Sen University
(Guangzhou, China).
(AAB) group;and(iv)
(Z-Leu-Leu-Leu-CHO,
Echocardiographic and hemodynamic measurements
Eight weeks after the surgery, each rat was weighed, and
transthoracic echocardiography was performed on each rat.
Rats were anesthetized with intraperitoneal pentobarbital
(50 mg/kg), and left atrial diameter, LV end-diastolic diam-
eter, fractional shortening, and septal and posterior wall
thickness were measured or analyzed by an echocardio-
graphic system (ATL-HDI5000; Philips Medical System,
Bothell, USA) equipped with a 10-MHz imaging transdu-
cer. All measurements were averaged for 10 consecutive
cardiac cycles.
Then, a catheter was introduced through the right carotid
artery into the LV for hemodynamic measurements in all
animals. Heart rate, LV systolic pressure, and LV end-
diastolic pressure were measured with a commercially
available analog-to-digital converter and BIOPAC acknowl-
edge analysis software (Goleta, USA).
Neurohormonal factors
Angiotensin II (Ang II) concentration and renin activity
levels were determined with commercially available kits
(Ambion, Austin, USA). Eight weeks after surgery, 5 ml of
blood collected from the right carotid artery was used to
measure the concentration of Ang II and renin activity by
radioimmunoassay (Northern Biot Co., Beijing, China)
according to the manufacturer’s instructions.
Histological analysis and cardiomyocyte
size measurements
Rats were sacrificed at 8 weeks after AAB. Hearts were
arrested in diastole with KCl (30 mM). Hearts and lungs
were dissected and weighed in all cases. LV samples were
frozen in liquid nitrogen and then stored at 2808C or fixed
with 10% formalin. Fixed hearts were embedded in
paraffin and sectioned to 4-mm thickness. Samples were
stained with hematoxylin–eosin for overall morphology.
Myocyte size from each group was evaluated by measuring
the cross-sectional area of cells using the Image-Pro Plus
system. Mean myocyte area was calculated by measuring
100 cells from sections stained with hematoxylin–eosin.
Western blot analysis
Heart tissue was lysed in RIPA buffer. Total protein con-
centration was determined using the bicinchoninic acid
protein assay (Pierce, Rockford, USA). Fifty micrograms
of total proteins were electrophoresed by 10% SDS–poly-
acrylamide gel and then transferred onto a PVDF mem-
brane. The membrane was blocked for 2 h at room
temperature in blocking solutions, incubated overnight at
48C with anti-atrial natriuretic peptide (anti-ANP, 1:400
dilution; Santa Cruz, Santa Cruz, USA), anti-B-type
natriuretic peptide (anti-BNP, 1:400 dilution; Santa Cruz),
anti-p-ERK1/2 (1:800 dilution; Cell Signal Technology,
Beverly, USA), anti-ERK1/2
Signal Technology),anti-p-JNK1
Cell Signal Technology), anti-JNK1 (1:1000 dilution; Cell
Signal Technology), anti-p-p38 (1:800 dilution; Cell Signal
Technology),anti-p38 (1:600
Technology), andanti-GAPDH
Kangcheng Inc., Shanghai, China) primary antibodies. The
membrane was then washed with TBS-T (10 mM Tris–
HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) and
incubated with horseradish peroxidase-conjugated anti-
mouse or anti-rabbit secondary antibody (Boshide Inc.,
Wuhan, China) at 378C for 1 h. The immune complex was
detected with enhanced chemiluminescence (Millipore,
Billerica, USA) system, exposed to X-ray film, and ana-
lyzed using NIH Image software. GAPDH was used as a
control.
(1:1000dilution;Cell
(1:1000dilution;
dilution;
(1:40,000
Cell Signal
dilution;
Statistical analysis
Data are expressed as the mean+SEM. Differences among
groups were tested by one-way ANOVA. Comparisons
between two groups were performed by unpaired Student’s
t-test. A value of P , 0.05 was considered statistically sig-
nificant. Statistical analyses were performed using SPSS
16.0 statistics software.
Results
Mortality after surgery
In our study, all animals in the sham group survived, and
the mortality in AAB rats 8 weeks after operation was
25%. Of all 40 rats, the final surviving number was 10 in
the vehicle-treated sham group, 10 in the MG132-treated
sham group, 7 in the vehicle-treated AAB group, and 8 in
the MG132-treated AAB group.
MG132 attenuates cardiac hypertrophy
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Morphometric characterization
As shown in Table 1, the LV weight/body weight (LVW/
BW) and lung weight/BW ratios of AAB rats were signifi-
cantly increased compared with vehicle-treated sham rats
(P, 0.01 and
,0.05, respectively). Compared with
vehicle-treated AAB rats, the LVW/BW and lung weight/
BW ratios in MG132-treated AAB rats were markedly
decreased (P , 0.05, respectively).
Hemodynamic measurements
After surgery, an increased LV systolic pressure was
observed in AAB rats (P, 0.01 vs. sham group). The LV
systolic pressure gradient was similar in vehicle- or
MG132-treated AAB rats (P . 0.05). The LV end-diastolic
pressure was higher in vehicle-treated AAB rats compared
with vehicle-treated sham rats (P, 0.01). MG132 treat-
ment significantly prevented the increase in LV end-
diastolic pressure in AAB rats (P, 0.01) (Table 1).
Echocardiographic data
As shown in Table 2, we observed an enlargement of the
LV end-diastolic diameter and left atrial diameter as well
as an increase in the septal and posterior wall thickness in
the vehicle-treated AAB group when compared with the
vehicle-treated sham rats (P , 0.05 and ,0.01, respect-
ively). Fractional shortening was significantly lower in the
vehicle-treated AAB group compared with the vehicle-
treated sham group (P, 0.01). In the MG132-treated
AAB group, LV end-diastolic diameter, left atrial diam-
eter, septal and posterior wall thickness, and fractional
shortening were significantly improved compared with the
vehicle-treated AAB group
respectively).
(P, 0.05and
,0.01,
Measurements of neurohormonal factors
Ang II and renin are established markers of cardiac hyper-
trophy. Therefore, we measured them in blood samples
from the animals. The plasma Ang II concentration and
renin activity were significantly increased in the vehicle-
treated AAB group compared with the vehicle-treated sham
group (P , 0.01, respectively). MG132 treatment markedly
prevented these alterations induced by AAB (P, 0.01,
respectively) (Table 2).
Table 1 Effects of AAB and MG132 on body weight, organ weights, and hemodynamic measurements
Parameters ShamAAB
Vehicle (n ¼ 10) MG132 (n ¼ 10) Vehicle (n ¼ 7) MG132 (n ¼ 8)
HR (b.p.m.)
LVW/BW (mg/g)
Lung wt/BW (mg/g)
LVSP (mmHg)
LVEDP (mmHg)
382+16
2.13+0.10
5.44+0.22
103.8+2.9
9.32+0.56
396+18
1.99+0.07
5.39+0.18
99.4+3.5
8.82+0.63
395+15
2.64+0.12**
6.26+0.21*
160.1+3.7**
23.47+1.07**
411+18
2.35+0.12#
5.64+0.15#
156.9+2.5
16.85+0.85##
HR, heart rate; LVW, left ventricular weight; BW, body weight; lung wt, lung weight; LVSP, left ventricular systolic pressure; LVEDP, left ventricular
end-diastolic pressure. Data are expressed as the mean+SEM. *P , 0.05 and **P , 0.01 vs. vehicle-treated sham group, and#P , 0.05 and##P , 0.01
vs. vehicle-treated AAB group.
Table 2 Echocardiographic assessment and neurohormonal factors
Parameters ShamAAB
Vehicle (n ¼ 10) MG132 (n ¼ 10)Vehicle (n ¼ 7)MG132 (n ¼ 8)
IVSd (mm)
PWd (mm)
LVDd (mm)
LA (mm)
FS (%)
Ang II (pg/ml)
Renin activity (ng/ml/h)
1.91+0.05
1.86+0.04
4.97+0.14
3.21+0.12
54.88+1.84
325+16
1.84+0.08
1.89+0.06
1.84+0.05
4.87+0.13
3.01+0.13
55.79+2.15
362+22
1.72+0.08
2.14+0.05*
2.16+0.08**
5.97+0.15**
4.09+0.11**
44.94+1.72**
1954+88**
6.70+0.27**
1.97+0.04#
1.97+0.04#
5.23+0.18##
3.65+0.13#
51.08+2.00#
887+40##
3.62+0.14##
IVSd, end-diastolic interventricular septal thickness; PWd, end-diastolic posterior wall thickness; LVDd, left ventricular end-diastolic diameter; LA,
left atrium; FS, fractional shortening; Ang II, angiotensin II. Data are expressed as the mean+SEM. *P , 0.05 and **P , 0.01 vs. vehicle-treated sham
group, and#P , 0.05 and##P , 0.01 vs. vehicle-treated AAB group.
MG132 attenuates cardiac hypertrophy
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Histological analysis
The cross-sectional area of LV myocytes was measured in
the different groups. Compared with the vehicle-treated
sham group, the mean myocyte area of the vehicle-treated
AABgroupwassignificantly
Treatment of AAB rats with MG132 markedly reduced the
mean myocyte area compared with the AAB rats without
MG132 treatment (P, 0.01) (Fig. 1).
increased(P, 0.01).
Effects of MG132 on the expression of ANP
and BNP protein
Cardiac hypertrophy is characterized by induction of ‘fetal’
gene expression. As shown in Fig. 2, ANP and BNP
protein levels were significantly higher in the vehicle-
treated AAB group compared with those in the vehicle-
treated sham group (P , 0.01, respectively). MG132 treat-
ment reversed the AAB-induced increases in ANP and
BNP protein expression (P , 0.01, respectively).
Effects of MG132 on mitogen-activated protein
kinase signaling pathways
Previous studiesdemonstrated
protein kinases (MAPKs) are involved in the regulation of
cardiac hypertrophy. To determine whether AAB and
MG132 treatment affect the activity of MAPKs, we
measured changes in the phosphorylation levels of ERK1/
2, JNK1, and p38. As shown in Fig. 3, the phosphorylation
of ERK1/2, JNK1, and p38 were significantly enhanced in
the vehicle-treated AAB group (P, 0.01 vs. vehicle-
treated sham, respectively). Long-term injection of MG132
had different impacts on them during the process of hyper-
trophic growth. Specifically, MG132 treatment resulted in
decreasedERK1/2 and
AAB-induced cardiac hypertrophy (P , 0.01, respectively),
whereas no significant difference was shown in the phos-
phorylation of p38 between vehicle- and MG132-treated
thatmitogen-activated
JNK1 phosphorylation in
AAB groups (P . 0.05). The observed effects of MG132
on AAB-mediated MAPK activation were not due to the
changes in total ERK1/2, JNK1, and p38 protein levels.
Discussion
Although much has been learned about the protective prop-
erties of MG132 on the heart, the role of MG132 treatment
on cardiac hypertrophy in vivo has not been established.
The present study for the first time elucidates the effects of
MG132 on cardiac hypertrophy in vivo and, based on the
Figure 1 Effects of MG132 on mean cardiac myocyte area
Bar ¼ 30 mm. (B) Average value of the cross-sectional area of 100 cardiomyocytes from sections was measured. Data are expressed as the
mean+SEM. **P, 0.01 vs. vehicle-treated sham group and##P, 0.01 vs. vehicle-treated AAB group.
(A) Representative hematoxylin- and eosin-stained LVs from different groups.
Figure 2 Effects of MG132 on ANP and BNP protein expression
(A) Western blot for ANP and BNP protein levels. (B) Densitometric
analysis of relative levels of ANP and BNP protein. Data are expressed as
the mean+SEM. **P, 0.01 vs. vehicle-treated sham group and##P,
0.01 vs. vehicle-treated AAB group.
MG132 attenuates cardiac hypertrophy
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data reported here, supports the notion that MG132 could
be an effective preventive and therapeutic agent against
cardiac hypertrophy. The cardioprotection of MG132 is
caused, at least in part, by direct or indirect interruption of
the activation of MAPK signaling transduction pathways.
There is a dynamic state of continual degradation and
resynthesis of proteins in cardiomyocytes [20,21]. The pro-
teasome plays a key role in heart muscle homeostasis by
affecting the level of specific proteins and adapting the
expression of signaling proteins [4]. Proteasome dysfunc-
tion is observed in patients with heart disease [20].
Although proteasome inhibitor MG132 has been shown to
protect cardiomyocytes against oxidative stress [22,23] and
effectively block artery restenosis [14], its role in the
process of cardiac hypertrophy has also been the subject of
speculation. In the present study, we used a rat model of
AAB to determine the effects of MG132 treatment on
cardiac hypertrophy and function. We showed that long-
termtreatmentwithMG132
pressure-overload-induced cardiac hypertrophy in AAB
rats, which is evidenced by decreased LVW/BW ratio,
septal and posterior wall thickness, myocyte area, as well
as ANP and BNP protein levels. Cardiac function was
markedly improved by MG132 treatment as determined by
increased LV fractional shortening, attenuated LV end-
diastolic pressure, and decreased lung weight/BW ratio. On
the basis of morphological and molecular evidence, we
suggested that chronic treatment with MG132 significantly
reduces cardiac hypertrophy and improves cardiac function
in the AAB rats. In the present study, we also found that
MG132 has no effect on blood pressure. Therefore, we
suggested that the inhibitory effect of MG132 on cardiac
hypertrophy is independent of blood pressure.
To understand the molecular determinants of the hyper-
trophic response, recent investigation has focused on char-
acterizing intracellular signal transduction pathways in the
heart. One of the major systems participating in the
significantly attenuated
transduction of signal from the cell membrane to nuclear
and other intracellular targets are MAPK signaling path-
ways. Involvement of all three classical MAPK pathways
has been implicated in the mechanisms of cardiac hypertro-
phy [24]. Numerous pathological mediators of cardiac
hypertrophy have been shown to activate different MAPK
pathways [25,26]. Considerable evidences pointed to the
key role of ERK1/2 and JNK1 MAPKs in contributing to
the hypertrophic growth [27–29]. Pharmacological inter-
ventions related to these signaling pathways have been
expected to become promising therapeutic options in treat-
ing cardiac hypertrophy [30]. Our results demonstrated that
MG132 treatment markedly decreased the activity of
ERK1/2 and JNK1 in AAB-induced cardiac hypertrophy.
As inhibition of ERK1/2 and JNK1 activation inhibits the
development of cardiomyocyte hypertrophy in vitro and in
vivo, it is well conceivable that MG132-mediated down-
regulation of these central signaling pathways contributes
to suppression of hypertrophic growth of cardiomyocytes.
However, the precise molecular mechanisms by which
MG132 inhibits ERK1/2 and JNK1 activation remain
unclear at this point and need further investigations.
Because proteasome is considered to play a critical role in
intracellular protein degradation, including inhibitor of
hypertrophy, we speculated that MG132 might block
degradation of upstream phosphatase that is dephosphory-
lating ERK1/2, and JNK1 by inhibiting proteasome. In this
study, we found that MG132 did not affect the activity of
p38 in vivo. Unexpectedly, recent evidence demonstrated
that MG132 is capable of activating p38 in cultured cardio-
myocytes [23]. Since the modulation of signaling transduc-
tion pathways in vivo is more complicated than that in
vitro. Therefore, we speculated that the differences in
experimental models might explain the discrepancy regard-
ing the effect of MG132 on activation of p38.
In summary, the present work for the first time demon-
strated that long-term treatment with MG132 attenuates
Figure 3 Effects of MG132 on ERK1/2, JNK1, and p38 activation
(B) Densitometric analysis of phospho-/total ERK1/2, JNK1, and p38. Data are expressed as the mean+SEM. **P, 0.01 vs. vehicle-treated sham
group and##P, 0.01 vs. vehicle-treated AAB group.
(A) Western blot for total and phosphorylated ERK1/2, JNK1, and p38.
MG132 attenuates cardiac hypertrophy
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