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ORIGINAL ARTICLE
Resveratrol Ameliorates Cardiac Dysfunction by
Inhibiting Apoptosis via the PI3K/Akt/FoxO3a Pathway in
a Rat Model of Diabetic Cardiomyopathy
Zhiye Wu, MD,*Anqing Huang, MD,*Jianyun Yan, PhD,†Bei Liu, PhD,‡Qicai Liu, PhD,*
Jianwu Zhang, PhD,§ Xiuli Zhang, MD,*Caiwen Ou, PhD,*and Minsheng Chen, PhD, MD*
Abstract: The aim of this study was to explore the effect and
mechanism of action of resveratrol (RSV) on cardiac function in
diabetic cardiomyopathy (DCM). Hyperglycemia-induced apoptosis
contributes to the pathogenic changes in DCM. RSV treatment
inhibited high glucose–induced apoptosis of neonatal rat ventricular
myocytes. Additionally, high glucose decreased cell viability, pre-
vented serine–threonine kinase (Akt) and FoxO3a phosphorylation,
and suppressed cytoplasmic translocation of FoxO3a. However,
these effects of apoptosis were reversed by 10 mM of RSV. The
PI3K inhibitor LY294002 abolished the RSV protective effect
in vitro. RSV (5 or 50 mg$kg
21
$d
21
orally for 8 weeks) prevented
the deterioration of cardiac function and structural cardiomyopathy
in a streptozotocin-induced rat model of diabetes and reduced
apoptosis in diabetic myocardium. Furthermore, it restored
streptozotocin-impaired phosphorylation of Akt and FoxO3a (p-Akt
and p-FoxO3a) and suppressed nuclear translocation of FoxO3a
in vivo. Together, these data indicate that RSV has therapeutic
potential against DCM by inhibiting apoptosis via the PI3K/Akt/
FoxO3a pathway.
Key Words: resveratrol, diabetic cardiomyopathy, apoptosis, PI3K,
FoxO3a
(J Cardiovasc Pharmacol 2017;70:184–193)
INTRODUCTION
Diabetic patients have a 50%–80% risk of dying from
diabetic cardiovascular complications.
1
Diabetic cardiomyop-
athy (DCM) is a serious complication, and its mechanism
remains incompletely understood.
2,3
Hyperglycemia-induced
apoptosis contributes to the pathogenic changes in DCM.
4
Sustained hyperglycemia is a major cause of excess reactive
oxygen species (ROS) formation, disturbance of lipid metabo-
lism, insulin resistance, and dysregulation of cytoplasmic cal-
cium, which leads to cell death and results in left ventricular
(LV) remodeling and subsequent heart failure.
3
The phospha-
tidylinositol 3-kinase (PI3K) pathway is an important anti-
apoptotic pathway. PI3K activates serine–threonine kinase
(Akt), which induces the phosphorylation of several down-
stream substrates, such as the type 4 glucose transporter
(GLUT4),
5,6
mammalian target of rapamycin (mTOR), and the
proapoptotic forkhead box O3 (FoxO) transcription factors.
7,8
Resveratrol (RSV), found in grape skin and red wine,
has become well known for the “French paradox,”exerting
protective effect against inflammation,
9
aging,
10,11
cancer,
12
cardiovascular diseases,
13
ROS, and diabetes.
14–16
A range of
subcellular mechanisms are involved in the protective effects
of RSV in diabetes, such as activation of superoxide dismu-
tase, nitric oxide synthase, sirtuins, protein kinase C, and
peroxisome proliferator–activated receptor a, and anti-
inflammatory and autophagy mechanisms.
17,18
RSV has been
reported to be closely associated with the PI3K/Akt signal
pathway
7,8
and to activate the insulin receptor and FoxO3a
signaling pathways.
14
It was recently shown that enhancing
PI3K activity can prevent DCM.
19
However, whether RSV
ameliorates DCM by inhibiting inappropriate apoptosis via
the PI3K/Akt/FoxO3a pathway has not been addressed yet.
Here, we evaluate whether RSV ameliorates DCM by regu-
lating the PI3K/Akt/FoxO3a pathway both in vitro and in the
streptozotocin (STZ)-induced rat model of diabetes.
MATERIALS AND METHODS
Animals and Treatment
All animal experiments were performed in accordance
with the Guide for the Care and Use of Laboratory
Animals (NIH Publication No. 85–23, revised 1985). All
investigations were approved by the Bioethics Committee
of Southern Medical University, Guangzhou, China. Male
Received for publication December 25, 2016; accepted April 26, 2017.
From the *Department of Cardiology, Guangdong Provincial Biomedical Engi-
neering Technology Research Center for Cardiovascular Diseases,
Zhujiang Hospital of Southern Medical University, Guangzhou, P. R. China;
†Department of Histology and Embryology, Southern Medical University,
Guangzhou, P. R. China; ‡Department of Cardiology, Shanghai General
Hospital, Shanghai, P. R. China; and §Department of Cardiology, Nanfang
Hospital of Southern Medical University, Guangzhou, P. R. China.
Supported by grants from the National Natural Science Foundation of China
(31400858, 31671025, 81470488).
The authors report no conflicts of interest.
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF
versions of this article on the journal’s Web site (www.jcvp.org).
Z. Wu and A. Huang contributed equally to this work.
Reprints: Caiwen Ou, PhD or Minsheng Chen, PhD, MD, Department of
Cardiology, Guangdong Provincial Biomedical Engineering Technology,
Research Center for Cardiovascular Diseases, Zhujiang Hospital of
Southern Medical University, No. 1023 Industrial Rd, Guangzhou,
510280, P. R. China (e-mail: caiwensmu@163.com or minshengsmu@
163.com).
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184 |www.jcvp.org J Cardiovasc PharmacolVolume 70, Number 3, September 2017
Copyright Ó2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Sprague-Dawley rats, weighing 250–300 g, were obtained
from the animal center of Southern Medical University.
All rats were housed in a temperature-controlled room at
25 618C under a 12-hour light/dark cycle.
Type 1 diabetes mellitus was induced with a single
intravenous injection of STZ (50 mg/kg).
20
Rats in the control
group received an equivalent volume of normal saline in the
same way. Plasma glucose concentrations were measured 2–3
times on days 7 and 14 after injection. Rats injected with STZ
showed polyuria, polyphagia, and body weight loss; those
with plasma glucose concentrations $16.65 mmol/L were
considered to be diabetic.
All diabetic rats were randomly allocated to 3 groups:
DM (diabetic models), DM + R5 (5 mg$kg
21
$d
21
RSV;
Sigma), and DM + R50 (50 mg$kg
21
$d
21
RSV). RSV
was suspended in 0.9% saline containing 0.5% carboxy-
methylcellulose (Sigma) and administered by gavage every
day for 8 weeks. Diabetic controls and nondiabetic animals
received vehicle. Body weight and blood glucose were re-
corded every 2 weeks posttreatment. At the end of the exper-
iment, rats underwent echocardiography, and the hearts were
harvested under anesthesia with amobarbital sodium (30 mg/
kg, Sigma).
Echocardiography
After 8 weeks, all rats were weighed then anesthetized
using amobarbital sodium. Two-dimensional M-mode trans-
thoracic echocardiography was used with an IE33 echocardi-
ography system (Philips Medical Systems, the Netherlands).
The percentage of LV fractional shortening (FS), LV ejection
fraction (EF), maximal velocity through the LV outflow tract
(Vmax), cardiac output, LV pressure half-time, and LV internal
dimensions at diastole and systole (LVIDd and LVIDs) were
measured, and hemodynamics including LV end-diastolic
pressure, LV end-systolic pressure, and rate of LV pressure
rise and fall (+dP/dt max and 2dP/dt max, respectively).
Heart Weight and Histology
Harvested hearts were washed 3 times in phosphate-
buffered saline (PBS), and heart weight to body weight (HW/
BW) ratio and LV mass to body weight (LVm/BW) ratio
were calculated. The hearts were then fixed in 4% para-
formaldehyde for 24 hours, dehydrated, and embedded in
paraffin for sectioning. Tissue sections were stained using
hematoxylin and eosin and Masson’s trichrome and observed
under a light microscope (Leica, German). Image-Pro Plus
(v6.0; Media Cybernetics, Carlsbad, CA) was used to mea-
sure myocyte area and fibrosis.
Cell Culture
Neonatal rat ventricular myocytes were isolated from
the hearts of neonatal Sprague-Dawley rats (1–2 days old;
Laboratory Animal Center of Guangdong Province, Guangz-
hou, China) using trypsin and collagenase. The cells were
cultured in Dulbecco’s modified Eagle’s medium containing
10% fetal bovine serum and 1% penicillin/streptomycin and
incubated at 378C with 5% CO
2
in a humidified atmosphere.
The medium was changed once a day in the first 3 days and
then once every 3 days until day 6 when the cardiomyocytes
were used for subsequent experiments. To assess cultured cell
apoptosis and the mechanism involved, we designed the
following groups: cells exposed to normal glucose (5 mM)
for 24 hours (control group); high glucose (HG; 33 mM)
for 24 hours (HG group); RSV pretreatment for 60 minutes
(10 mM) before HG exposure for 24 hours (HG + RSV
group); and exposure to the PI3K inhibitor LY294002 for
60 minutes (50 mM; Abcam) before RSV for 60 minutes
(10 mM) followed by HG for 24 hours (HG + RSV +
LY294002 group).
MTT Assay
We used an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphen-
yl-2-H-tetrazolium bromide (MTT) assay kit (Invitrogen,
Grand Island, NY) to measure cell viability. Neonatal rat
ventricular myocytes cells were seeded in 96-well plates at
a density of 7000 cells per well allocated to the following
groups: normal glucose (5 mM) for 24 hours (control
group); high glucose (HG; 33 mM) for 24 hours (HG
group); RSV pretreatment for 60 minutes (1, 10, and
100 mM) before HG exposure for 24 hours (HG + RSV
1
,
HG + RSV
10
,andHG+RSV
100
groups); and exposure to the
PI3K inhibitor LY294002 for 60 minutes (50 mM; Abcam)
before RSV for 60 minutes (10 mM) followed by HG for 24
hours (LY group). The old medium was replaced with 100 mLof
fresh medium supplemented with MTT reagent (final concentra-
tion, 0.5 mg/mL), and the wells were incubated for 4 hours. The
medium was then replaced with 100 mL of dimethylsulfoxide
(DMSO). Optical density of the solution was measured at
490 mm using a microplate reader (iMark; Bio-Rad).
Assessment of Apoptosis
Tissue Sections
A TUNEL kit (KeyGen BioTech, China) was used to
detect apoptotic cells in tissue sections. In brief, sections were
incubated with proteinase K for 20 minutes at 378C to per-
meabilize the cells, then in 3% H
2
O
2
for 10 minutes at room
temperature to block nonspecific binding. This was followed
by incubation with terminal deoxynucleotidyl transferase for
60 minutes at 378C and streptavidin–HRP for 30 minutes at
378C in the dark. Apoptotic cells were visualized using 3,30-
diaminobenzidine, then counterstained with hematoxylin, and
viewed under a fluorescence microscope (EVOS FL Cell
Imaging System; Advanced Microscopy Group, Bothell,
WA) at ·200 magnification.
Cultured Cells
TUNEL Label Mix (Roche) was applied to reveal
apoptotic cells, and the results were analyzed by fluorescence
microscopy. In brief, cells were fixed in cold 4% para-
formaldehyde for 20 minutes at room temperature. PBS (0.01
M) with 0.3% Triton X-100 and 5% fetal bovine serum was
used to block nonspecific binding. After washing with PBS,
the samples were incubated with TUNEL reagent, terminal
deoxynucleotidyl transferase, and fluorescent isothiocyanate-
dUTP for 60 minutes at 378C in the dark. Nuclei were visu-
alized by incubating with 40,6-diamidino-2-phenylindole at
room temperature for 3 minutes. The samples were observed
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under a fluorescence microscope (BX50-FLA; Olympus, To-
kyo, Japan) and the results were analyzed using Image-Pro
Plus 6.0.
Western Blotting
Radio Immunoprecipitation Assay (RIPA) lysis buffer
(Beyotime, Guangzhou, China) and the CelLytic NuCLEAR
Extraction Kit (Sigma) were used to prepare total cell protein and
nucleoprotein in tissue sections and cultured cells, following the
instructions of manufacturers. Samples were stored at 2208C
after denaturing in water at 1008C. Proteins were separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis in
a 10% gel and were electrotransferred to polyvinylidene fluoride
(PVDF) membranes. The membranes were blocked in 5% milk
solution then incubated with primary antibodies (Bax, Bcl-2,
phospho-Akt, phospho-FoxO3a, Akt, FoxO3a, histone H3, and
GAPDH; all from Abcam) overnight at 48C and then with the
secondary antibody [goat anti-rabbit immunoglobulin G (IgG)
horseradish peroxidase (HRP), Bioworld Technology] for 2
hours at room temperature. Proteins were visualized using
enhanced chemiluminescence (Engreen, China) and an Image-
Quant LAS 500 imager (GE Healthcare). Image-Pro Plus 6.0 was
used to quantify the band intensities.
Statistical Analysis
Results are expressed as mean 6SD. Difference between
more than 2 groups was analyzed using a one-way analysis
of variance, followed by post-hoc Tukey’stest.P,0.05
was considered to indicate a significant difference between
groups.
RESULTS
RSV alleviates the symptoms of diabetes, increasing
body weight and decreasing blood glucose. Rats in the DM
group had markedly lower body weights and higher blood
glucose levels than those in the control group throughout the
experiment (Figs. 1A, B). Compared with the DM group, the
DM + R5 group had significantly higher body weights 4
weeks after treatment (P,0.01) and lower blood glucose
levels 6 weeks after treatment (P,0.01); the DM + R50
group showed the best regulation of diabetes symptoms,
with significant differences in weight and blood glucose
compared with the DM group observed from 2 weeks
(P,0.01) and 4 weeks (P,0.01), respectively. At the
end of the experiment (10 weeks), the DM + R50 group
showed significantly higher body weights (P,0.05) and
lower blood glucose levels (P,0.01) than the DM + R5
group. In addition, we found that RSV treatment signifi-
cantly increases the plasmatic insulin levels (P,0.01, see
Fig. 1,Supplemental Digital Content, http://links.lww.
com/JCVP/A266).
RSV Alleviates Cardiac Hypertrophy and
Fibrosis in Diabetic Rats
Analysis of hematoxylin and eosin staining revealed
that myocytes were enlarged in the DM group; this was
ameliorated by treatment with RSV (DM + R5, P,0.01 vs.
DM; DM + R50, P,0.01 vs. DM) (Figs. 2A, B). Fibrosis
among myocytes revealed by Masson’s staining was lower in
the RSV-treated groups than in the DM group (DM + R5, P
,0.01 vs. DM; DM + R50, P,0.01 vs. DM) (Figs. 2A, C).
RSV reduced HW/BW ratio (DM + R5, P,0.01; DM +
R50, P,0.01) and LVm/BW ratio (DM + R5 vs. DM, P,
0.05; DM + R50, P,0.01 vs. DM) (Fig. 2D).
RSV Alleviates LV Systolic Dysfunction in
Diabetic Heart
Cardiac performance and structural parameters derived
from echocardiography are shown in Figure 3. Impaired LV
function, evidenced by reduced LV ejection fraction (LVEF)
and fractional shortening (LVFS), was observed in the DM
group compared with the control group (P,0.01). LVEF
and LVFS were restored after RSV treatment (DM + R5, P,
0.01 vs. DM; DM + R50, P,0.05 vs. DM) in a dose-
dependent manner (P,0.05). LVIDd and LVIDs were also
elevated, but Vmax was lower in diabetic rats than in control
rats; this damage was reduced by RSV. Furthermore, hemo-
dynamic monitoring data were consistent with cardiac per-
formance parameters, indicating that RSV alleviates LV
systolic dysfunction in rats with STZ-induced diabetes (see
Table 1,Supplemental Digital Content, http://links.lww.
com/JCVP/A269).
RSV Inhibits Cardiomyocyte Apoptosis in
Diabetic Heart
There were significantly more TUNEL-positive (apo-
ptotic) cells in the DM group than in the control group
FIGURE 1. RSV alleviates body
weight and blood glucose symptoms
of diabetes. A, Body weight in non-
diabetic rats (N), diabetic rats (DM),
and diabetic rats fed with 5 or 50
mg$kg
21
$d
21
of RSV (DM + R5 and
DM + R50, respectively). B, Blood
glucose levels in the 4 groups. Data
are mean 6SD (n = 3–6).*P,0.05,
**P,0.01 versus DM group; #P,
0.05, ##P,0.01 versus DM + R5
group.
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(P,0.01) (Figs. 4A, B). However, groups treated with RSV
had fewer TUNEL-positive cells than the DM group (DM +
R5, P,0.05; DM + R50, P,0.01); this effect was dose-
dependent (P,0.01), but the higher dose of RSV did not
reduce the number of apoptotic cells to control levels. Bax
and Bcl-2 regulate apoptosis. Western blot results revealed
that the expression ratio of Bcl-2/Bax protein was markedly
lower in the DM group than in the control group (P,
0.01) and was significantly upregulated in both RSV-
treated groups (DM + R5, P,0.01; DM + R50, P,
0.01) (Figs. 4C, D). However, we did not find any change
of cardiomyocyte apoptosis and the expression ratio of Bcl-
2/Bax protein in normal controls after RSV treatment (see
Fig. 2,Supplemental Digital Content, http://links.lww.
com/JCVP/A267).
RSV Regulates the PI3K/Akt/FoxO3a Pathway
in Diabetic Heart
The PI3K/Akt pathway is one of the best-known
survival signals for inhibiting cell apoptosis. It can be
activated by RSV in our diabetes model. Western blotting
was performed to analyze the protein expression of p-Akt,
Akt, p-FoxO3a, and FoxO3a (Fig. 5). Akt (P,0.01) and
FoxO3a (P,0.01) phosphorylation was markedly lower in
the DM group than in the control group. Compared with the
DM group, both RSV-treated groups showed significant
upregulation of p-Akt (DM + R5, P,0.05; DM + R50,
P,0.01) and p-FoxO3a (DM + R5, P,0.01; DM + R50,
P,0.01) expression. High-dose RSV had a greater effect
on Akt (P,0.01) and FoxO3a (P,0.01) phosphorylation
than the low dose. RSV increased cytoplasmic FoxO3a
FIGURE 2. RSV alleviates cardiac
hypertrophy and fibrosis in diabetic
rats. A, Cardiomyocyte size and col-
lagen volume fraction: photomicro-
graphs of left ventricular (LV) tissue
sections stained using hematoxylin
and eosin (upper panel) and Masson
(lower panel). B, LV cardiomyocyte
area. C, LV collagen volume fraction.
D, Ratios of heart weight to body
weight (HW/BW) and LV mass to
body weight (LVm/BW). Data are
mean 6SD (n = 6). **P,0.01 ver-
sus group N; #P,0.05, ##P,0.01
versus DM group; $P,0.05, $$P,
0.01 versus DM + R5 group.
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expression at both doses (DM + R5, P,0.01; DM + R50,
P,0.01) and decreased nuclear expression (DM + R5,
P,0.01; DM + R50, P,0.01) compared with the DM
group.
RSV Inhibits HG-Induced Apoptosis of
Neonatal Rat Cardiomyocytes via PI3K
The viability of cardiomyocytes exposed to HG was
approximately half that of control cells (P,0.01). Cells
FIGURE 3. Effects of RSV on left ven-
tricular (LV) systolic function. A, Rep-
resentative echocardiography images.
B, Ejection fraction (LVEF) and frac-
tional shortening (LVFS). C, Maximal
velocity through LV outflow tract
(Vmax), LV internal dimensions at
diastole (LVIDd) and systole (LVIDs).
Data are mean 6SD (n = 6). **P,
0.01 versus group N; #P,0.05, ##P
,0.01 versus DM group; $P,0.05,
$$P,0.01 versus DM + R5 group.
FIGURE 4. RSV attenuates rat ven-
tricular myocyte apoptosis in diabetic
hearts. A, TUNEL-stained sections;
black arrowheads indicate TUNEL-
positive nuclei (brown). B, Quantita-
tive analysis of myocyte apoptosis in
diabetic hearts. C and D, Western
blot analysis and quantification of
Bcl-2 and Bax expression in my-
ocytes. Data are mean 6SD (n = 6).
*P,0.05, **P,0.01 versus group
N; ##P,0.01 versus DM group; $P
,0.05, $$P,0.01 versus DM + R5
group.
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pretreated with RSV (1, 10, and 100 mM) showed greater via-
bility than those in the HG group (P,0.05, P,0.01, P,
0.01, respectively; see Fig. 3,Supplemental Digital Content,
http://links.lww.com/JCVP/A268), indicating that RSV pro-
tected the cells from glucose toxicity. The higher concentrations
of RSV (10 and 100 mM) had a greater protective effect than
1mM RSV (both P,0.01), with no significant difference
between 10 and 100 mM RSV. However, this protective effect
was abolished by pre-exposure to the PI3K inhibitor LY294002
(10 mM; P,0.01 vs. 10 and 100 mMRSV).
There were significantly more apoptotic cells in the
HG group than in the control group (P,0.01; Figs. 6A–D).
RSV prevented apoptosis in cardiomyocytes exposed to
HG (P,0.01), and this effect was partly blocked by
LY294002 (P,0.01 vs. HG + RSV). Consistent with
these results, the ratio of the apoptosis-related proteins
Bcl-2/Bax in the HG group was lower than that in the
control group (P,0.01). RSV increased the Bcl-2/Bax
ratio in cardiomyocytes subjected to HG compared with
exposure to HG alone (P,0.05). LY294002 abolished this
protective effect of RSV, indicated by a greater level of
apoptosis and a lower Bcl-2/Bax ratio (P,0.05), suggest-
ing that PI3K is required for the anti-apoptotic effect of
RSV on rat cardiomyocytes.
RSV Activates Akt and FoxO3a
Phosphorylation and Reduces Nuclear
Localization of FoxO3a in Neonatal Rat
Cardiomyocytes Exposed to HG
HG decreased the expression of p-Akt (P,0.01)
and p-FoxO3a (P,0.01) (Figs. 7A–E). RSV enhanced
phosphorylation of Akt (P,0.01) and FoxO3a (P,
0.01) in cells exposed to HG compared with cells exposed
to HG without RSV pretreatment. Pretreatment with
LY294002 reversed the protective effect of RSV on the
phosphorylation of Akt (P,0.01) and FoxO3a (P,
0.01), indicating that PI3K is involved in the protective
mechanism of RSV. Moreover, the HG + RSV group
FIGURE 5. RSV regulates the PI3K/
Akt/FoxO3a pathway in diabetic rat
hearts. A, Phosphorylated p-Akt and
p-FoxO3a, Akt, FoxO3a, cytoplasmic
FoxO3a, and nuclear FoxO3a protein
expression (Western blot). B–E,
Quantification of p-Akt/total Akt (B),
p-FoxO3a/total FoxO3a (C), cyto-
plasmic FoxO3a/GAPDH (D), and
nuclear FoxO3a/histone H3 (E). Data
are mean 6SD (n = 6). **P,0.01
versus group N; #P,0.05, ##P,
0.01 versus DM group; $$P,0.01
versus DM + R5 group.
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showed cytoplasmic localization of p-FoxO3a, caused by
RSV (P,0.01), then blocked its pro-apoptotic effects.
HG enhanced the translocation of FoxO3a to the nucleus
and triggered apoptosis. Surprisingly, RSV reduced the
level of FoxO3a in the nucleus (P,0.01) and exerted
protection against apoptosis. LY294002 abolished the pro-
tective effect of RSV manifesting in downregulation in the
cytoplasmic expression of FoxO3a (P,0.01) and upre-
gulation of FoxO3a expression in the nucleus (P,0.01).
Together, these data indicate that RSV played a protective
FIGURE 6. RSV protects car-
diomyocytes from apoptosis after
exposure to HG (33 mmol/L), and
this effect is partly abolished by
LY294002. A, Fluorescence micro-
scope images (·200): green, TU-
NEL-positive cells; blue, cell nuclei.
B, Apoptotic index in car-
diomyocytes. C and D, Western
blot analysis of Bcl-2 and Bax
expression in neonatal rat ventric-
ular myocytes. Data are mean 6SD
(n = 6). *P,0.05, **P,0.01
versus control group; #P,0.05,
##P,0.01 versus rats in HG
group; $$P,0.01 versus HG + RSV
group.
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role under HG conditions, mediated partly via the PI3K/
Akt/FoxO3a pathway.
DISCUSSION
Diabetes is associated with high risk of complications
impairing many organs, such as the hearts, eyes, kidneys, and
nervous system. The pathophysiology of DCM is complex
and multifactorial, and it involves cardiac dysfunction,
cardiomyocyte hypertrophy, myocardial interstitial fibrosis,
apoptosis, and oxidative stress.
21
Previous studies have
shown a close correlation between LV diastolic dysfunction
and myocardial apoptosis, and controlling myocardial apopto-
sis is known to improve cardiac function.
5,14,22
In contrast,
elevated myocardial apoptosis causes a loss of contractile
units and cardiac remodeling, leading to cardiac dysfunction.
Hyperglycemia acts as one of the main drivers of the meta-
bolic, functional, and structural alterations in the diabetic
hearts. The pathogenic mechanisms of DCM include impaired
cardiac insulin and glucose homeostasis, lipotoxicity,
impaired cellular and mitochondrial calcium regulation, over-
expression of ROS, and mitochondrial uncoupling.
21
All
these also contribute to cell death and tissue fibrosis.
Together, the present results indicate that diabetic rats have
impaired cardiac function, cardiomyocyte hypertrophy, myo-
cardial fibrosis, and cardiomyocyte apoptosis and that these
unfavorable changes were alleviated or revered by RSV.
Here, we observed that RSV reduced expression of Bax,
which inserted into the mitochondrial membrane to cause
mitochondria-mediated apoptosis by releasing pro-apoptotic
factors, such as cytochrome c. In addition, RSV increased the
anti-apoptotic factor Bcl-2, which prevents Bax
FIGURE 7. RSV induces Akt and
FoxO3a phosphorylation and re-
duces nuclear localization of FoxO3a
in neonatal rat cardiomyocytes in
response to HG. A, Expression of
phosphorylated p-Akt and p-FoxO3a,
Akt, FoxO3a, cytoplasmic FoxO3a,
and nuclear FoxO3a in diabetic
hearts (Western blot). B–E, Quantifi-
cation of p-Akt/total Akt (B), p-Fox-
O3a/total FoxO3a (C), cytoplasmic
FoxO3a/GAPDH (D), and nuclear
FoxO3a/histone H3 (E). Data are
mean 6SD (n = 6). *P,0.05, **P,
0.01 versus control group; ##P,0.01
versus rats in HG group; $$P,0.01
versus HG + RSV group.
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oligomerization onto mitochondrial membrane to induce cell
apoptosis. This upregulation of the Bcl-2/Bax ratio was
shown both in vivo and in vitro, providing strong evidence
of its involvement in the cardioprotective effect of RSV.
Accumulating studies have shown that RSV reduces the
blood glucose levels in STZ-treated type 1 diabetic animals.
23
Consistent with these previous findings, our data also show
that RSV reduces blood glucose levels in diabetic rats. How-
ever, some other studies have suggested that RSV has no
effect on decreasing blood glucose levels.
24,25
Differences in
dosage and duration of RSV treatment may account for this
discrepancy. PI3K/Akt has played an important role in the
anti-hyperglycemic effect of RSV in diabetic rats.
26
RSV
and its derivative piceatannol treatment can activate the insulin
receptor signal and stimulate the translocation of GLUT4 from
the cytoplasm to the plasma membrane.
6,27,28
RSV exerts an
insulin-like effect to improve insulin sensitivity, reverse insu-
lin resistance,
29
and enhance FoxO3 phosphorylation,
8
subse-
quently improving hyperglycemia in diabetic rats.
RSV exerts cardioprotective effects via its anti-
inflammation, anti-hypertrophy, ROS reduction, and
anti-apoptosis effects.
14,23
The present in vivo and in vitro
experiments indicate that RSV attenuates cardiac dysfunction
induced by hyperglycemia via activation of phosphorylation
of Akt. Our study demonstrates that STZ-induced diabetes
triggers the inflammatory process in cardiac tissue, evidenced
by enlarged cardiomyocytes, increased interstitial fibrosis,
and structural distortion. RSV treatment partially reversed
these irregularities and improved cardiac function.
PI3K/Akt is one of the most important survival signals.
PI3K recruits and activates Akt/protein kinase B, 3’-phos-
phoinositide-dependent kinase 1 (PDK1), and monomeric
G-proteins, leading to the activation of several downstream
targets, including glycogen synthase kinase-3
b
, mTOR, endo-
thelial nitric oxide synthase, p70S6 kinase, and several anti-
apoptotic effectors.
30
PI3K protects against a number of
cardiac stressors, including pressure overload, dilated cardio-
myopathy, atrial fibrillation, and myocardial infarction, as
well as exerting the protective effect on the hearts in STZ-
induced type 1 diabetes mouse models.
22
Decreased PI3K
activity in diabetes directly exacerbates DCM.
19
HG induces
apoptosis by reducing PI3K/Akt pathway activation.
31,32
Here, we show that the phosphorylation of Akt and FoxO3a
was weak in diabetic hearts, but dose-dependently increased
by RSV. A similar outcome was detected in vitro, with RSV
correcting the decrease in p-Akt and p-FoxO3a in neonatal rat
ventricular myocytes exposed to HG. Furthermore,
LY294002 blocked this protective effect of RSV. This indi-
cates that PI3K/Akt responds to RSV treatment, which might
contribute to the mechanism of action of RSV in DCM and in
elucidating the pathogenic mechanism of DCM.
The FoxO3a transcription factor, activated by the
phosphorylation of Akt at threonine 32, serine 253, and serine
315, subsequently translocates to the cytoplasm and inhibits
apoptosis by inhibiting Bim and Fas-L.
33,34
Therefore, Akt
phosphorylation, and subsequent inhibition of FoxO3a trans-
location to the nucleus, can be considered cardioprotective
actions as they improve cardiac function and promote cell
survival. Indeed, p-FoxO3a mediates HG-induced apoptosis
in neonatal rat ventricular myocytes.
33
In the present study,
RSV increased p-FoxO3a translocation to the cytoplasm in
diabetic hearts and ventricular myocytes exposed to HG. The
protective effect of RSV was blocked by the application of
LY294002. Together, these data indicate that the PI3K/Akt/
FoxO3a pathway may be responsible for in vivo and in vitro
RSV inhibition of HG-induced cardiac injury.
Although the protective effects of RSV and the involve-
ment of the PI3K/Akt pathway in diabetes have been reported
here and elsewhere, the pleuripotency of RSV may also
associate with multiple signaling pathways. Interestingly, hyper-
glycemia enhances the function and differentiation of adult rat
cardiac fibroblasts,
35
and RSV inhibits myocardial fibroblast
proliferation via PI3K/Akt.
36,37
Further studies are needed to
explore the detailed mechanisms underlying the effects of
RSV on myocardial fibroblasts and other myocardial cells in
diabetic hearts and to address the cross-talk between them.
CONCLUSIONS
We demonstrate that RSV ameliorates DCM by inhib-
iting apoptosis via the PI3K/Akt/FoxO3a pathway in vitro and
vivo. These findings indicate that RSV has therapeutic
potential to ameliorate LV remodeling and improve the
cardiac function in DCM. Additional studies are necessary
to investigate the potential clinical implications in the future.
REFERENCES
1. Wang ZV, Hill JA. Diabetic cardiomyopathy: catabolism driving metab-
olism. Circulation. 2015;131:771–773.
2. Farag YMK, Gaballa MR. Diabesity: an overview of a rising epidemic.
Nephrol Dial Transplant. 2011;26:28–35.
3. Huynh K, Bernardo BC, McMullen JR, et al. Diabetic cardiomyopathy:
mechanisms and new treatment strategies targeting antioxidant signaling
pathways. Pharmacol Ther. 2014;142:375–415.
4. van Empel VP, De Windt LJ. Myocyte hypertrophy and apoptosis: a bal-
ancing act. Cardiovasc Res. 2004;63:487–499.
5. Gencoglu H, Tuzcu M, Hayirli A, et al. Protective effects of resveratrol
against streptozotocin-induced diabetes in rats by modulation of visfatin/
sirtuin-1 pathway and glucose transporters. Int J Food Sci Nutr. 2015;66:
314–320.
6. Penumathsa SV, Thirunavukkarasu M, Zhan L, et al. Resveratrol enhan-
ces Glut-4 translocation to the caveolar lipid raft fractions through ampk/
akt/enos signalling pathway in diabetic myocardium. J Cell Mol Med.
2008;12:2350–2361.
7. Liu MH, Yuan C, He J et al. Resveratrol protects Pc12 cells from high
glucose-induced neurotoxicity via Pi3k/akt/Foxo3a pathway. Cell Mol
Neurobiol. 2015;35:513–522.
8. Lin CH, Lin CC, Ting WJ, et al. Resveratrol enhanced Foxo3 phosphor-
ylation via synergetic activation of Sirt1 and Pi3k/akt signaling to
improve the effects of exercise in elderly rat hearts. Age (Dordr).
2014;36:9705.
9. Jimenez-Gomez Y, Mattison JA, Pearson KJ, et al. Resveratrol improves
adipose insulin signaling and reduces the inflammatory response in adi-
pose tissue of rhesus monkeys on high-fat, high-sugar diet. Cell Metab.
2013;18:533–545.
10. Park SJ, Ahmad F, Philp A, et al. Resveratrol ameliorates aging-related
metabolic phenotypes by inhibiting camp phosphodiesterases. Cell.
2012;148:421–433.
11. Pearson KJ, Baur JA, Lewis KN, et al. Resveratrol delays age-related
deterioration and mimics transcriptional aspects of dietary restriction
without extending life span. Cell Metab. 2008;8:157–168.
12. Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of re-
sveratrol, a natural product derived from grapes. Science. 1997;275:218–220.
13. Petrovski G, Gurusamy N, Das DK. Resveratrol in cardiovascular health
and disease. Ann N Y Acad Sci. 2011;1215:22–33.
Wu et al J Cardiovasc PharmacolVolume 70, Number 3, September 2017
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Copyright Ó2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
14. Sadi G, Bilgehan Pektas M, Bugra Koca H, et al. Resveratrol improves
hepatic insulin signaling and reduces the inflammatory response in
streptozotocin-induced diabetes. Gene. 2015;570:213–220.
15. Vallianou NG, Evangelopoulos A, Kazazis C. Resveratrol and diabetes.
Rev Diabet Stud. 2013;10:236–242.
16. Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo
evidence. Nat Rev Drug Discov. 2006;5:493–506.
17. Wang B, Yang Q, Sun YY, et al. Resveratrol-enhanced autophagic flux
ameliorates myocardial oxidative stress injury in diabetic mice. J Cell
Mol Med. 2014;18:1599–1611.
18. Turan B, Tuncay E, Vassort G. Resveratrol and diabetic cardiac function:
focus on recent in vitro and in vivo studies. J Bioenerg Biomembr. 2012;
44:281–296.
19. Ritchie RH, Love JE, Huynh K, et al. Enhanced phosphoinositide 3-
kinase(P110alpha) activity prevents diabetes-induced cardiomyopathy
and superoxide generation in a mouse model of diabetes. Diabetologia.
2012;55:3369–3381.
20. Forman LJ, Estilow S, Mead J, et al. Eight weeks of streptozotocin-induced
diabetes influences the effects of cold stress on immunoreactive beta-
endorphin levels in female rats. Horm Metab Res. 1988;20:555–558.
21. Sung MM, Hamza SM, Dyck JR. Myocardial metabolism in diabetic
cardiomyopathy: potential therapeutic targets. Antioxid Redox Signal.
2015;22:1606–1630.
22. Zhou X, An G, Lu X. Hydrogen sulfide attenuates the development of
diabetic cardiomyopathy. Clin Sci. 2015;128:325–335.
23. Szkudelski T, Szkudelska K. Anti-diabetic effects of resveratrol. Ann N Y
Acad Sci. 2011;1215:34–39.
24. Schmatz R, Schetinger MR, Spanevello RM, et al. Effects of resveratrol
on nucleotide degrading enzymes in streptozotocin-induced diabetic rats.
Life Sci. 2009;84:345–350.
25. Schmatz R, Mazzanti CM, Spanevello R, et al. Ectonucleotidase and
acetylcholinesterase activities in synaptosomes from the cerebral cortex
of streptozotocin-induced diabetic rats and treated with resveratrol. Brain
Res Bull. 2009;80:371–376.
26. Chi TC, Chen WP, Chi TL, et al. Phosphatidylinositol-3-Kinase is
involved in the antihyperglycemic effect induced by resveratrol in
streptozotocin-induced diabetic rats. Life Sci. 2007;80:1713–1720.
27. Minakawa M, Miura Y, Yagasaki K. Piceatannol, a resveratrol deriva-
tive, promotes glucose uptake through glucose transporter 4 translocation
to plasma membrane in L6 myocytes and suppresses blood glucose levels
in type 2 diabetic model Db/Db mice. Biochem Biophys Res Commun.
2012;422:469–475.
28. Michael LF, Wu Z, Cheatham RB, et al. Restoration of insulin-sensitive
glucose transporter (Glut4) gene expression in muscle cells by the tran-
scriptional coactivator Pgc-1. Proc Natl Acad Sci USA. 2001;98:
3820–3825.
29. Cote CD, Rasmussen BA, Duca FA, et al. Resveratrol activates duodenal
Sirt1 to reverse insulin resistance in rats through a neuronal network. Nat
Med. 2015;21:498–505.
30. Oudit GY, Sun H, Kerfant BG, et al. The role of phosphoinositide-3
kinase and Pten in cardiovascular physiology and disease. J Mol Cell
Cardiol. 2004;37:449–471.
31. Yu W, Zha W, Ke Z, et al. Curcumin protects neonatal rat cardiomyo-
cytes against high glucose-induced apoptosis via Pi3k/akt signalling
pathway. J Diabetes Res. 2016;2016:4158591.
32. Tsai CY, Wang CC, Lai TY, et al. Antioxidant effects of diallyl
trisulfide on high glucose-induced apoptosis are mediated by the
Pi3k/akt-dependent activation of Nrf2 in cardiomyocytes. Int J Cardi-
ol. 2013;168:1286–1297.
33. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by
phosphorylating and inhibiting a forkhead transcription factor. Cell.
1999;96:857–868.
34. Maiese K. Foxo transcription factors and regenerative pathways in dia-
betes mellitus. Curr Neurovasc Res. 2015;12:404–413.
35. Shamhart PE, Luther DJ, Adapala RK, et al. Hyperglycemia enhances
function and differentiation of adult rat cardiac fibroblasts. Can J Physiol
Pharmacol. 2014;92:598–604.
36. Olson ER, Naugle JE, Zhang X, et al. Inhibition of cardiac fibroblast
proliferation and myofibroblast differentiation by resveratrol. Am J Phys-
iol Heart Circ Physiol. 2005;288:H1131–H1138.
37. Venkatachalam K, Mummidi S, Cortez DM, et al. Resveratrol inhibits
high glucose-induced Pi3k/akt/erk-dependent Interleukin-17 expression
in primary mouse cardiac fibroblasts. Am J Physiol Heart Circ Physiol.
2008;294:H2078–H87.
J Cardiovasc PharmacolVolume 70, Number 3, September 2017 Resveratrol Ameliorates Cardiac Dysfunction
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