Diab Cvd Gene

Am J Physiol Heart Circ Physiol 304: H567–H578, 2013.
First published December 7, 2012; doi:10.1152/ajpheart.00650.2012.

Therapeutic effect of MG-132 on diabetic cardiomyopathy is associated with
its suppression of proteasomal activities: roles of Nrf2 and NF-␬B
Yuehui Wang,1 Weixia Sun,2,3 Bing Du,2 Xiao Miao,1,3 Yang Bai,2,3 Ying Xin,3,4 Yi Tan,3,5
Wenpeng Cui,1,3 Bin Liu,1 Taixing Cui,5,6 Paul N. Epstein,3,7 Yaowen Fu,2 and Lu Cai3,5,7
1

The Second Hospital, Jilin University, Jilin, China; 2The First Hospital, Jilin University, Jilin, China; 3Kosair Children’s Hospital
Research Institute, Department of Pediatrics, University of Louisville, Louisville, Kentucky; 4Norman Bethune Medical College,
Jilin University, Jilin, China; 5Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical College,
Wenzhou, China; 6Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia,
South Carolina; and 7Departments of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky
Submitted 31 August 2012; accepted in final form 4 December 2012

Wang Y, Sun W, Du B, Miao X, Bai Y, Xin Y, Tan Y, Cui W,
Liu B, Cui T, Epstein PN, Fu Y, Cai L. Therapeutic effect of
MG-132 on diabetic cardiomyopathy is associated with its suppression of proteasomal activities: roles of Nrf2 and NF-␬B. Am J Physiol
Heart Circ Physiol 304: H567–H578, 2013. First published December
7, 2012; doi:10.1152/ajpheart.00650.2012.—MG-132, a proteasome
inhibitor, can upregulate nuclear factor (NF) erythroid 2-related factor
2 (Nrf2)-mediated antioxidative function and downregulate NF␬B-mediated inflammation. The present study investigated whether
through the above two mechanisms MG-132 could provide a therapeutic effect on diabetic cardiomyopathy in the OVE26 type 1 diabetic mouse model. OVE26 mice develop hyperglycemia at 2–3 wk
after birth and exhibit albuminuria and cardiac dysfunction at 3 mo of
age. Therefore, 3-mo-old OVE26 diabetic and age-matched control
mice were intraperitoneally treated with MG-132 at 10 ␮g/kg daily for
3 mo. Before and after MG-132 treatment, cardiac function was
measured by echocardiography, and cardiac tissues were then subjected to pathological and biochemical examination. Diabetic mice
showed significant cardiac dysfunction, including increased left ventricular systolic diameter and wall thickness and decreased left ventricular ejection fraction with an increase of the heart weight-to-tibia
length ratio. Diabetic hearts exhibited structural derangement and
remodeling (fibrosis and hypertrophy). In diabetic mice, there was
also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic changes were reversed by MG-132
treatment. MG-132 treatment significantly increased the cardiac expression of Nrf2 and its downstream antioxidant genes with a significant increase of total antioxidant capacity and also significantly
decreased the expression of I␬B and the nuclear accumulation and
DNA-binding activity of NF-␬B in the heart. These results suggest
that MG-132 has a therapeutic effect on diabetic cardiomyopathy in
OVE26 diabetic mice, possibly through the upregulation of Nrf2dependent antioxidative function and downregulation of NF-␬B-mediated inflammation.
diabetic cardiomyopathy; nuclear factor erythroid 2-related factor 2;
nuclear factor-␬B; proteasome inhibitor; MG132; therapeutic effect

(UPS) represents the major
nonlysosomal pathway for the degradation of ubiquitinated
proteins (26). In addition to the removal of misfolded or
damaged proteins, the UPS also plays a crucial role in the
regulation of many cellular processes, such as the cell cycle,
apoptosis, transcriptional control, and responses to stress. Recent studies (26, 36) have indicated that alterations in the UPS
THE UBIQUITIN-PROTEASOME SYSTEM

Address for reprint requests and other correspondence: L. Cai, Dept. of
Pediatrics, Univ. of Louisville, 570 S. Preston St., Baxter I, Suite 321B or
304F, Louisville, KY 40202 (e-mail: [email protected]).
http://www.ajpheart.org

contribute to the pathogenesis and progression of several cardiac diseases, and inhibition of proteasome activity has become
an interesting approach to prevent various diseases, including
cardiac diseases.
Bortezomib (velcadew) was the first proteasome inhibitor
approved by the Federal Drug Administration in 2003 (8).
However, side effects of Bortezomib and the development of
resistance in some patients with tumors prompted the study of
new proteasome inhibitors (8). The cell-permeable MG-132
tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde
proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. Reportedly nontoxic concentrations of MG-132 inhibit Nrf2 proteasomal degradation and
stimulate nuclear factor (NF) erythroid 2-related factor 2
(Nrf2) translocation into the nucleus (5, 28). By blocking the
proteasome, this tripeptide has been shown to induce the
expression of cell-protective proteins, such as heat shock
proteins, in vitro and in vitro. MG-132 was found to play an
important role in cardiovascular protection from various
stresses and pathogeneses (4, 19, 28).
One mechanism responsible for the cardiac protection by
MG-132 is its upregulation of antioxidants via activation of
Nrf2 (28). Nrf2 is a key transcription factor in the regulation of
multiple antioxidants, including NADPH-quinone oxidoreductase (NQO)-1, heme oxygenase (HO)-1, catalase (CAT), SOD,
glutathione peroxidase, and glutamate-cysteine ligase. The
actin-tethered protein kelch-like ECH-associated protein 1 is a
cytosolic repressor that binds to and retains Nrf2 in the cytoplasm, which promotes Nrf2 proteasomal degradation and
results in prevention of Nrf2 transcription (14, 30). Therefore,
inhibition of proteasome activity is one method to maintain
cytoplasm Nrf2 available to nuclear translocation and activation in response to oxidative stress (15).
Another important mechanism underlying MG-132 cardiovascular protection is its inactivation of NF-␬B, a nuclear
transcription factor that regulates proinflammatory cytokine
expression. Activation of NF-␬B has been documented in
myocardial ischemia-reperfusion (13), and specific inhibition
of NF-␬B is cardioprotective (25). NF-␬B usually is inhibited
via I␬B by forming a complex with NF-␬B. However, I␬B is
degraded by proteasome ubiquitination, resulting in the release
of NF-␬B and nuclear translocation to turn on inflammatory
genes. Therefore, proteasomal inhibitors such as MG-132 can
inhibit proteasome activity to provide anti-inflammation function, leading to cardiac protection from ischemic damage.

0363-6135/13 Copyright © 2013 the American Physiological Society

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THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

Diabetic cardiomyopathy is also associated with early cardiac
oxidative stress and inflammation, which initiates the late development of cardiac remodeling and dysfunction (1, 12, 27, 34). A
previous report (16) has shown that diabetes increased cardiac
proteasome function (11, 22), which was associated with diabetic
complications. Therefore, we hypothesized that MG-132 could be
one of the proteasome inhibitors that may be potentially used for
the prevention of diabetic cardiomyopathy via upregulation of
Nrf2 to reduce oxidative stress and downregulation of NF-␬Bmediated inflammation (16). A few studies (3, 4, 17, 19, 28, 31)
have shown cardiac and renal protection by proteasome inhibitor
from other pathogeneses. Dreger et al. (9) demonstrated the
protective effect of low-dose MG-132 on H2O2-induced oxidative
stress and damage in cardiomyocytes and also showed increased
sensitivity in myocytes from Nrf2 knockout mice, suggesting the
important role of Nrf2 in cardiac protection. Using MG-132,
several studies (16, 32) have also shown the preventive effect of
inhibition of proteasome activity on diabetic vascular injury.
Results from a pilot study (18) indicated that a proteasomal
inhibitor that upregulates Nrf2 activity and inhibits NF-␬Bmediated inflammation provided a preventive effect on diabetesinduced renal dysfunction.
From a clinical setting, there is an urgent need for an efficient
approach that can provide a therapeutic effect rather than only
prevention, since it is not easily accepted that diabetic patients will
receive preventive medical treatement once diagnosed as diabetic.
So far, however, there is no report regarding the therapeutic effect
of MG-132 on diabetic complications; in the present study, therefore, we tried to address the question of whether MG-132 can
provide a therapeutic effect on diabetic cardiomyopathy by testing
if MG-132 could reserve or slow the progression of established
cardiac dysfunction in the OVE26 mouse model of severe type 1
diabetes. In addition, mechanistic experiments were focused on
the upregulation of Nrf2-mediated antioxidants and downregulation of NF-␬B-mediated inflammation.
MATERIALS AND METHODS

Four mice from both control and DM groups were killed at 3 mo of
age, and other mice (n ⫽ 6) were treated with MG-132 for 3 mo and
killed at 6 mo of age. After heart weight and tibia length had been
measured, whole heart tissue was harvested for protein and mRNA
analysis. Histopathological observations were predominantly based on
the left ventricle (LV).
Echocardiography
To assess cardiac function, echocardiography was performed in
mice using a Visual Sonics Vevo 770 high-resolution imaging system,
as previously described (29, 39). Under sedation with Avertin (2,2,2tribromoethanol, 240 mg/kg ip), mice were placed in the supine
position on a heating pad to maintain body temperature at 36 –37°C.
Heart rates were kept 400 –550 beats/min. Two-dimensional and
M-mode echocardiography were used to assess wall motion, chamber
dimensions, and cardiac function. Directly measured indexes included
LV internal dimensions (LVID) at diastole and systole, LV posterior
wall thickness (LVPW) at diastole and systole, and interventricular
septal thickness (IVS) at diastole and systole. LV fractional shortening
(FS) was determined as follows: FS ⫽ [(LVID at diastole ⫺ LVID at
systole)/LVID at diastole] ⫻ 100. LV ejection fraction (EF) was
determined as follows: EF ⫽ [(LV volume at diastole ⫺ LV volume
at systole)/LV volume at diastole] ⫻ 100.
Western Blot Analysis
Western blot analysis was performed as described in our previous
studies (2, 39). The primary antibodies used included 3-nitrotyrosine
(3-NT; 1:1,000 dilution), 4-hydroxy-2-nonenal (4-HNE; 1:1,000 dilution), TNF-␣ (1:500 dilution), ICAM-1 (1:500 dilution), plasminogen activator inhibitor (PAI)-1 (1:1,000 dilution), transforming
growth factor (TGF)-␤1 (1:500 dilution), fibronectin (1:500 dilution),
Nrf2 (1:500 dilution), NQO-1 (1:500 dilution), HO-1 (1:500 dilution),
CAT (1:5,000 dilution), atrial natriuretic peptide (ANP; 1:1,000 dilution), NF-␬B (1:1,000 dilution), I␬B-␣ (1:1,000 dilution), ␣-tubulin
(1:2,000 dilution), and ␤-actin (1:2,000 dilution), all of which were
purchased from Santa Cruz Biotechnology except for TNF-␣ (Abcam), 3-NT (Millipore), NF-␬B, I␬B-␣, and ␣-tubulin (Cell Signaling), and 4-HNE (Alpha Diagnostic). All antibodies were polyclonal
antibodies except for TNF-␣ and PAI-1 antibodies, which were monoclonal.

Animals
The transgenic type 1 diabetic OVE26 mouse model on a FVB background has been characterized in a previous study (38). All mice were
housed at the University of Louisville Research Resources Center at 22°C
with a 12:12-h light-dark cycle and provided with free access to standard
rodent chow and tap water. All animal procedures were approved by the
Institutional Animal Care and Use Committee, which is certified by the
American Association for Accreditation of Laboratory Animal Care.
OVE26 mice normally develop severe hyperglycemia before 3 wk
of age and develop albuminuria by 3 mo of the age, particularly in
female OVE26 mice (38). Sixteen 3-mo-old female OVE26 mice were
randomly divided into two groups: a diabetic group (DM group; n ⫽
10) and a MG-132-treated OVE26 group (DM/MG-132 group; n ⫽ 6).
Sixteen age- and sex-matched nondiabetic FVB mice were randomly
divided into two groups: a control group (n ⫽ 10) and a MG-132treated group (MG-132 group; n ⫽ 6). MG-132 (Sigma-Aldich, St.
Louis, MO) was dissolved in DMSO at a concentration of 0.0025
␮g/ml and diluted with saline for injection. For MG-132 and DM/
MG-132 mice, MG-132 was given intraperitoneally at a dose of 10
␮g/kg body wt daily for 3 mo, based on a recent study (18), starting
at 3 mo old in OVE26 mice when these mice already displayed
significant renal dysfunction. For control and DM mice, equal
amounts of physiological saline solution containing 0.0025 ␮g
DMSO/ml were given. MG-132 treatment for 3 mo in OVE26 diabetic
mice did not affect their blood glucose levels (7).

Isolation of RNA and Real-Time RT-PCR
Isolation of RNA and real-time RT-PCR were performed as described
in our previous study (39) for Nrf2 (primer: Mm00477784_m1), ANP
(primer: Mm01255748_g1), ␤-myosin heavy chain (␤-MHC; primer:
Mm00600555_m1), HO-1 (primer: Mm00516005_m1), NQO-1 (primer:
Mm253561_m1), CAT (primer: Mm00437229_m1), and the housekeeping gene ␤-actin (primer: Mm00607939_s1). All primers were purchased
from Applied Biosystems (Foster City, CA). Total RNA was extracted
from whole heart tissues using TRIzol reagent (RNA STAT 60 Tel-Test,
Ambion, Austin, TX). RNA concentration and purity were quantified
using a Nanodrop ND-1000 spectrophotometer (Biolab, Ontario, CA).
First-strand cDNA was synthesized from total RNA according to the
RNA PCR kit (Promega, Madison, WI) following the manufacturer’s
protocol.
Cardiac Histopathological Examination and
Immunohistochemical Staining
Whole cardiac tissue was fixed overnight in 10% phosphate buffered formalin, dehydrated in a graded alcohol series, cleared with
xylene, embedded in paraffin, and then sectioned at 5 ␮m thickness
for pathological and immunohistochemical staining. Paraffin sections
were dewaxed followed by an incubation in 1⫻ target retrieval
solution (Dako, Carpinteria, CA) for 15 min at 98°C for antigen

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

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THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

2 h at room temperature. For the development of color, sections were
treated with the peroxidase substrate 3,3-diaminobenzidine in the
developing system (Vector Laboratories, Burlingame, CA).

retrieval. Sections were then treated with 3% H2O2 for 15 min at room
temperature followed by blocking with 5% BSA for 30 min.
Cardiac sections were stained with hematoxylin and eosin and
Sirius red, respectively, as previously described (2, 39). For immunohistochemical staining, sections were incubated with the primary
antibodies of TGF-␤1 (1:100 dilution, Santa Cruz Biotechnology) and
PAI-1 (1:100 dilution, BD Biosciences) overnight at 4°C. After being
washed with PBS, sections were incubated with horseradish peroxidase-conjugated secondary antibody (1:300 – 400 dilutions in PBS) for

LVID;s

LVID;d

3.6

3

*#

2.7
1.8

2

90

*

*

*

#

#

60

*

*

40

60

1

#

*

FS%

4.5

Ctrl
MG132
DM
DM/MG132

The 20S proteasome, the catalytic core of the 26S proteasome
complex, is responsible for the breakdown of short-lived regulatory
proteins, including Nrf2 and NF-␬B (6, 9). Since MG-132 mainly

EF%

A

Proteasome Activity

20

30

0.9

3m

1.0

*#

0.5

0.0

0

6m

*

0.9

LVPW;d

IVS;d

*

3m

3m

0

6m

*#

120

0.6

0.3

6m

3m

#

1.2

*
IVS;s

LVPW;s

0.8

0.4

1.2

*

0.6

0.0

0

*

6m

3m

*#

6m

*#

50

0.0
3m

3m

100

#

*

*

6m

40

6m

1.8

3m

80

0.0
3m

LV Mass corrected(mg)

B

0

6m

LV Mass(mg)

0.0

6m

0

3m

6m

Fig. 1. Therapeutic effects of MG-132 on diabetes-induced cardiac dysfunction. Three-mo-old female OVE26 mice and FVB control mice were given MG-132
(10 ␮g/kg) or an equal volume of physiological saline solution daily for 3 mo. Mice were divided into the following groups: control FVB mice (Ctrl group),
control mice with MG-132 treatment (MG-132 group), diabetic OVE26 mice (DM group), and diabetic mice with MG-132 treatment (DM/MG-132 group).
Cardiac functional (A) and structural (B) changes were evaluated by echocardiography. LVID;d and LVID;s, left ventricular (LV) internal dimension at diastole
and at systole, respectively; EF%, ejection fraction (in %); FS%, fractional shortening (in %); IVS;d and IVS;s, interventricular septal thickness at diastole and
at systole, respectively; LVPW;d and LVPW;s, LV posterior wall thickness at diastole and at systole, respectively; 3m and 6m, 3 mo and 6 mo, respectively.
Data are presented as means ⫾ SD. *P ⬍ 0.05 vs. the Ctrl group; #P ⬍ 0.05 vs. the DM group.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

inhibits proteasome chymotrypsin-like activity (24), we determined
20S proteasome activity by quantifying the hydrolysis of the SLLVYAMC-a fluorogenic substrate for chymotrypsin-like activity. 20S
proteasome activity was measured with the 20S proteasome activity
assay kit (Millipore).

(Invitrogen, Camarillo, CA), as previously described (21). The low
detection limits were 3 and 9 pg/ml for serum TNF-␣ and MCP-1,
respectively. Nuclear p65 DNA-binding activity was determined by
an ELISA-based NF-␬B activity assay using cardiac tissue from the
whole heart (Cayman Chemical, Ann Arbor, MI), according to manufacturer instructions.

Isolation of Nuclei

Total Antioxidant Capacity Assay

Cardiac nuclei were isolated using the nuclei isolation kit (SigmaAldich). Whole cardiac tissue (60 mg) from each mouse was homogenized for 45 s with 300 ␮l cold lysis buffer containing 1 ␮l DTT and
0.1% Triton X-100. After that, 600 ␮l of cold 1.8 mol/l cushion
solution [sucrose cushion solution-sucrose cushion buffer-DTT (900:
100:1)] were added to the lysis solution. The mixture was transferred
to a new tube preloaded with 300 ␮l of 1.8 mol/l sucrose cushion
solution followed by centrifugation at 13,000 rpm for 45 min. The
supernatant contained the cytosolic components, and nuclei were
visible as a thin pellet at the bottom of the tube.

The cardiac total antioxidant capacity (TAC) assay was performed
using a commercially available assay kit (Cell Biolabs, San Diego,
CA) according to the manufacturer’s instructions. Briefly, whole heart
tissues were homogenized in cold PBS and then centrifuged at 10,000
g at 4°C for 10 min to collect the supernatant for the TAC assay.
Values were calculated using optical density at 490 nm and expressed
as micromoles per gram of protein for TAC.
Statistical Analysis
Data were expressed as means ⫾ SD. For statistical analysis,
one-way ANOVA was used as appropriate. An overall F-test was
performed to test the significance of the ANOVA models. The
significance of the interactions and main effects were taken into
consideration, and multiple comparisons were then performed with a

Measurements of Serum TNF-␣ and Monocyte Chemoattractant
Protein-1 and Cardiac Nuclear NF-␬B DNA-Binding Activity
Serum levels of TNF-␣ and monocyte chemoattractant protein
(MCP)-1 were performed using mouse TNF-␣ and MCP-1 ELISA kits

A

B
Ctrl
MG132
DM
DM/MG132

*&#

*

MG132

Ctrl

5

DM
0
6m

C

D
8

*

6
4

#

2
0

l
Ctr G132
M

DM G132
/M
DM

E

β-MHC/β-Actin mRNA
(fold to control)

3m

ANP/β-Actin mRNA
(fold to control)

Fig. 2. Therapeutic effect of MG-132 on diabetes-induced cardiac hypertrophy. A: at both 3
and 6 mo of diabetes, the ratio of heart weight to
tibia length was measured and calculated after
mice had been euthanized. B: cardiac structural
derangement was examined by morphological
examination with hematoxylin and eosin staining. The blue arrow indicates myocardial hypertrophy; the black arrow indicates myocardial
cells degeneration; the brown arrow indicates the
proliferation of interstitial collagen fibers. Magnification: ⫻400. Bar ⫽ 50 ␮m. C–E: mRNA
expression of the cardiac hypertrophic markers
atrial natriuretic peptide (ANP; C) and ␤-myosin
heavy chain (␤-MHC; D) as well as the protein
expression of ANP (E) were measured by realtime quantitative PCR and Western blot analysis. Data are presented as means ⫾ SD. *P ⬍
0.05 vs. the Ctrl group; &P ⬍ 0.05 vs. the 3-m
DM group; #P ⬍ 0.05 vs. the DM group.

Heart/Tibia (mg/mm)

10

Saline

4

*

3

#

2
1
0

l
Ctr G132
M

2.4

trl

C

132
MG DM

32
G1
M
/
DM

ANP
β-Actin

ANP/β-Actin

H570

DM G132
/M
DM

*

1.6

#

0.8
0.0

l
Ctr G132
M

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

DM G132
/M
DM

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

Bonferroni test. P values of ⬍0.05 were considered as statistically
significant.
RESULTS

Therapeutic Effect of MG-132 on Diabetes-Induced Cardiac
Dysfunction and Hypertrophy
Transgenic OVE26 type 1 DM mice at 3 mo showed increased
LVID at systole and decreased EF and FS percentages, suggesting the exist of cardiac dilation and decreased systolic function
(Fig. 1A). Hypertrophic measurements of IVS, LVPW, and LV
mass were significantly increased in these mice, with a progressive manner from 3 to 6 mo (P ⬍ 0.05; Fig. 1B). However, when
some of the DM mice at 3 mo of age were treated with low-dose
MG-132 for 3 mo (DM/MG-132 group), diabetes-induced cardiac
dysfunction was almost completely reversed (P ⬍ 0.05; Fig. 1).
The therapeutic effects of MG-132 on diabetes-induced
cardiac hypertrophy were also reflected by the ratio of heart
weight to tibia length (Fig. 2A), which was significantly in-

A

Saline

H571

creased in an age-dependent manner in the DM group from 3
to 6 mo but not in the DM/MG-132 group.
To further confirm cardiac hypertrophy, cardiac morphology
and molecular hypertrophy makers were also examined. Hematoxylin and eosin staining showed that, compared with the
control and MG-132 groups, the main pathological changes in
the DM group included 1) myocardial hypertrophy, 2) a few
cardiac cells that showed features of degeneration and 3) proliferation of interstitial collagen fibers. Those pathological
changes were alleviated or not observed in DM/MG-132 mice
(Fig. 2B). Real-time PCR analysis showed that DM induced a
significant increase of the cardiac mRNA expression of ANP
(Fig. 2C) and ␤-MHC (Fig. 2D), an effect that was completely
abolished by MG-132 treatment in the DM/MG-132 group.
Consistent with the ANP mRNA findings, Western blot analysis showed a significant increase of cardiac ANP protein
expression at 6 mo of age in DM mice but not in DM/MG-132
mice (Fig. 2E). These results suggest that 3-mo treatment with
low-dose MG-132 can significantly or even completely pre-

B

MG132

*

Collgen content
(fold to control)

3
Ctrl

DM

2

#

1

0

C

132
132M
/MG
l
r
G
t
M
C
D
M
D
TGF-β1
Fibronectin
PAI-1
β-Actin

D
Ctrl

MG132

Relative protein expression

l
Ctr G132 DMG132
M
/M
DM
2.4
1.8

Ctrl
MG132
DM
DM/MG132

*
#

*#

1.2

*#

0.6
0.0

in
ta1
ect
-be
n
F
o
r
TG
Fib
DM

I-1
PA

Fig. 3. Therapeutic effect of MG-132 on diabetes-induced cardiac fibrosis. A: cardiac tissue
was stained with Sirius red for collagen. Arrows
indicate the proliferation of interstitial collagen
fibers. Original magnification: ⫻200. Bar ⫽ 50
␮m. B: semiquantitative analysis was done by a
computer imaging system. C: cardiac expression of transforming growth factor (TGF)-␤1,
fibronectin, and plasminogen activator inhibitor
(PAI)-1 was examined by Western blot analysis.
D: cardiac expression of TGF-␤1 and PAI-1 was
also tested by immunohistochemical staining.
Arrows indicate positive staining. Data are presented as means ⫾ SD. *P ⬍ 0.05 vs. the Ctrl
group; #P ⬍ 0.05 vs. the DM group.

DM/MG132

TGF-β1

PAI-1

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

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THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

Cardiac tissue was examined with Sirius red staining for
collagen (Fig. 3A) followed by semiquantitative analysis with
a computer imaging system (Fig. 3B), which showed a significant increase in collagen accumulation in the DM group but
not in the DM/MG-132 group. Western blot analyses of TGF␤1, fibronectin, and PAI-1 as important profibrotic mediators
confirmed the Sirius red staining results: there was a significant
increase in cardiac fibrosis in the DM group but not in the
DM/MG-132 group (Fig. 3C). Furthermore, the increased expression of TGF-␤1 and PAI-1 was supported by immunohistochemical staining results (Fig. 3D).

and in the heart by Western blot assay (Fig. 4, C and D). Serum
TNF-␣ and MCP-1 levels were progressively elevated from 3 to
6 mo in the DM group and were completely prevented by
MG-132 treatment in the DM/MG-132 group. Increased cardiac
TNF-␣ and MCP-1 contents were also observed only in the DM
group and not in the DM/MG-132 group.
Considering that inflammation in target organs often causes
oxidative stress and that oxidative stress is also able to induce
inflammation, we examined oxidative stress by measuring
3-NT accumulation as an index of protein nitration (Fig. 5A)
and 4-HNE as an index of lipid peroxidation (a measure of
oxidative damage; Fig. 5B). Both 3-NT and 4-HNE contents
were significantly increased in the hearts of DM mice but not
DM/MG-132 mice. TAC in heart tissue (Fig. 5C) was slightly
decreased (P ⬍ 0.05) in the DM group at both 3 and 6 mo but
was not changed in the DM/MG-132 group at 6 mo.

Therapeutic Effect of MG-132 on Diabetes-Induced Cardiac
Inflammation and Oxidative Stress

Possible Mechanisms for the Therapeutic Effect of MG-132
on Diabetic Cardiomyopathy

Since PAI-1 acts as both a profibrotic and inflammatory mediator, its increase in the hearts of DM mice suggests possible
cardiac inflammation. Thus, we next examined the protein expression of TNF-␣ (Fig. 4A) and MCP-1(Fig. 4B) in serum by ELISA

Diabetes increases cardiac proteasomal activity, an effect
prevented by MG-132. Since tetrahydrobiopterin deficiency
has been reported to uncouple the enzymatic activity of endothelial nitric oxide synthase in DM hearts triggered by DM-

Therapeutic Effect of MG-132 on Diabetes-Induced
Cardiac Fibrosis

Serum TNF-α (pg/ml)

A

B

Ctrl
MG132
DM
DM/MG132

120

*

*

80

150

#

40

0
6m

l

132
/MG
M
D

C

*

100

#

50

0
6m

D
Ctr

132 M
MG
D

l
Ctr

TNF-α

ICAM-1

β-Actin

β-Actin

1.8

TNF-α/β-Actin

*

3m

*

1.2

#

0.6
0.0

l
Ctr G132
M

1 32 M
MG
D

2.0

DM G132
/M
DM

ICAM-1/β-Actin

Fig. 4. Therapeutic effect of MG-132 on diabetesinduced inflammatory cytokines. A and B: TNF-␣
(A) and MCP-1 (B) levels in serum were determined by ELISA. C and D: cardiac tissue was
subjected to Western blots for TNF-␣ (C) and
ICAM-1 (D). Data are presented as means ⫾ SD.
*P ⬍ 0.05 vs. the Ctrl group; #P ⬍ 0.05 vs. the
DM group.

3m

Serum MCP-1 (pg/ml)

vent the progression of the cardiac hypertrophy induced by diabetes.

32
G1
M
/
DM

*

1.5

#

1.0
0.5
0.0

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

l
Ctr G132
M

DM G132
/M
DM

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

l
Ctr

A

132
/MG
M
D

13 2
MG DM

l
Ctr

B

3-NT

4HNE

β-Actin

β-Actin

*

#

1.2

0.6

0.0

l
Ctr G132
M

TAC μmol/g protein

C
8

132
/MG
M
D

*

1.8

4-HNE/β-Actin

3-NT/β-Actin

1.8

13 2
MG DM

1.2

#

0.6

0.0

DM G132
/M
DM

H573

l
Ctr G132
M

DM G132
/M
DM

Fig. 5. Therapeutic effect of MG-132 on diabetesinduced cardiac oxidative stress. A and B: cardiac
tissue was subjected to Western blots for 3-nitrotyrosine (3-NT; A) and 4-hydroxy-2-nonenal (4-HNE;
B). C: total antioxidant capacity (TAC) in cardiac
tissue was detected and expressed as micromoles
per gram of protein. Data are presented as means ⫾
SD. *P ⬍ 0.05 vs. the Ctrl group; #P ⬍ 0.05 vs. the
DM group.

Ctrl
MG132
DM
DM/MG132

*

6

*

4

#

*

2
0
3m

6m

increased proteasome-dependent mechanisms (35), we examined whether cardiac proteasome activity was increased in
diabetic mice. The results shown in Fig. 6 demonstrate that
MG-132 decreased, and diabetes increased, cardiac 20S pro-

20S proteasome activity
(fold to control)

2.4

*

1.6

#

*

0.8

0.0

Ctr

l
MG

132

DM
D

G
M/M

132

Fig. 6. Effect of MG-132 on cardiac activity of the proteasome. Cardiac proteasome activity was measured using a 20S proteasome activity kit. Data are
presented as means ⫾ SD. *P ⬍ 0.05 vs. the Ctrl group; #P ⬍ 0.05 vs. the DM
group.

teasomal activity. Compared with the DM group, cardiac 20S
proteasomal activity was significantly reduced, even to control
levels, in the DM/MG-132 group (45% DM, P ⬍ 0.05 vs. the
DM group).
MG-132 inhibits cardiac proteasomal activity, resulting in an
upregulation of cardiac Nrf2 and its downstream antioxidants.
Proteasomal degradation of Nrf2 has been delineated as the
major of mechanism responsible for Nrf2’s negative regulation
(14, 30). Our finding that MG-132 completely inhibited diabetes-upregulated proteasomal activity implies a possibility for
MG-132 treatment to decrease the degradation of Nrf2 protein.
Therefore, expression of Nrf2 at mRNA and protein levels was
examined with real-time PCR and Western blot assays, respectively. We found that MG-132 had no impact on, but diabetes
had a significant increase in, the mRNA expression of Nrf2
(Fig. 7A). The DM/MG-132 group had a similar expression of
Nrf2 mRNA as in the DM group (Fig. 7A). The results shown
in Fig. 7B demonstrate that both MG-132 and DM increased
the cardiac expression of Nrf2 protein, so that Nrf2 protein
expression was synergistically increased in the DM/MG-132
group.
Furthermore, the upregulated Nrf2 transcription activity was
reflected by the increase of several downstream antioxidant
genes (Fig. 7, C and D). MG-132 treatment in control mice
significantly increased the expression of NQO-1, HO-1, and

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

H574

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

B
4

1
0

2.7

DM G132
/M
DM

0.9

l
Ctr G132
M

6

4

#

DM G132
/M
DM

#
#

*

**

NQO1

HO-1

*

* *

0.0

Ctrl
MG132
DM
DM/MG132

2

#

1.8

C
Relative mRNA levels

Fig. 7. Effect of MG-132 on cardiac expression and
function of nuclear factor (NF) erythroid 2-related
factor 2 (Nrf2). A and B: cardiac expression of Nrf2
mRNA (A) and protein (B) levels was examined
with real-time PCR and Western blot assays, respectively. C and D: Nrf2 function was measured
by determining the expression of Nrf2 downstream
genes [NADPH-quinone oxidoreductase (NQO)-1,
heme oxygenase (HO)-1, and catalase (CAT)] at
mRNA (C) and protein (D) levels. Data are presented as means ⫾ SD. *P ⬍ 0.05 vs. the Ctrl
group; #P ⬍ 0.05 vs. the DM group.

32
G1
M
/
DM

β-Actin

*

2

l
Ctr G132
M

132 M
MG
D

Nrf2

*

3

l
Ctr

Nrf2/β-Actin

Nrf2/β-Actin mRNA
(fold to control)

A

*

*

0

#
1 32
MG DM

32
G1
M
/
DM

NQO1
HO-1
CAT
β-Actin

CAT at the mRNA and protein levels compared with the
control group. Diabetes also increased the expression of these
antioxidants at the mRNA and protein levels. Expression of
these Nrf2 downstream antioxidant genes in the hearts of
DM/MG-132 mice was synergistically increased at both the
mRNA and protein levels compared with both MG-132 or DM
mice.
MG-132 inhibits cardiac proteasomal activity, resulting in a
reduction of cardiac inflammation by preventing NF-␬B nuclear translocation. Since NF-␬B is bound by I␬B-␣ in the
cytoplasm, inhibiting NF-␬B nuclear translocation to transcriptionally upregulate inflammatory cytokines, a reduction of
I␬B-␣ will indirectly upregulate NF-␬B-mediated inflammation. Western blot analysis showed that the expression of
I␬B-␣ was significantly decreased in hearts of the DM group
but not the DM/MG-132 group (Fig. 8A). This suggests that
MG-132 inhibits the proteasomal degradation of I␬B-␣. To

Relative protein levels

D
l
Ctr

CAT

4

#

3
2

#

*

**

**

*

NQO1

HO-1

CAT

1
0

define whether preservation of a normal level of I␬B-␣ is able
to prevent NF-␬B nuclear translocation, cardiac proteins were
separated into nuclear and cytoplasmic parts, which showed
that the nuclear accumulation of NF-␬B was significantly
increased in the DM group but not in the DM/MG-132 group
(Fig. 8B). Furthermore, the increased cardiac nuclear NF-␬B
DNA-binding activity only in the hearts of DM mice at both 3
and 6 mo was further confirmed by an ELISA-based NF-kB
activity assay (Fig. 8C).
DISCUSSION

The present study is the first to report the therapeutic effects
of chronic treatment with low-dose MG-132 on diabetesinduced cardiomyopathy using the OVE26 diabetic mouse
model. We demonstrated that the therapeutic effect of MG-132
on diabetic cardiomyopathy is associated with its suppression

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

l

Ctr

1.6

132
/MG
M
D

13 2
MG DM

IκB-α/β-Actin

A

H575

IκB-α
β-Actin

1.2

#

0.8

*

0.4
0.0

l
Ctr G132
M

B
l
Ctr

13 2
MG DM

32
G1
M
/
DM

l
Ctr

NF-κB(c)

α-Tubulin

β-Actin

#
1.4
0.7

132
MG DM

132
/MG
M
D

* *

2.1
1.4
0.7

Fig. 8. Effect of MG-132 on the cardiac expression of NF-␬B and I␬B-␣ as well as NF-␬B
activity. A and B: I␬B-␣ protein expression (A)
and NF-␬B protein expression in the nuclear
and cytosolic portion (B) were examined with a
Western blot assay. C: nuclear p65 DNAbinding activity was determined by an ELISAbased NF-kB activity assay. Data are presented
as means ⫾ SD. *P ⬍ 0.05 vs. the Ctrl group;
#P ⬍ 0.05 vs. the DM group.

0.0

0.0

l
Ctr G132
M

DM G132
/M
DM

3

(fold to control)

l
Ctr G132
M

C

NF-κB(c)/β-Actin

*

2.1

NF-κB DNA binding activity

NF-κB(n)/α-Tubulin

NF-κB(n)

DM G132
/M
DM

Ctrl
MG132
DM
DM/MG132

*

2

DM G132
/M
DM

*
#

1

0
3m

6m

of diabetic upregulation of proteasome activity, which may
increase the proteasomal degradation of I␬B-␣ and Nrf2.
Increased degradation of I␬B-␣ would release its binding and
restricting NF-␬B in the cytoplasm, leading to an increase of
NF-␬B nuclear translocation to generate inflammatory cytokines, as shown in Fig. 9. These cytokines then initiate an
overgeneration of ROS/reactive nitrogen species, cardiac oxidative stress and damage, and remodeling and dysfunction. In
addition, increased proteasomal degradation of Nrf2 will reduce the transcription of Nrf2 to generate its downstream
antioxidants, which will exacerbate cardiac pathogenic alterations, leading to an acceleration of cardiomyopathy development, as shown in Fig. 9.
Several in vivo and in vitro studies have provided evidence
for the increase in proteasomes under diabetic conditions. For
example, exposure of human umbilical vein endothelial cells to

a high level of glucose significantly increased 26S proteasome
activity (35). Proteasomal activity was also found to be increased in the hearts of type 1 diabetic mice (11, 22) and in the
gastrocnemius muscles of spontaneous type 2 diabetic (db/db)
mice (33). Measurements of proteasomal activity in the kidneys of diabetic rats showed an increase of 26S proteasomal
activity in streptozotocin-induced diabetic rats (18).
An in vivo pilot study (18) has shown the preventive effect
of MG-132 on diabetes-induced renal damage. In that study,
shortly after the induction of diabetes, rats were treated with
MG-132 at 10 ␮g/kg daily for 3 mo. This regimen produced
renal prevention, as indicated by reductions in proteinuria,
basement membrane thickening, and glomerular mesangial
expansion. MG-132 also reduced kidney markers of oxidative
stress and increased protein levels of Nrf2 and several antioxidant enzymes. These experiments demonstrated a potential for

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

H576

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

Diabetes

Proteasome activity

MG132

NF-κB

IκBα

KEAP1

Nrf2

Cytoplasm
Fig. 9. Illustration of possible mechanisms by which
MG-132 provides therapeutic effects on diabetic cardiomyopathy. Diabetes increases cardiac proteasomal activity, leading to the degradation of Nrf2 and I␬B-␣ in the
cytoplasm. The increased degradation of Nrf2 results in a
decrease of Nrf2 nuclear translocation and transcriptional
upregulation of its downstream antioxidants. The increased degradation of I␬B-␣ results in a release of
NF-␬B from I␬B-␣ inhibition and an increase of NF-␬B
nuclear translocation to transcriptional upregulation of
inflammatory cytokines. All these cause cardiac oxidative damage, remodeling, and dysfunction (cardiomyopathy). MG-132 could prevent Nrf2 degradation and
NF-␬B nuclear translocation by inhibiting the proteasome. KEAP1, kelch-like ECH-associated protein 1.

Nucleus
NF-κB
Nrf2
Inflammation
cytokines

Antioxidant
genes

Oxidative damage

Cardiac
Remodeling

Cardiomyopathy
(Cardiac Dysfunction)

MG-132 to prevent the development of diabetic complications.
However, whether Nrf2 activators have therapeutic effects on
the heart and/or kidney of diabetic subjects remains unknown.
Here, we report, for the first time, the therapeutic effects of
MG-132 on diabetic cardiomyopathy in the transgenic type 1
diabetic OVE26 mouse model. When these diabetic mice at 3
mo old began to exhibit albuminuria as an index of renal
dysfunction (38) and cardiac dysfunction (Fig. 1), some of
them were treated with MG-132 at a very low dose for 3 mo,
which offered a significant therapeutic outcome, as indicated
by the completely prevention of diabetes-induced cardiac inflammation and oxidative stress and damage, leading to a
complete reverse of cardiac remodeling (fibrosis and hypertrophy) and dysfunction. Diabetic mice without treatment with
MG-132 showed the progressive development of cardiac structural remodeling and functional abnormalities.

In terms of the mechanisms underlying the therapeutic
effects of MG-132 on diabetic cardiomyopathy, we assumed
that they are associated with the significant upregulation of
Nrf2 expression and transcriptional increases of its downstream antioxidants and the complete prevention of diabetesreduced I␬B content and diabetes-increased NF-␬B nuclear
accumulation, as shown in Fig. 9.
Reportedly nontoxic concentrations of MG-132 inhibited the
proteasomal degradation of Nrf2 to stimulate Nrf2 translocation into the nucleus (5, 28). An in vitro study (9) has shown
that treatment with 0.5 ␮M MG-132 for 48 h protected neonatal rat cardiac myocytes against H2O2-mediated oxidative
stress. This was correlated with reduced levels of intracellular
ROS and significant upregulation of superoxide, HO-1, and
CAT expression. This demonstrated that nontoxic concentrations of MG-132 could upregulate Nrf2-mediated antioxidant

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00650.2012 • www.ajpheart.org

THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

enzymes to confer cardiomyocyte protection (9). We (10) have
demonstrated the high susceptibility of cardiomyocytes from
Nrf2-null mice to high-glucose-induced ROS generation and
damage compared with those from Nrf2 wild-type mice.
Therefore, we assumed that the preventive effect of the proteasomal inhibitor MG-132 was due to elevated Nrf2 protein
content, which increased the expression of multiple downstream antioxidant enzymes.
RT-PCR analysis revealed that diabetes, but not MG-132,
significantly increased Nrf2 mRNA expression (Fig. 7A), suggesting the existence of a compensatory response of the heart
to diabetes-induced oxidative stress by upregulation of Nrf2
mRNA expression. However, diabetes also increased 20S proteasome activity, which should have reduce Nrf2 protein levels
and resulted in less nuclear translocation; therefore, the final
outcome of Nrf2 protein expression in the diabetic heart
remained at a relatively high level compared with the control
group. In contrast to the DM group, DM/MG-132 mice showed
increased Nrf2 mRNA expression, which should be attributed
to an effect of diabetes (Fig. 7A), and also synergistically
increased Nrf2 protein levels, which should be attributed to
both an effect of diabetes on mRNA expression as well as the
inhibitory effect of MG-132 on proteasomal degradation of the
Nrf2 protein level. Expression profiles of Nrf2 downstream
antioxidants at the mRNA and protein levels (Fig. 7, C and D)
were similar to the profile of Nrf2 protein expression among
the groups (Fig. 7B). These upregulated Nrf2-mediated antioxidants seem to be responsible for the therapeutic effects on
the heart, based on a previous study (37) reporting that upregulated Nrf2 expression in the heart or kidney provided significant prevention of diabetes-induced damage.
However, due to the fact that the expression of Nrf2 and its
downstream antioxidants was not significantly decreased in the
hearts of DM mice compared with control mice, we assumed
that the increased expression of Nrf2 and its downstream
antioxidants may be not the sole mechanism underlying the
therapeutic effects of MG-132 on diabetic cardiomyopathy.
The role of an excessive inflammatory response in the
initiation of diabetic cardiomyopathy has been discussed recently (27, 34). As a key transcription factor to control inflammation, NF-␬B activation was found to play a pivotal role in
the development of cardiomyopathy (20). Recent studies (19,
23) have demonstrated the protection of MG-132 in the other
organs via inhibition of NF-␬B. Here, we demonstrated the
activation of NF-␬B in the hearts of OVE26 diabetic mice,
which was significantly inhibited by treatment with MG-132 in
the DM/MG-132 group. This suggests that the effective inhibition of proteasome activity by MG-132 may be attributed to
its effective inhibition of cardiac inflammation, as we observed
in this study. As shown in Fig. 9, NF-␬B triggers the transcription of many inflammation cytokines. These inflammatory
cytokines stimulate the generation of ROS and/or reactive
nitrogen species, which induce cardiac oxidative stress and
damage and remodeling, leading to the development of diabetic cardiomyopathy. Therefore, inhibition of cardiac inflammation activation should provide cardiac protection from diabetes. We demonstrated that diabetes significantly suppressed
the expression of I␬B-␣ and increased the nuclear accumulation of NF-␬B and DNA-binding capacity. All these effects
were almost completely abolished by MG-132 treatment,

H577

which was accompanied with a complete reverse of diabetesinduced cardiac inflammation and oxidative damage.
A potential limitation of the present study may be the route
(intraperitoneal injection) by which MG-132 was given, which
may not directly applicable to clinics. In the present study, we
used intraperitoneal injection because we wanted to ensure the
precise dose given for the low dose of MG-132. Although we
cannot directly expect that what we do in this study will be directly extrapolated to clinics, the eventual oral administration of
the MG-132 for diabetic patients with the appearance of albuminuria will be easily established if we confirm the therapeutic effect,
safety, and underlying mechanisms. The latter two will be further
investigated in future studies.
In summary, the present study demonstrated that the proteasomal inhibitor MG-132 at a low dose (10 ␮g/kg) can provide
a therapeutic effect on diabetic cardiomyopathy. Here, we used
the transgenic type 1 diabetic OVE26 mouse model. When
these diabetic mice began to exhibit both albuminuria as an
index of renal dysfunction and cardiac dysfunction by echocardiography, some of them were treated with MG-132 at a
very low dose for 3 mo, which offered a significant therapeutic
outcome, as indicated by the complete prevention of diabetesinduced cardiac inflammation and oxidative damage, leading to
a reversal of cardiac remodeling and dysfunction. In contrast,
diabetic mice without treatment with MG-132 showed the
progressive development of cardiac inflammation, structural
remodeling, and dysfunction. These therapeutic changes were
associated with both significant upregulation of Nrf2 expression and transcriptional increases of its downstream antioxidants and significant suppression of NF-␬B-mediated inflammation (Fig. 9). Therefore, MG-132 has great potential as a
therapeutic agent for diabetic patients, including those with
diabetic cardiomyopathy.
GRANTS
This work was supported in part by American Diabetes Association Basic
Research Award 1-11-BA-17 (to L. Cai), a Chinese-American Research Institute
for Diabetic Complications from Wenzhou Medical College Starting-Up Fund (to
L. Cai and Y. Tan), National Natural Science Foundation of China Grants
81070189 (to Y. Wang) and 81273509 (to Y. Tan), and Jilin University Bethune
Foundation Grant 2012221 (to Y. Wang).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: Y.W., B.L., T.C., Y.F., and L.C. conception and
design of research; Y.W., W.S., B.D., Y.B., Y.X., Y.T., W.C., and B.L.
performed experiments; Y.W., W.S., B.D., X.M., Y.B., Y.X., Y.T., and W.C.
analyzed data; Y.W., W.S., X.M., Y.T., B.L., T.C., Y.F., and L.C. interpreted
results of experiments; Y.W., W.S., B.D., and W.C. prepared figures; Y.W.,
Y.F., and L.C. drafted manuscript; Y.W., W.S., B.D., X.M., Y.B., Y.X., Y.T.,
W.C., B.L., T.C., P.N.E., Y.F., and L.C. approved final version of manuscript;
T.C., P.N.E., Y.F., and L.C. edited and revised manuscript.
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