From the First Department of Internal Medicine, Fukushima Medical
College, Hikarigaoka 1, Fukushima 960–12, Japan.
Correspondence to Yukio Maruyama, MD, Professor and Chairman, First Department of Internal Medicine, Fukushima Medical College, Hikarigaoka 1, Fukushima, 960–12, Japan. E-mail yaoita{at}cc.fmu.ac.jp
Methods and Results—Sprague-Dawley rats were subjected to a
30-minute coronary occlusion followed by a 24-hour reperfusion.
An inert vehicle (dimethylsulfoxide; group 1, n=8) or ZVAD-fmk, at a
total dose of 3.3 mg/kg (group 2, n=8), was administered
intravenously every 6 hours starting at 30 minutes before
coronary occlusion until 24 hours of reperfusion. At this
24-hour point, hemodynamics were assessed by means of
cardiac catheterization; then, the rats were killed,
and the left ventricle was excised and sliced. The myocardial infarct
size/ischemic area at risk and the count of presumed
apoptotic cardiomyocytes (terminal
deoxynucleotidyl transferase–mediated dUTP-biotin
nick end labeling [TUNEL]-positive cells) within the ischemic
area at risk were assessed through
triphenyltetrazolium chloride staining and
TUNEL methods, respectively. Peak positive left ventricular
dP/dt was higher (P=.02) and left
ventricular end-diastolic pressure was lower
(P=.04) in group 2 than in group 1. The infarct
size/ischemic area at risk of group 2 (52.4±4.0%) was smaller
(P=.02) than that of group 1 (66.6±3.7%), and
TUNEL-positive cells were fewer (P=.0002) (group 2,
3.1±0.9%; group 1, 11.1±1.0%). Agarose gel electrophoresis revealed
DNA laddering in the border zone myocardium of group 1, but
DNA ladder formation was attenuated in group 2.
Conclusions—ZVAD-fmk was effective in reducing myocardial
reperfusion injury, which could at least be partially attributed to the
attenuation of cardiomyocyte apoptosis.
The caspase inhibitors, that is, ICE-like protease
inhibitors,11 interfere with
apoptosis at a point subsequent to the initiation of the
proapoptotic process in cells that have already received
apoptosis-promoting signals. As opposed to reducing the
exposure of cardiomyocytes to injurious stimuli,
apoptosis of these cells is attenuated through modulation of
the caspase-related proapoptotic process, and this may allow
ischemic myocardium to survive even after receiving
significant injury. ZVAD-fmk (fluoro-methylketone), a tripeptide
inhibitor of the caspase, is reported to attenuate
cardiomyocyte apoptosis in
vitro.6 In the present study, we investigated
whether ZVAD-fmk lowers the extent of experimental myocardial
reperfusion injury in vivo by attenuating cardiomyocyte
apoptosis. In a rat model for myocardial reperfusion injury,
infarct size and the appearance of presumed apoptotic
cardiomyocytes were assessed in two groups that were or
were not administered this protease inhibitor.
Animal Model
One milligram of ZVAD-fmk (Enzyme Systems Products) was dissolved
in 107 µL of DMSO (Wako Pure Chemicals). In group 2 animals (n=8),
ZVAD-fmk, at one fourth of a total dose of 3.3 mg/kg body weight, was
administered as a bolus into the tail vein four times during the study
(first 30 minutes before coronary occlusion and then three
times every 6 hours after reperfusion). The same amount of an inert
vehicle (DMSO) was administered in the same manner to rats of group 1
(n=8).
To assess whether the amount of DMSO used as a vehicle would have a
toxic effect in vivo, one fourth of a total DMSO volume of 353 µL/kg
body weight (n=5) or the same volume of saline (n=5) was administered
four times to sham-operated rats in the same manner as to groups 1 and
2.
Leukocytes are known to be involved in the formation of myocardial
reperfusion injury.12 As a positive control for
this model of coronary reperfusion injury, 4 rats were
administered absorbed polyclonal rabbit anti-rat PMN antisera at a dose
of 3 mL/kg (Inter-Cell Technologies) 36 hours before coronary
occlusion. Each was subjected to the 30-minute coronary
occlusion and 24-hour reperfusion protocol, and 0.5 mL of blood was
taken before occlusion and just before death.
Hemodynamic Assessment
Assessment of Infarcted Area and Detection of TUNEL-Positive
Cardiomyocytes
Using an eyepiece for the point-counting method (Integration No. 1,
Zweiss), which was performed under a light microscope at a
magnification of 400x, we determined the count ratio of the area of
cardiomyocytes with TdT-stained nuclei with that of total
cardiomyocytes (TUNEL-positive cardiomyocytes)
within the ischemic area at risk. The entire area was searched
through an orderly shifting of the visual field using the outer grids
of the eyepiece for orientation. TUNEL-positive
cardiomyocytes were carefully distinguished from
TUNEL-positive noncardiomyocytes, such as
macrophages.
To assess the distribution of the infarcted area and TUNEL-positive
cardiomyocytes in the left ventricular wall, we
subdivided the ischemic area at risk into three transmural
stratified layers of equal thickness (epicardial, middle, and
endocardial) in each slice mentioned above (Fig 1
Using some of the paraffin sections of groups 1 and 2, hematoxylin and
eosin staining was also performed for confirmation of myocardial
reperfusion injury, such as myocardial cell coagulation, contraction
bands, bleeding, and inflammatory cell infiltration.
Genomic DNA Extraction and Agarose Gel Electrophoresis
Statistical Analysis
Positive Control for the Rat Model of Reperfusion Injury
Hemodynamics
For the sham-operated rats, administration of DMSO or saline resulted
in no differences in LVSP/EDP or LV dP/dt value or in the heart
rate.
Myocardial Infarct Size and TUNEL-Positive Cardiomyocytes
We confirmed that all nuclei of cardiomyocytes on
sections subjected to DNase treatment (as a positive control for the
TUNEL method) were stained dark brown each time the TUNEL method was
performed. The concentration of the TUNEL-positive
cardiomyocytes of group 2 (3.1±0.9%) was significantly
(P=.0002) less than that of group 1 (11.1±1.0%) (Fig 2
Neither TTC-negative zones nor TUNEL-positive
cardiomyocytes were detected in the sham-operated rats
administered DMSO or saline.
Agarose Gel Electrophoresis
ZVAD-fmk achieved
To date, nothing is known about the fate of cardiomyocytes
that have been exposed to ZVAD-fmk but have not undergone a
proapoptotic process, such as initiation of apoptotic
signal transduction via TNF receptor. These cardiomyocytes
may continue to survive or may undergo an early death because of injury
already sustained. Myocardial infarct size was assessed only at 24
hours after reperfusion in the present study. Future studies will
be needed to evaluate the viability of cardiomyocytes that
escape apoptosis through assessment of infarct extension in the
later phase of reperfusion.
Received July 21, 1997;
revision received October 2, 1997;
accepted October 2, 1997.
2.
Ambrosio G, Flaherty JT, Duilio C, Tritto I, Santoro
G, Elia PP, Condorelli M, Chiariello M. Oxygen radicals generated at
reflow induce peroxidation of membrane lipids in reperfused hearts.
J Clin Invest. 1991;87:2056–2066.
3.
Bolli R, Zughaib M, Li XY, Tang XL, Sun JZ, Triana JF,
McCay PB. Recurrent ischemia in the canine heart causes
recurrent bursts of free radical production that have a
cumulative effect on contractile function: a
pathophysiological basis for chronic myocardial
`stunning.' J Clin Invest. 1995;96:1066–1084.
4.
Kukielka GL, Smith CW, Manning AM, Youker KA, Michael
LH, Entman ML. Induction of interleukin-6 synthesis in the
myocardium: potential role in postreperfusion inflammatory
injury. Circulation. 1995;92:1866–1875.
5.
Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V,
Comstock KL, Glembotski CC, Quintana PJ, Sabbadini RA. Tumor necrosis
factor alpha-induced apoptosis in cardiac myocytes: involvement
of the sphingolipid signaling cascade in cardiac cell death.
J Clin Invest. 1996;98:2854–2865.[Medline]
[Order article via Infotrieve]
6.
Gottlieb RA, Gruol DL, Zhu JY, Engler RL.
Preconditioning rabbit cardiomyocytes: role of pH, vacuolar
proton ATPase, and apoptosis. J Clin Invest. 1996;97:2391–2398.[Medline]
[Order article via Infotrieve]
7.
Przyklenk K, Zhao L, Kloner RA, Elliott GT.
Cardioprotection with ischemic preconditioning and MLA: role of
adenosine-regulating enzymes? Am J Physiol.. 1996;271:H1004–1014.
8.
Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition
of nitric oxide synthesis reduces infarct size by an
adenosine-dependent mechanism. Circulation.. 1995;91:1545–1551.
9.
Yao Z, Gross GJ. A comparison of
adenosine-induced cardioprotection and ischemic
preconditioning in dogs: efficacy, time course, and role of KATP
channels. Circulation. 1994;89:1229–1236.
10.
Homeister JW, Hoff PT, Fletcher DD, Lucchesi BR.
Combined adenosine and lidocaine administration limits
myocardial reperfusion injury. Circulation. 1990;82:595–608.
11.
Tatsuta T, Cheng J, Mountz JD. Intracellular IL-1 beta
is an inhibitor of Fas-mediated apoptosis.
J Immunol. 1996;157:3949–3957.[Abstract]
12.
Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA,
Lucchesi BR. Reduction of the extent of ischemic myocardial
injury by neutrophil depletion in the dog. Circulation. 1983;67:1016–1023.
13.
Vivaldi MT, Kloner RA, Schoen FJ.
Triphenyltetrazolium staining of
irreversible ischemic injury following coronary
occlusion in rats. Am J Pathol. 1985;121:522–530.[Abstract]
14.
Weibel ER. Principles and methods for the morphometric
study of the lung and other organs. Lab Invest. 1963;12:131–155.[Medline]
[Order article via Infotrieve]
15.
Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of
programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J Cell Biol. 1992;119:493–501.
16.
Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y,
Matsuda H, Tsujimoto Y. Bcl-2 expression prevents activation of the ICE
protease cascade. Oncogene. 1996;12:2251–2257.[Medline]
[Order article via Infotrieve]
17.
Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y,
Matsuda H, Tsujimoto Y. Retardation of chemical hypoxia-induced
necrotic cell death by Bcl-2 and ICE inhibitors: possible
involvement of common mediators in apoptotic and necrotic
signal transductions. Oncogene. 1996;12:2045–2050.[Medline]
[Order article via Infotrieve]
18.
Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y,
Matsuda H, Tsujimoto Y. Bcl-2 blocks loss of mitochondrial membrane
potential while ICE inhibitors act at a different step
during inhibition of death induced by respiratory chain
inhibitors. Oncogene. 1996;13:21–29.[Medline]
[Order article via Infotrieve]
19.
Russell SW, Rosenau W, Lee JC. Cytolysis induced by
human lymphotoxin. Am J Pathol. 1972;69:103–111.[Medline]
[Order article via Infotrieve]
20.
Perlman H, Maillard L, Krasinski K, Walsh K. Evidence
for the rapid onset of apoptosis in medial smooth muscle cells
after balloon injury. Circulation. 1997;95:981–987.
© 1998 American Heart Association, Inc.
Basic Science Reports
Attenuation of Ischemia/Reperfusion Injury in Rats by a Caspase Inhibitor
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Z-Val-Ala-Asp(OMe)-CH2F
(ZVAD-fmk), a tripeptide inhibitor of the caspase
interleukin-1ß–converting enzyme family of cysteine proteases, may
reduce myocardial reperfusion injury in vivo by attenuating
cardiomyocyte apoptosis within the ischemic
area at risk.
Key Words: myocardium • reperfusion • apoptosis • caspase
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Attempts to reduce
the extent of myocardial reperfusion injury have included lowering the
risk posed by certain injurious factors and potentiating various
aspects of cardioprotection relating to ischemic
duration,1 oxygen free
radicals,2 3 proinflammatory
cytokines,4 5 and
preconditioning.6 7 8 9 10 It has been reported that
apoptosis is a significant contributor to myocardial cell death
as a result of reperfusion injury.1 Therefore, it
might be hypothesized that this type of injury could be attenuated if a
portion of the injured myocardial cells could be rescued from an
apoptotic death.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
This study was carried out under the supervision of the Animal
Research Committee in accordance with the Guideline on Animal
Experiments of Fukushima Medical College and Japanese Government Animal
Protection and Management Law (No. 105).
Twenty-six of 36 adult male (290 to 310 g body weight)
Sprague-Dawley rats were anesthetized through
intraperitoneal administration of 30 mg/kg sodium
pentobarbital. Under artificial ventilation with a rodent ventilator, a
left thoracotomy was performed. The proximal portion of the left
coronary artery was surgically occluded for 30 minutes through
ligation with a suture (size 6.0) followed by coronary
reperfusion through release of the tie. Coronary occlusion was
confirmed through elevation of the ST segment on the ECG obtained from
a limb lead. Transient ventricular arrhythmias were
evoked in all rats 
5 minutes after coronary occlusion, but
these usually disappeared after 10 minutes of occlusion. After
coronary reperfusion, the tie was left loose on the surface of
the heart, the chest was closed, and the intratracheal tube and ECG
electrodes were removed. The rats were returned to their cages, where
they awakened, and they were allowed free access to food and water
until they were killed 24 hours later.
Twenty-four hours after coronary reperfusion, rats were
anesthetized again through intraperitoneal
administration of 30 mg/kg sodium pentobarbital. ECG readings were
monitored, and a polyethylene tube (PE 50; Becton-Dickinson) was
inserted into the left ventricular cavity via the right
carotid artery. LVSP, LVEDP, and (±)-LV dP/dt were measured using a
polygraph system (AP601G; Nihon Koden).
After hemodynamics were assessed at 24 hours of
coronary reperfusion, 0.5 mL of blood was obtained from the
catheter for measurement of blood cells. Then, an intratracheal tube
was inserted, and the chest was reopened under artificial ventilation.
The coronary artery was again briefly occluded through ligation
of the tie that remained at the site of the previous occlusion.
Immediately after the ligation, 1% Evans blue solution was infused
through the catheter into the beating left ventricular
cavity to delineate the ischemic area at risk (underperfused
and then reperfused area) of the left ventricle. After administration
of an excessive dose of sodium pentobarbital into the left
ventricular cavity, the heart was excised and
cross-sectioned from the apex to the atrioventricular
groove into five specimens of 2 mm in thickness with the use of
a stereoscope. Because there may be some anatomic differences in the
left coronary artery of each rat, the three middle slices were
prepared for morphometry to determine the ischemic area at
risk. These slices were incubated with a 4%
TTC13 solution for 30 minutes at 37°C in a dark
room. Then, ischemic but viable (TTC-stained) and infarcted
(TTC-unstained) zones within the underperfused and then reperfused area
(Evans blue–unstained) and the nonischemic area (Evans
blue–stained) were stereoscopically measured using the point-counting
method of Weibel14 with an eyepiece equipped with
a 25-square grid (Integration No. 1; Zweiss) under 100x magnification,
and I/R was calculated. These slices were then fixed in 10%
neutral-buffered formalin. Using paraffin sections that were 4
µm thick, TUNEL was performed as described
previously15 with minor modifications. Briefly,
nuclei of tissue sections were stripped of proteins through incubation
with 20 µg/mL proteinase K (Sigma Chemical) for 15 minutes at room
temperature. The slides were incubated with 2%
H2O2 for 5 minutes to allow
inactivation of endogenous peroxidase and then incubated
for 60 minutes at 37°C with 0.3 EU/µL TdT (Takara Schuzo Co) and
0.04 nmol/µL biotinylated dUTP (Boehringer-Mannheim
Biochemica) in TdT buffer containing 30 mmol/L Tris-HCl, pH 7.2,
140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride.
The reaction was terminated with buffer containing 300 mmol/L NaCl
and 30 mmol/L sodium citrate. The slides were coated with
avidin-conjugated peroxidase (Medical and Biological Laboratories)
diluted 1:3000 in PBS and visualized with the use of chromogen
3,3'-diaminobenzidine (Dojindo) and
H2O2. Counterstaining was
performed with 2% methyl green. Using this method, each
cardiomyocyte could be defined, and TdT-positive or
-negative nuclei were stained dark brown or light green, respectively,
under light microscopy. When the TUNEL method was performed, positive
controls were always included. For DNase treatment in
situ,15 sections were processed with proteinase
K, and peroxidase inactivation was carried out as described above.
Next, the sections were pretreated with DN buffer (30 mmol/L
Tris-HCl, pH 7.2, 140 mmol/L K cacodylate, 4 mmol/L
MgCl2, and 0.1 mmol/L dithiothreitol); then,
DNase I (Sigma) at 100 ng/mL was dissolved in this buffer and used to
cover each section. After a 15-minute incubation at room temperature,
the slides were washed extensively with double-distilled water, and DNA
nick end labeling was carried out. 
). We also divided the ischemic
area at risk into five radial segments, and then these five radial
segments were rearranged as (Fig 1) a right lateral border segment
adjacent to the interventricular septum; a total of three
central segments; and a left lateral border segment adjacent to the
left ventricular posterior wall. For each of the segments
or layers, I/R and TUNEL-positive cardiomyocytes were
calculated, as well as for the entire ischemic area at
risk.
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Figure 1. Myocardial segments and layers for assessment of
distribution of the infarcted area and TUNEL-positive
cardiomyocytes. The three middle slices of the five left
ventricular slices of each heart were analyzed for
measurements of the infarcted area and TUNEL-positive
cardiomyocytes. The entire ischemic
area at risk was first divided into five radial segments and then
classified into three segments (right lateral border, central three
radial segments, and left lateral border segment). The
ischemic area at risk (R) was also divided into
three myocardial layers (endocardial, middle, and epicardial layers).
The measurements of I/R and TUNEL-positive cardiomyocytes
were done in the entire ischemic area at risk, as well as in
each ischemic portion.
Rats subjected to the same occlusion and reperfusion protocol as
groups 1 and 2, respectively (n=3 each group), had their hearts excised
at 24 hours after reperfusion, and underperfused myocardium
was delineated using Evans blue. The excised heart was sliced
immediately as described above. Because TUNEL-positive
cardiomyocytes were found mainly in the lateral border
zones and the endocardial side of the ischemic area at risk (as
noted in "Results"), we isolated fresh myocardial specimens from
these zones and from the core of infarcted zones (mainly corresponding
to the zone of the central segment, including middle and epicardial
layers in Fig 1
) for DNA extraction. Each myocardial specimen weighed
0.2 mg and was minced in homogenization buffer
(10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 10 mmol/L
EDTA, pH 8.0) at 0°C and homogenized for 15 seconds at
10 000 rpm using a Polytron homogenizer (Kinematica
AG). The homogenate was then treated with 100 µg/mL
proteinase K and 0.1% SDS for 90 minutes at 50°C. The DNA was
extracted with phenol and chloroform followed by ethanol precipitation.
The pellet was resuspended in TE buffer (10 mmol/L Tris-HCl, pH
8.0, and 1 mmol/L EDTA) and treated with DNase-free RNase
(Boehringer-Mannheim) for 2 hours at 37°C. The concentration
of DNA was measured through spectrophotometry, and 10 µg of each DNA
sample was then electrophoretically fractionated on a 1.5% agarose gel
containing ethidium bromide at a concentration of 0.4 µg/mL. DNA was
visualized with a UV (302 nm) transilluminator, and the gel was
photographed with the use of a Polaroid camera.
Data are expressed as mean±SEM. To compare group 1 (control
ischemia/reperfusion) with group 2 or to compare the two groups
of sham-operated rats, an unpaired t test was performed. For
comparisons between the positive anti-PMN control and the other groups,
one-way ANOVA followed by Fisher's posthoc comparison was carried out.
For comparisons in I/R and TUNEL-positive cardiomyocytes
among different myocardial portions, two-way ANOVA followed by
Fisher's posthoc comparison was carried out. A value of
P<.05 was considered statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Hemograms
White blood cell counts just before death revealed no difference
between group 1 (9502±351/µL) and group 2 (9350±435/µL). In
anti-PMN–treated rats, white blood cell counts were 956±132/µL
(P<.0001 versus group 1 and group 2) before
coronary occlusion and 1081±156/µL (P<.0001
versus group 1 and group 2) just before death. In this positive control
group, lymphocytes made up most of the white blood cells (>99%).
The ischemic area at risk was 53.0±2.5% (NS versus group
1 and group 2), and the I/R was 51.0±1.7% (P<.05 versus
group 1, NS versus group 2).
Although the LVSP did not differ between groups 1 and 2, the LVEDP
of group 2 was lower (P=.04) than that of group 1 (Table).
The positive LV dP/dt of group 2 was greater (P=.02) than
that of group 1, but the heart rates of the two groups did not
differ.
View this table:
[in a new window]
Table 1. Hemodynamics Before Death
The ischemic areas at risk of groups 1 and 2
were similar (53.9±2.9% in group 1 and 55.4±3.0% in group 2, NS).
In the entire ischemic area at risk, the I/R of group 2
(52.4±4.0%) was significantly (P=.02) smaller than that of
group 1 (66.6±3.7%) (Fig 2, left). The
I/Rs of left and right lateral border segments or endocardial and
epicardial layers were smaller (P<.05, <.05, or
P<.05, <.01, respectively) than that of the central
segment or that of the middle layer in group 1 (Fig 3). In group 2, the I/Rs of all of three
myocardial segments and all of three layers were smaller
(P<.05, each) than those of group 1.
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Figure 2. Infarct size and TUNEL-positive
cardiomyocytes in the entire underperfused and then
reperfused area. Left, I/R. Right, TUNEL-positive
cardiomyocytes in the ischemic area at risk. The
column representing the infarcted area was lower
(P=.02) for group 2 than for group 1. The counts of
TUNEL-positive cardiomyocytes in group 2 were lower
(P=.0002) than in group 1. Group 1, infarcted rats (n=8)
administered vehicle; group 2, infarcted rats (n=8) treated with
ZVAD-fmk at a total dose of 3.3 mg/kg.
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Figure 3. The I/R in myocardial segments or myocardial
layers. In group 1, the I/R was smaller in left and right lateral
border segments (P<.05, respectively) than the central
segment (left). Furthermore, in this group, the I/R was smaller in
endocardial and epicardial layers (P<.05, <.01,
respectively) than the middle layer (right). In group 2, the I/R of
three myocardial segments (left) and of three myocardial layers (right)
was smaller than that of the corresponding segments or layers of group
1 (P<.05, respectively). , right). In group 1, TUNEL-positive cardiomyocytes were
greater in left and right lateral segments (P<.05, <.01,
respectively) than in the central segment and greater in the
endocardial layer (P<.01) but smaller in the epicardial
layer (P<.01) than in the middle layer (Fig 4). In group 2, TUNEL-positive
cardiomyocytes of all of three segments (P<.01,
each) and of endocardial, middle, and epicardial layers
(P<.01, <.01, <.05, respectively) were smaller than those
of group 1 (Figs 4 and 5). Therefore,
there were no significant differences of TUNEL-positive
cardiomyocytes in group 2 among the three myocardial
segments or three myocardial layers (Fig 4).
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Figure 4. The TUNEL-positive cardiomyocytes in
myocardial segments or myocardial layers within the ischemic
area at risk. In group 1, TUNEL-positive cardiomyocytes of
left and right lateral border segments (left) were greater than that of
the central segment (P<.05, <.01, respectively). In
this group, TUNEL-positive cardiomyocytes of the
endocardial layer or those of the epicardial layer were greater
(P<.01) or smaller (P<.01) than those
of the middle layer, respectively (right). In group 2, TUNEL-positive
cardiomyocytes were smaller than those of group 1 in all of
three myocardial segments and three myocardial layers
(P<.05 or <.01).
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Figure 5. Light microscopic findings on the
distribution of TUNEL-positive cardiomyocytes at 24 hours
after reperfusion. A, TUNEL-positive cardiomyocytes
(arrows) in the endocardial layer of ischemic area at risk on
the section from group 1 at a high magnification. B, Possibly necrotic
cardiomyocytes (top to middle) and a TUNEL-positive
cardiomyocyte (bottom) in the middle layer on the section
from group 2. TUNEL-positive cardiomyocytes were frequently
detected in the endocardial layer in group 1 (control
ischemia/reperfusion) (A). In this group, TUNEL-positive
cardiomyocytes were widely spread close to endocardium. In
contrast, only a few TUNEL-positive cardiomyocytes were
detected mainly in the endocardial side close to the core of infarction
in group 2 (ischemia/reperfusion with administration of
ZVAD-fmk). The core of infarction consisted of possibly necrotic
cardiomyocytes with appearance of disappeared nuclei and
degenerated cytoplasm (B). TUNEL-positive cardiomyocytes
did not coexist with the mass of these degenerated
cardiomyocytes but were present in the surrounding area
within central segments or middle layers.
DNA laddering indicative of fragmented DNA was clearly
demonstrated in myocardial specimens sampled from the lateral border
zones and the endocardial side of the ischemic area at
risk in group 1 (lane 4) but was attenuated in group 2 (lane 3),
as shown in Fig 6. DNA laddering in the
core of infarction was attenuated in group 1 (lane 2) and was absent in
group 2 (lane 1).
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Figure 6. Agarose gel electrophoresis. S indicates
size marker (bp). Lane 1, sampled from the core of infarction in
myocardium with the same experimental protocol as group 2;
lane 2, sampled from the core of infarction in myocardium
with the same experimental protocol as group 1; lane 3, sampled from
endocardial and lateral border zones of the ischemic area at
risk in myocardium with the same experimental protocol as
group 2; and lane 4, sampled from endocardial and lateral border zones
of the ischemic area at risk in myocardium with the
same experimental protocol as group 1. DNA laddering was well defined
in the sample from peripheral zone of the ischemic
area at risk in control ischemia/reperfusion (lane 4), but DNA
ladder formation was attenuated in peripheral zone of the
ischemic area at risk in the ZVAD-fmk–treated
ischemia/reperfusion (lane 3) or in the core of infarction in
control ischemia/reperfusion (lane 2). DNA ladder was not
detected in the core of infarction in ZVAD-fmk–treated
ischemia/reperfusion (lane 1).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study revealed that administration of
ZVAD-fmk reduced both the size of the myocardial infarct, as assessed
through TTC staining, and the number of TUNEL-positive
cardiomyocytes, with significant
hemodynamic improvement in vivo in rats that underwent
the 30-minute coronary occlusion and 24-hour reperfusion
procedure. TUNEL-positive cardiomyocytes appeared to be
apoptotic in this study because well-defined (group 1) or
attenuated (group 2) DNA laddering on electrophoresis was
consistent with a higher or lower value of TUNEL-positive
cardiomyocytes, respectively, in the ischemic area
at risk of the two groups. In a preliminary study using frozen
sections, we confirmed that none of the TUNEL-positive
cardiomyocytes were stained with TTC. Therefore, a
reduction in their number appeared to contribute to a reduction in the
myocardial infarct size. These results suggested that ZVAD-fmk was
effective in reducing myocardial reperfusion injury, which could be at
least partially attributed to the attenuation of
cardiomyocyte apoptosis. 21% decrease in the I/R and 72% decrease in
TUNEL-positive cardiomyocytes, as ratios compared with the
control ischemia/reperfusion. However, the absolute value for
decrease in TTC unstained area (14%) appeared somewhat greater than
that of TUNEL-positive cardiomyocytes (8%) (Fig 2); we
must be careful to simply compare the absolute values of TUNEL-positive
cardiomyocytes with the I/R because the methodology for
quantification was not the same between TTC staining (histochemical
area measurement on myocardial slices) and the TUNEL method
(histological cell counting on paraffin sections).
Furthermore, we cannot exclude the possibility that ZVAD-fmk interferes
with myocardial necrotic process as well as the apoptotic
process.16 17 18 Tsujimoto and
colleagues17 18 recently revealed that ICE
inhibitors retarded necrotic cell death as well as
apoptotic cell death in their in vitro system of chemical
hypoxia. The authors speculated that there was possible
involvement of common mediators in apoptotic and necrotic
signal transductions, although their detailed mechanisms remain to be
determined. In the present study, we might have observed effects of
ZVAD-fmk on these possible but undetermined common mediators. However,
our examination in an in vivo system is not suited for approach to
signal transductions of these two forms of cell death. The third
possibility is the difference in time from initiation of cellular
change until elimination between apoptosis and other types of
cell death, both forming the infarction. Apoptotic cells are
eliminated through phagocytosis in a few minutes in an in vitro
condition19 and in a few hours in an in vivo
condition.20 In contrast, necrotic
cardiomyocytes are eliminated much slowly by infiltrating
inflammatory cells. Although a turnover of apoptotic
cardiomyocytes in vivo has not been clarified so far, it
may be speculated that the amount of TUNEL-positive cells quantified at
a death stage may not equal the total amount of apoptotic
cardiomyocytes that appear during a 24-hour reperfusion
period.
Selected Abbreviations and Acronyms
DMSO
=
dimethylsulfoxide
ICE
=
interleukin-1ß–converting enzyme
I/R
=
infarct size/ischemic area at risk
(±)-LV dP/dt
=
peak positive (+) and negative (-) first derivatives of left
ventricular pressure
LVEDP
=
left ventricular end-diastolic pressure
LVSP
=
left ventricular peak systolic pressure
PMN
=
polymorphonuclear leukocyte
TdT
=
terminal deoxynucleotidyl transferase
TTC
=
triphenyltetrazolium chloride
TUNEL
=
terminal deoxynucleotidyl transferase–mediated
dUTP-biotin nick end labeling
ZVAD-fmk
=
Z-Val-Ala-Asp(OMe)-CH2F
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Fliss H, Gattinger D. Apoptosis in
ischemic and reperfused rat myocardium. Circ
Res. 1996;79:949–956.
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|
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|
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|
|
|
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|
|
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|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 Q Z Feng, T D Li, L X Wei, X Qiao, J Yi, L Wang, and T S Yang Tempero-spatial dissociation between the expression of Fas and apoptosis after coronary occlusion Mol. Pathol., December 1, 2003; 56(6): 362 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
 |
|
 M. Singh and H. K. Saini Resident Cardiac Mast Cells and Ischemia-Reperfusion Injury Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 135 - 148. [Abstract] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
E. Hochhauser, S. Kivity, D. Offen, N. Maulik, H. Otani, Y. Barhum, H. Pannet, V. Shneyvays, A. Shainberg, V. Goldshtaub, et al. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2351 - H2359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 J. Narula and H. W. Strauss P.S. I Love You: Implications of Phosphatidyl Serine (PS) Reversal in Acute Ischemic Syndromes J. Nucl. Med., March 1, 2003; 44(3): 397 - 399. [Full Text] [PDF]
|
|
|
|
 |
|
 M. de Perrot, M. Liu, T. K. Waddell, and S. Keshavjee Ischemia-Reperfusion-induced Lung Injury Am. J. Respir. Crit. Care Med., February 15, 2003; 167(4): 490 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
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|
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 A. Iwata, J. M. Harlan, N. B. Vedder, and R. K. Winn The caspase inhibitor z-VAD is more effective than CD18 adhesion blockade in reducing muscle ischemia-reperfusion injury: implication for clinical trials Blood, August 28, 2002; 100(6): 2077 - 2080. [Abstract] [Full Text] [PDF]
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W. Chao, Y. Shen, L. Li, and A. Rosenzweig Importance of FADD Signaling in Serum Deprivation- and Hypoxia-induced Cardiomyocyte Apoptosis J. Biol. Chem., August 23, 2002; 277(35): 31639 - 31645. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
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M. Chen, D.-J. Won, S. Krajewski, and R. A. Gottlieb Calpain and Mitochondria in Ischemia/Reperfusion Injury J. Biol. Chem., August 2, 2002; 277(32): 29181 - 29186. [Abstract] [Full Text] [PDF]
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T. Date, A. J Belanger, S. Mochizuki, J. A Sullivan, L. X Liu, A. Scaria, S. H Cheng, R. J Gregory, and C. Jiang Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity Cardiovasc Res, August 1, 2002; 55(2): 309 - 319. [Abstract] [Full Text] [PDF]
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G. Yaniv, M. Shilkrut, R. Lotan, G. Berke, S. Larisch, and O. Binah Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes Cardiovasc Res, June 1, 2002; 54(3): 611 - 623. [Abstract] [Full Text] [PDF]
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 L. K. Chang and E. M. Johnson Jr. Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition J. Cell Biol., May 28, 2002; 157(5): 771 - 781. [Abstract] [Full Text] [PDF]
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M. Shimizu, K. Fukuo, S. Nagata, T. Suhara, M. Okuro, K. Fujii, Y. Higashino, M. Mogi, Y. Hatanaka, and T. Ogihara Increased plasma levels of the soluble form of fas ligand in patients with acute myocardial infarction and unstable angina pectoris J. Am. Coll. Cardiol., February 20, 2002; 39(4): 585 - 590. [Abstract] [Full Text] [PDF]
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C. GILL, R. MESTRIL, and A. SAMALI Losing heart: the role of apoptosis in heart disease--a novel therapeutic target? FASEB J, February 1, 2002; 16(2): 135 - 146. [Abstract] [Full Text] [PDF]
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G. Horstick, O. Berg, A. Heimann, O. Gotze, M. Loos, G. Hafner, B. Bierbach, S. Petersen, S. Bhakdi, H. Darius, et al. Application of C1-Esterase Inhibitor During Reperfusion of Ischemic Myocardium: Dose-Related Beneficial Versus Detrimental Effects Circulation, December 18, 2001; 104(25): 3125 - 3131. [Abstract] [Full Text] [PDF]
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H. Ruetten, C. Badorff, C. Ihling, A. M. Zeiher, and S. Dimmeler Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects J. Am. Coll. Cardiol., December 1, 2001; 38(7): 2063 - 2070. [Abstract] [Full Text] [PDF]
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 C. R Holleyman and D. F Larson Apoptosis in the ischemic reperfused myocardium Perfusion, December 1, 2001; 16(6): 491 - 502. [Abstract] [PDF]
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J. Grunenfelder, D. N. Miniati, S. Murata, V. Falk, E. G. Hoyt, M. Kown, M. L. Koransky, and R. C. Robbins Upregulation of Bcl-2 Through Caspase-3 Inhibition Ameliorates Ischemia/Reperfusion Injury in Rat Cardiac Allografts Circulation, September 18, 2001; 104 (2009): I-202 - I-206. [Abstract] [Full Text] [PDF]
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B. Stadler, J. Phillips, Y. Toyoda, M. Federman, S. Levitsky, and J. D. McCully Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia-reperfusion Ann. Thorac. Surg., August 1, 2001; 72(2): 555 - 563. [Abstract] [Full Text] [PDF]
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T. Matsui, J. Tao, F. del Monte, K.-H. Lee, L. Li, M. Picard, T. L. Force, T. F. Franke, R. J. Hajjar, and A. Rosenzweig Akt Activation Preserves Cardiac Function and Prevents Injury After Transient Cardiac Ischemia In Vivo Circulation, July 17, 2001; 104(3): 330 - 335. [Abstract] [Full Text] [PDF]
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 Z. Zhou, X. Sun, and Y. J. Kang Ethanol-Induced Apoptosis in Mouse Liver : Fas- and Cytochrome c-Mediated Caspase-3 Activation Pathway Am. J. Pathol., July 1, 2001; 159(1): 329 - 338. [Abstract] [Full Text] [PDF]
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F. W. Bowen, T. Hattori, N. Narula, I. S. Salgo, T. Plappert, M. G. St. John Sutton, and L. H. Edmunds Jr Reappearance of myocytes in ovine infarcts produced by six hours of complete ischemia followed by reperfusion Ann. Thorac. Surg., June 1, 2001; 71(6): 1845 - 1855. [Abstract] [Full Text] [PDF]
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H. Fauvel, P. Marchetti, C. Chopin, P. Formstecher, and R. Neviere Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1608 - H1614. [Abstract] [Full Text] [PDF]
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T. Yamamura, H. Otani, Y. Nakao, R. Hattori, M. Osako, and H. Imamura IGF-I differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1191 - H1200. [Abstract] [Full Text] [PDF]
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 G. D. Dispersyn and M. Borgers Apoptosis in the Heart: About Programmed Cell Death and Survival Physiology, February 1, 2001; 16(1): 41 - 47. [Abstract] [Full Text] [PDF]
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 K. Kuwano, R. Kunitake, T. Maeyama, N. Hagimoto, M. Kawasaki, T. Matsuba, M. Yoshimi, I. Inoshima, K. Yoshida, and N. Hara Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor Am J Physiol Lung Cell Mol Physiol, February 1, 2001; 280(2): L316 - L325. [Abstract] [Full Text] [PDF]
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S. Negoro, H. Oh, E. Tone, K. Kunisada, Y. Fujio, K. Walsh, T. Kishimoto, and K. Yamauchi-Takihara Glycoprotein 130 Regulates Cardiac Myocyte Survival in Doxorubicin-Induced Apoptosis Through Phosphatidylinositol 3-Kinase/Akt Phosphorylation and Bcl-xL/Caspase-3 Interaction Circulation, January 30, 2001; 103(4): 555 - 561. [Abstract] [Full Text] [PDF]
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R. NEVIÈRE, H. FAUVEL, C. CHOPIN, P. FORMSTECHER, and P. MARCHETTI Caspase Inhibition Prevents Cardiac Dysfunction and Heart Apoptosis in a Rat Model of Sepsis Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 218 - 225. [Abstract] [Full Text]
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 J R Thiagarajah, P Gourmelon, N M Griffiths, F Lebrun, R J Naftalin, and K C Pedley Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts Gut, November 1, 2000; 47(5): 675 - 684. [Abstract] [Full Text] [PDF]
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T. E. McDonald, M. N. Grinman, C. M. Carthy, and K. R. Walley Endotoxin infusion in rats induces apoptotic and survival pathways in hearts Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2053 - H2061. [Abstract] [Full Text] [PDF]
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