Myocardial autophagy variation during acute myocardial infarction in rats: the effects of carvedilol.

Abstract

The loss of cardiac myocytes is one of the mechanisms involved in acute myocardial infarction (AMI)-related heart failure. Autophagy is a common biological process in eukaryote cells. The relationship between cardiac myocyte loss and autophagy after AMI is still unclear. Carvedilol, a non-selective alpha1- and beta-receptor blocker, also suppresses cardiac myocyte necrosis and apoptosis induced by ischemia. However, the association between the therapeutic effects of carvedilol and autophagy is still not well understood. The aim of the present study was to establish a rat model of AMI and observe changes in autophagy in different zones of the myocardium and the effects of carvedilol on autophagy in AMI rats.
The animals were randomly assigned to a sham group, an AMI group, a chloroquine intervention group and a carvedilol group. The AMI rat model was established by ligating the left anterior descending coronary artery. The hearts were harvested at 40 minutes, 2 hours, 24 hours and 2 weeks after ligation in the AMI group, at 40 minutes in the chloroquine intervention group and at 2 weeks in other groups. Presence of autophagic vacuoles (AV) in the myocytes was observed by electron microscopy. The expression of autophagy-, anti-apoptotic- and apoptotic-related proteins, MAPLC-3, Beclin-1, Bcl-xl and Bax, were detected by immunohistochemical staining and Western blotting.
AVs were not observed in necrotic regions of the myocardium 40 minutes after ligation of the coronary artery. A large number of AVs were found in the region bordering the infarction. Compared with the infarction region and the normal region, the formation of AV was significantly increased in the region bordering the infarction (P < 0.05). The expression of autophagy- and anti-apoptotic-related proteins was significantly increased in the region bordering the infarction. Meanwhile, the expression of apoptotic-related proteins was significantly increased in the infarction region. In the chloroquine intervention group, a large number of initiated AVs (AVis) were found in the necrotic myocardial region. At 2 weeks after AMI, AVs were frequently observed in myocardial cells in the AMI group, the carvedilol group and the sham group, and the number of AVs was significantly increased in the carvedilol group compared with both the AMI group and the sham group (P < 0.05). The expression of autophagy- and anti-apoptotic-related proteins was significantly increased in the carvedilol group compared with that in the AMI group, and the positive expression located in the infarction region and the region bordering the infarction.
AMI induces the formation of AV in the myocardium. The expression of anti-apoptosis-related proteins increases in response to upregulation of autophagy. Carvedilol increases the formation of AVs and upregulates autophagy and anti-apoptosis of the cardiac myocytes after AMI.

Full text

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Chin Med J 2009;122(19):2372-2379
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Original article

Myocardial autophagy variation during acute myocardial
infarction in rats: the effects of carvedilol

ZHANG Jing-lan, LU Jia-kai, CHEN Dong, CAI Qing, LI Tong-xun, WU Li-song and WU Xue-si

Keywords: acute myocardial infarction; myocardial autophagy; carvedilol; rats

Background The loss of cardiac myocytes is one of the mechanisms involved in acute myocardial infarction
(AMI)-related heart failure. Autophagy is a common biological process in eukaryote cells. The relationship between
cardiac myocyte loss and autophagy after AMI is still unclear. Carvedilol, a non-selective α1- and β-receptor blocker, also
suppresses cardiac myocyte necrosis and apoptosis induced by ischemia. However, the association between the
therapeutic effects of carvedilol and autophagy is still not well understood. The aim of the present study was to establish
a rat model of AMI and observe changes in autophagy in different zones of the myocardium and the effects of carvedilol
on autophagy in AMI rats.
Methods The animals were randomly assigned to a sham group, an AMI group, a chloroquine intervention group and a
carvedilol group. The AMI rat model was established by ligating the left anterior descending coronary artery. The hearts
were harvested at 40 minutes, 2 hours, 24 hours and 2 weeks after ligation in the AMI group, at 40 minutes in the
chloroquine intervention group and at 2 weeks in other groups. Presence of autophagic vacuoles (AV) in the myocytes
was observed by electron microscopy. The expression of autophagy-, anti-apoptotic- and apoptotic-related proteins,
MAPLC-3, Beclin-1, Bcl-xl and Bax, were detected by immunohistochemical staining and Western blotting.
Results AVs were not observed in necrotic regions of the myocardium 40 minutes after ligation of the coronary artery. A
large number of AVs were found in the region bordering the infarction. Compared with the infarction region and the
normal region, the formation of AV was significantly increased in the region bordering the infarction (P <0.05). The
expression of autophagy- and anti-apoptotic-related proteins was significantly increased in the region bordering the
infarction. Meanwhile, the expression of apoptotic-related proteins was significantly increased in the infarction region. In
the chloroquine intervention group, a large number of initiated AVs (AVis) were found in the necrotic myocardial region. At
2 weeks after AMI, AVs were frequently observed in myocardial cells in the AMI group, the carvedilol group and the sham
group, and the number of AVs was significantly increased in the carvedilol group compared with both the AMI group and
the sham group (P <0.05). The expression of autophagy- and anti-apoptotic-related proteins was significantly increased
in the carvedilol group compared with that in the AMI group, and the positive expression located in the infarction region
and the region bordering the infarction.
Conclusions AMI induces the formation of AV in the myocardium. The expression of anti-apoptosis-related proteins
increases in response to upregulation of autophagy. Carvedilol increases the formation of AVs and upregulates
autophagy and anti-apoptosis of the cardiac myocytes after AMI.
Chin Med J 2009;122(19):2372-2379

A
utophagy is a process by which cytoplasmic proteins
and organelles are degraded and recycled by
lysosomes, and it is a common phenomenon in eukaryotic
cells. Autophagy is initiated by the emergence of an
isolation membrane, which envelops cytosolic proteins
and organelles to form the autophagosome, which has a
tight double-membrane structure. The autophagosome is
delivered to the lysosome to form an autophagolysosome
for subsequent degradation of their content by lysosomal
hydrolases to nucleotides, amino acids and free fatty
acids.1 The nutrients freed by the processes above can be
recycled by the cells. The process can not only provide
nutrients to renew the bioactivity of macromolecular
materials, but also degrade the harmful contents in the
cell.2,3 Autophagy is a physiologic process that is
necessary for cell survival and maintains stable levels of
nutrients to sustain cellular homeostasis.4 Besides
managing the recycling of cellular contents, autophagy is
also involved in various pathophysiological processes,
and shows increased activity in response to extracellular
and intracellular stimulation such as nutrient starvation
and hypoxia. Autophagy increases in response to acute
myocardial ischemia (AMI), chronic myocardial ischemia,
heart failure and cardiomyopathy degeneration.5
DOI: 10.3760/cma.j.issn.0366-6999.2009.19.033
Department of Surgical Intensive Care Unit (Zhang JL, Li TX and
Wu LS), Department of Anesthesiology (Lu JK), Department of
Pathology (Chen D), Department of Cardiac Medicine (Wu XS),
Beijing Anzhen Hospital, Capital Medical University, Beijing
100029, China
Medical Experiment & Testing Center, Capital Medical University,
Beijing 100069, China (Cai Q)
Correspondence to: Dr. WU Xue-si, Department of Cardiac
Medicine, Beijing Anzhen Hospital, Capital Medical University,
Beijing 100029, China (Tel:
wuxuesi@126.net)
86-10-64456971. Email:

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Down-regulation of autophagy can lead to tumor and
heart disease.6 According to previous studies, excessive
autophagy can lead to type II programmed cell death,
which is independent of caspase proteins.7 In other
studies, inhibition of autophagy can protect the cells from
death.8 Whether autophagy is the cause of cell death or a
compensatory mechanism to protect the cells from death
is still controversial. Prior research suggests that
autophagy has two opposing effects in impaired cardiac
myocytes; one is its protection of cells from death, the
other is that it can promote cell death.9

Histological changes in response to AMI include myocyte
death and scar formation in the infarction zone and
cellular hypertrophy in the non-infarction zone. The
pathological foundation of heart failure after AMI is the
loss of cardiac myocytes, which is recognized to be a
result of apoptosis.10 However, the relationship between
cell loss of cardiac myocytes after AMI and autophagy is
still unclear. Carvedilol, a third generation β-blocker, can
block α1- and β-receptors, has antioxidative properties
and also suppresses the cardiac myocyte necrosis and
apoptosis induced by ischemia.11,12 Several clinical trials
have demonstrated that long-term administration of
carvedilol can reduce the area of a myocardial infarction
and improve the prognosis of patients in the acute stage
of AMI. However, the relationship between the
therapeutic effects of carvedilol and autophagy is not well
understood.

The aim of the present study was to establish an AMI
model in rats and determine the level of autophagy in
different zones of the myocardium and analyze the effects
of carvedilol on autophagy after AMI.

METHODS

Animals and grouping
A total of 105 male Wistar rats (Academy of Military
Medical Sciences, Beijing, China), 6 weeks age and
weighting 200–250 g, were assigned in a random blind
fashion to four groups as follows: sham group (n=15);
AMI group (n=60); chloroquine group (n=15): rats were
administered with 10 mg/kg chloroquine (Applichem
BioChemica, Germany) via an intra-peritoneal injection,
as described by Iwai-kanai et al,13 2 hours before
establishing the AMI model; and carvedilol group
(n=15): rats were administered with 2 mg·kg-1·d-1
carvedilol (ROCHE S.P.A, Switzerland) orally, 2 hours
after establishing the AMI model. Animals were
anesthetized by intra-peritoneal injection of sodium
pentobarbital (50 mg/kg) and AMI was established as
described by Palojoki et al.14 After implanting the
electrocardiogram monitor, the rat was connected to a
respirator through a tracheotomy, and the heart was
rapidly exteriorized through a left thoracotomy and
pericardial incision. The left anterior descending coronary
artery was ligated. Successful establishment of AMI was
confirmed with a pale appearance of the anterior wall of
the left ventricular and apex, a weakened pulse and S-T
segment elevation. The sham-operated group rats
underwent the same procedure except for the ligation of
the coronary artery. The hearts were harvested 40 minutes,
2 hours, 24 hours or 2 weeks after ligation of the coronary
artery in the AMI group, 40 minutes after AMI in the
chloroquine group, and 2 weeks after AMI in other
groups.

Light microscopy
Animals were sacrificed after anesthesia, and the hearts
were excised and washed with cold saline. The hearts
were cut along the long axis cross-section of the left
ventricle, and infarcted tissue, border infarcted tissue and
healthy tissue were isolated. The tissues were fixed with
4% paraformaldehyde solution, embedded in paraffin and
sectioned.

Immunohistochemistry
The myocardial specimen slides were deparaffinized,
heat-induced antigens were retrieved using 0.01 mol/L
citrate buffer (pH 6.0), endogenous peroxidase activity
was quenched and slides
phosphate-buffered saline (PBS). The tissues were then
permeabilized in 0.2%–1.0% Triton-X 100, and blocked
with bovine serum. Slides were incubated with diluted
primary antibodies against Beclin-1 (Cell Signaling,
USA), MAPLC-3 (Abcam, UK), Bcl-xl (Abcam) and Bax
(Abcam) and incubated at 4°C overnight. The sections
were then washed with PBS containing 1% bovine serum
albumin three times before incubation with biotinylated
secondary antibody (Santa Cruz Biotechnology, USA) for
30 minutes at 37°C and labeled with peroxidase avidin
using diaminobenzidine as the chromogen. The sections
were counterstained with hematoxylin and then blocked.
A V-PMTVC microscope (OLYMPUS company, Japan)
was used for qualitative analysis of the section (×400).
Ten sections were obtained from each group and the
expression of Beclin-1, MAPLC-3, Bcl-xl and Bax was
assessed.

Western blotting analysis
After extraction of myocardium protein, the protein
samples (30 µg of total protein) were denatured in
SDS-PAGE gel loading buffer supplemented with 5%
mercaptoethanol for 5 minutes. After electrophoresis on
10% SDS-polyacrylamide gels, the proteins were
transferred to nitrocellulose membranes. The membranes
were blocked with Tris-buffered saline supplemented
with Tween 20 containing 5% skimmed milk powder for
2 hours at room temperature. The membranes were
incubated with primary antibodies against Beclin-1, Bcl-xl
and β-actin (Santa Cruz) at 4°C overnight. Finally, the
membranes were hybridized with a secondary antibody
peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz).
The bound antibodies were detected using an enhanced
chemiluminescence system (Pierce, Rockford, USA) and
were washed in

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Chin Med J 2009;122(19):2372-2379
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hours in the AMI group. D: Region bordering the infarction at 24 hours in AMI group. E: Infarction region at 40 minutes in the
chloroquine group. F: Sham group. G: The number of AVs in cardiac myocytes in different regions at 24 hours after AMI in the AMI
group, *P <0.05. AVi: initiated autophagic vacuoles; AVd: degraded autophagic vacuoles; arrows: autophagic vacuoles.

high-performance chemilu- minescence film (Pierce).

Transmission electron microscopy (TEM)
Specimens were isolated from the infarcted region, the
region bordering the infarction and from healthy tissue in
the left ventricular myocardium. The specimens were
fixed with 3% glutaraldehyde PBS, 1% osmium, and 0.1
mol/L tetroxide PBS, dehydrated with an acetone gradient,
embedded in EPON-812 resin at 35°C overnight, and
repolymerized at 60°C for 24–58 hours. Semi-thin
sections (1–2 µm) were then cut into 60–80 nm ultra-thin
slices, with positioning control. The sections were double
electronic color-stained with uranyl acetate and lead
citrate, and observed and photographed with a
perspective electron microscope (JEM-1230 type, Japan
JEOL) (×12 000–×15 000).

Statistical analysis
Statistical analyses were performed using SPSS 15.0
software. All data are expressed as means±standard
deviation (SD). Differences between the groups were
determined using one-way analysis of variance (ANOVA)
followed by the Student-Neuman-Keul′s post hoc test.
Differences with a value of P <0.05 were considered
statistically significant.

RESULTS

Autophagic vacuoles (AV) presented in the region
bordering the infarction in the AMI group:
Transmission electron microscope (TEM) results
At 40 minutes, 2 hours and 24 hours after coronary artery
ligation, cardiac myocytes in the infarction region
exhibited varying degrees of ischemia injury. Cardiac
myocyte necrosis was observed in severely injured areas
and AV was not seen in these cardiac myocytes (Figure
1A and 1C). A large number of primary initial autophagic
vacuoles (AVi) and the degraded autophagic vacuoles
(AVd) were observed in the region bordering the
infarction (Figure 1B and 1D) and non-necrotic cardiac
myocytes in the infarction region. A few AVs (Figure 1F)
were also found in cardiac myocytes in the sham group.

At 24 hours after coronary artery ligation, the number of
AVs in cardiac myocytes in the region bordering the
infarction was significantly greater compared with that in
the infarction region and the normal region (P <0.05;
Figure 1G). The results indicate that AMI increased the
number of AVs in cardiac myocytes in the ischemic area.

To investigate whether AMI can induce the formation of
autophagosomes, we administered chloroquine, a
lysosomal inhibitor, 2 hours before coronary artery
ligation. Forty minutes after AMI, we found that the
cardiac myocyte injury in the infarction region was more
severe compared with the AMI group at 40 minutes and at
2 hours after coronary artery ligation, and a large number
of AVi were observed in necrotic cardiac myocytes
(Figure 1E). There was a large number of AVs in cardiac
myocytes in the region bordering the infarction. The
results demonstrated that AMI could induce AV formation
in myocardial cells.

Magnitude of myocardial damage in the AMI and
chloroquine groups
Immunohistochemical results
In this study, the expression of MAPLC-3 (Figure 2A),
Beclin-1 (Figure 2C) and Bcl-xl (Figure 2E) was increased
in the region bordering the infarction, and the expression of
Bax (Figure 2G) was increased in the infarction region
(Table 1) at 24 hours after AMI in the AMI group. The
positive expression was localized to the cytoplasm. The
results indicate that autophagy and anti-apoptotic activity
were increased in cardiac myocytes after AMI.
Figure 1. Cardiac
myocytes at different
time points after AMI
( T E M , o r i g i n a l
magnification ×12 000).
A: Infarction region at
40 minutes in the AMI
group. B: Region
bordering the infarction
at 40 minutes in the
A M I g r o u p . C :
Infarction region at 24

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Figure 2. Myocardial tissue at 24 hours after AMI in the AMI group (immunohistochemistry, original magnification ×400). A: MAPLC-3
expression in the region bordering the infarction. B: MAPLC-3 expression in the normal region. C: Beclin-1 expression in the region
bordering the infarction. D: Beclin-1 expression in the normal region. E: Bcl-xl expression in the region bordering the infarction. F:
Bcl-xl expression in the normal region. G: Bax expression in the infarction region. H: Bax expression in the normal region.



Table 1. Immunohistochemical evaluation of the expression of
MAPLC-3, Beclin-1, Bcl-xl and Bax 24 hours after AMI in
myocardial tissues (index, n=15)
Regions MAPLC-3 Beclin-1
Infarction 130.8±64.2 77.2±22.3
Border 560.2±95.5*† 458.8±89.5*†
Normal 362.2±49.2 214.6±19.3
F values 95.773 63.081
*P <0.01 compared with the infarction region. †P <0.01 compared with the
normal region.

The expression of Bcl-xl (Figure 3A and 3B) in the region
bordering the infarction and the expression of Bax
(Figure 3D and 3E) in the infarction region increased at
40 minutes after coronary artery ligation in the AMI and
chloroquine groups (Table 2). The positive staining was
localized to the cytoplasm. The results indicate that the
anti-apoptotic activity was markedly increased in the
region bordering the infarction.

Results of Western blotting
The expression of Bcl-xl antibody was increased in the
Bcl-xl
314.6±49.4
424.6±130.6*† 356.0±44.8*†
98.0±51.3
47.464
Bax
573.2±27.6
111.6±38.5
187.849
Table 2. Immunohistochemical evaluation of the expression of
Bcl-xl and Bax at 40 minutes after AMI in two groups (n=15)
Groups Bcl-xl
AMI

I-R 350.2±44.5
B-R 566.6±47.4*†
N-R 98.0±51.3
F values 120.261
Chloroquine

I-R 401.4±55.6
B-R 613.8±40.0*†
N-R 98.0±51.3
F values 155.377
I-R: infarction region; B-R: border region; N-R: normal region. *P <0.01
compared with the normal region; †P <0.01 compared with the infarction region;
‡P <0.05 compared with the infarction region; F: value F in analysis of variance.
No significant difference in the two groups.

region bordering the infarction in both the AMI group and
in the chloroquine group at 40 minutes and 24 hours, as
was the expression of the Beclin-1 antibody in the AMI
group (Figure 4) at 24 hours. The results indicated that
the anti-apoptotic activity and autophagy were increased
in the region bordering the infarction after AMI.
Bax

510.8±20.1
274.8±51.7*‡
154.4±60.0
74.255

501.2±46.5
336.2±40.6*‡
174.8±87.3
34.988
Figure 3. Myocardial tissues taken at 40 minutes after AMI
(immunohistochemistry, original magnification ×400). The
positive expression was located to the cytoplasm. A: Bcl-xl
expression in the region bordering the infarction region in the
AMI group. B: Bcl-xl expression in the region bordering the
infarction in the chloroquine group. C: Bcl-xl expression in the
normal region. D: Bax expression in the infarction region in the
AMI group. E: Bax expression in the infarction region in the
chloroquine group. F: Bax expression in the normal region.

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Figure 6. Myocardial tissue taken at 2 weeks after AMI. The positive expression was localized to the cytoplasm. (A–E: Bcl-xl
immunohistochemistry, original magnification ×400; F–H: Bax immunohistochemistry, original magnification ×400). A: Infarction region
in the AMI group. B: Region bordering the infarction in the AMI group. C: Infarction region in the carvedilol group. D: Region bordering
the infarction in the carvedilol group. E: Sham group. F: Infarction region in the AMI group. G: Infarction region in the carvedilol group.
H: Sham group.


Figure 4. Protein expression was evaluated by Western blotting
at different time in the infarction region (Lanes 1, 3 and 5), the
region bordering the infarction (Lanes 2, 4 and 6) and the
normal region (Lanes 7 and 8). Chl: chloroquine group.

Autophagy in the AMI, carvedilol and sham groups
TEM results
AVs were observed in surviving cardiac myocytes in
three different regions in each group at 2 weeks after
coronary artery ligation. The volume of AVs was
significantly higher in the AMI group (Figure 5A and 5B)
and in the carvedilol group (Figure 5C and 5D) than in
the sham group (Figure 5E). The number of AVs was
significantly greater in the carvedilol group compared
with the AMI group (Figure 5F). The AVs were mainly
localized to the region bordering the infarction in the
AMI group and in the carvedilol group. The number of
AVis was greater in the carvedilol group than in the AVd,
while the volumes of AVis and AVds in the AMI group
were similar. In the AMI group, the cardiac myocytes in
the infarction region exhibited disordered arrangement
and had a dispersed appearance. In the carvedilol group,
the cardiac myocytes in the infarction region were broken
and had a locally dispersed appearance. In the sham
group, the cardiac myocytes exhibited no significant
Figure 5. Cardiac myocytes at 2
weeks after coronary artery banding
(EM; original magnification ×15 000).
A: Infarction region in the AMI
group. B: Region bordering the
infarction in the AMI group. C:
Infarction region in the carvedilol
group. D: Region bordering the
infarction in the carvedilol group. E:
Sham group. F: The number of AVs 2
weeks after coronary artery ligation.
*P <0.05 compaied with AMI group.
Arrows: AVs.