Extract
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adjusted to 7.4. The hearts were kept in a glass water-heated
jacket, which kept the surrounding air temperature at 37°C.
The basic perfusion pressure was 90
cmH2O, and the low-flow perfusion pressure was 15
cmH2O. Coronary flow was measured by using timed collections of the coronary
effluent with graded glass cylinders throughout the protocol,
and aliquots of perfusate were collected for lactate
dehydrogenase (LDH) analysis. Samples for the estimation of LDH
were collected in chilled tubes and stored at -20 °C until
assayed by colorimetry.
All animals were provided by the Experimental Animal
Center of Tongji College, Huazhong University of Science
and Technology, and received humane care in compliance
with the principles outlined in the Guide for the Care and Use
of Laboratory Animals.
Protocol After equilibrium was reached in the
20-min perfusions, hearts were divided into 8 groups as follows:
group I: hearts were perfused under a constant pressure of
90 cmH2O for 150 min; group II: hearts were switched to the
low-flow ischemia mode, and reperfused for 30 min after 120
min of low-flow ischemia; group III: hearts were switched to
low-flow ischemia and a dose of 1×10_6 mol/L dobutamine in 100 µL of
water[5] was injected into a side arm immediately
above the aortic cannula after 120 min of low-flow ischemia;
group IV (NS group): 0.9% sodium chloride (7 mL/kg, ip, 24 h
before experiment)-treated hearts were switched to the
low-flow ischemia mode and after 120 min of low-flow ischemia,
the apical part of each heart was cut off on ice and
immediately put into fixation solution, and the remaining part was
freeze-clamped and stored in liquid nitrogen for biochemical
analysis; group V (reserpine group): reserpinized hearts
(reserpine, 7 mg/kg, ip, 24 h before experiment; Guangdong
Banmin Pharmaceutical Co) were studied as in group IV; group
VI (NE group): hearts were studied as in group IV except for
the presence of norepinephrine L-bitartrate (75 ng/mL,
Sigma-Aldrich, St Louis, USA) in perfusion. Depletion of NE stores
in the hearts of reserpinized rats was measured by testing
the effect of tyramine (5 µg/mL, Sigma-Aldrich, St Louis,
USA) in the perfusion buffer on the heart rates of
spontaneously beating hearts (group
VII)[6]. The same measurements were carried out for the hearts of non-reserpinized control
animals (group VIII).
Myocardial ultrastructure
examination The apical part of each heart was cut down on ice and immediately put into
a 2.5% glutaraldehyde fixation solution at 4 °C. The
subendocardium was subdivided into tissue blocks of 1
mm3, which were again put into the fixation solution for 30-min
oscillation and fixation. Electron microscope slices were
produced according to routine methods for the preparation of
transmission electron microscope specimens. Ultrathin
sections were stained with uranyl acetate and lead citrate for
examination on a transmission electron microscope.
In situ terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling The left ventricle was
immersion-fixed in 10% neutral formalin and embedded in paraffin.
Serial sections of 3-µm thickness were produced. Apoptosis
was detected by using the terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL) technique
with an in situ cell death detection kit (AP, Roche, Germany)
according to the manufacturer¡¯s instructions. The numbers
of TUNEL-positive cardiomyocytes were counted by 2 blinded
observers using microscopes with an eyepiece grid (magnification, ×400) using the HPIAS-2 000 image
analysis system. Five different visual fields were counted in each
slice. The cardiomyocytes that had blue-stained nuclei were
TUNEL-positive cardiomyocytes. TUNEL-positive nuclei
that could not be definitively confirmed to have myocyte
origins were excluded. The extent of apoptosis was expressed
by normalizing the results to the number of myocyte nuclei
per visual field in each sample.
In situ immunohistochemistry for Bcl-2 and Bax expression Each specimen was cut into 5-mm sections. The
sections were deparaffinized with two 5-min changes of xylene
and two 3-min changes of 100% alcohol. The slides were
then washed in distilled water, and intrinsic peroxidase
activity was inhibited with 3% hydrogen peroxide. After being
rinsed, the slides were treated with 5% normal serum in 0.01 mol/L phosphate buffered saline for 10 min to block
nonspecific binding. The sections were then incubated at
room temperature overnight with monoclonal mouse
anti-human Bcl-2 oncoprotein or monoclonal mouse anti-human
Bax oncoprotein (Beijing Zhongshan Biotechnology Co) at
dilutions of 1:50 (for both). The sections were then rinsed
with phosphate buffered saline and incubated with
biotin-conjugated goat antimouse IgG (Beijing Zhongshan
Biotechnology Co) for 30 min. After being rinsed, the sections were
incubated for 25 min, then after further rinsing, they were
incubated for 30 min with a S-A/HRP work solution at 37 °C,
then stained with diaminobenzidine (DAB) solution. Each
section was photographed under a microscope with an eye
piece grid (magnification, ×400) using the HPIAS-2 000
image analysis system. Twenty randomly chosen visual fields
for each group were used for computer-based automatic
calculation of the mean absorbance of areas of immunopositive
expression.
Biochemical analysis Adenosine 5¡¯-triphosphate (ATP)
concentration in myocardium was determined by using a
bioluminescence method as described by Sperlagh and
Krisztina et al[7,8]. A luminometric method based on the
luciferin-luciferase reaction was used to quantify ATP in
myocardium by using an ATP assay kit (Beyotime
Bio-technology). The concentration of phosphocreatine in
myocardium was determined by using the reverse phase-high
performance liquid chromatography (RP-HPLC) methods
described by Zhang et al[9]. The phosphocreatine standard
was from Sigma-Aldrich. The concentration of glycogen in
myocardium was determined with a chemistry colorimetry
method by using a glycogen detection kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China).
Detection of LDH LDH leakage from each heart was
measured in the coronary effluent with a linked-enzyme
spectrophotometric assay by using a lactate dehydrogenase kit
(Zhongsheng Beikong Bio-technology and Science), as
described by Bergmeyer[10]. LDH leakage was expressed as
international units of LDH released per min per gram wet
heart.
Data reduction and statistical analyses The
pressure-rate product (PRP), which is the product of heart rate and left
ventricular developed pressure, was calculated and used to
assess the mechanical function, because it provides a method
for quantification of the performance of a heart (for
comparison with the same heart at a different time or with other
hearts).
All data were expressed as mean±SD. Student¡¯s paired
or unpaired t-test was used to assess the differences
between any 2 groups, and analysis of variance was used to
assess differences among groups using SPSS 12.0 for
Win-dows. P<0.05 was considered statistically significant.
Results
Recovery of cardiac function In group I, the hearts were
perfused under a constant pressure of 90
cmH2O, and coronary flow, PRP, and LDH leakage were 11.9 mL/min, 23 236
mmHg·beat-1·min-1 and 64.25
mU·min-1·g-1, respectively,. There
was no significant difference throughout the experiment. In
group II, with the onset of ischemia, coronary flow, PRP, and
LDH leakage promptly fell to 8.5%, 12.2%, and 23.7% of their
pre-ischemic values, respectively, and there was no further
significant decline over the next 2 h of low-flow ischemia.
However, coronary flow and PRP promptly increased to
control levels after reperfusion. In group III, hearts were strongly
responsive to dobutamine: a bolus of dobutamine
(1×10-6 mol/L) elicited a marked increase in systolic pressure (from
20.5 to 65.0
mmHg·beat-1·min-1,
P<0.01) but a non-significant elevation in end-diastolic pressure (from 6.3 to 6.7 mmHg·beat-1·min-1, P>0.05).
There was no significant difference between the PRP
values of the NS group and the reserpine group before ischemia,
at the beginning of ischemia or after 120 min of ischemia. In
the NE group, the PRP values were comparable to those of
the other 2 groups after 20-min equilibrium perfusion, but
the PRP values increased significantly after the addition of
NE to the perfusion solution. With the onset of ischemia,
the PRP values immediately fell to the same level as observed
for the other 2 groups, then decreased progressively to
1005.1
mmHg·beat-1·min-1, which was significantly
less than the comparable values in the other 2 groups
(P<0.05)
(Table 1).
Tyramine test After addition of tyramine 5 µg /mL to the
perfusion buffer, the heart rate of spontaneously beating
hearts from non-reserpinized control rats was significantly
enhanced from 308.2 to 409.8 beat/min (P<0.05), but the heart
rate of spontaneously beating hearts from reserpinized rats
was not significantly affected by tyramine (from 275.8 to 285.7 beat/min; P>0.05), which indicated that myocardial
NE was depleted in reserpinized rats.
Myocardial ultrastructure Cardiac muscle fibers in the
NS group and reserpine group were abundant, with regular
arrays of myofibrils closely arranged within the sarcomere.
The mitochondria were intact and had no swelling or
disruption. The amount of glycogen in the mitochondria
decreased, but cardiac muscle fibers and mitochondria from
the NE group were significantly different from those in the
other groups. The differences included a loss of myofibrillar
content, myofibrillar disarray, and mitochondrial swelling,
disruption and vacuolation. The glycogen content in the
mitochondria decreased significantly (Figure 1, Table 3).
Injury to myocardial cell membranes LDH leakage was
used to assess injury sustained by the myocardial cell
membrane. The rate of LDH leakage was 14.8±3.0, 13.8±3.8,
and 22.5±5.4
mU·min-1·g-1 after 120-min ischemia in the NS
group, reserpine group, and NE group, respectively. There
was no significant difference between the former 2 values,
but compared with the NS group, the rate of LDH leakage
was significantly higher in the NE group (P<0.05).
Analysis of myocyte apoptosis As assessed by TUNEL,
there was no significant difference in the number of apoptotic myocytes between the NS group and the reserpine group,
but the number of apoptotic myocytes was significantly
greater in the NE group than in the NS group. The dark
yellow immunoreactive Bcl-2 or Bax products were found in
the cytoplasm of myocytes at various levels in the 3 groups.
There was no significant difference between the NS group
and reserpine group with respect to the mean absorbance of
areas of Bcl-2 and Bax immunopositive expression, but the
mean absorbance of areas of Bcl-2 and Bax immunopositive
expression were significantly lower and higher, respectively,
in the NE group than those in the NS group (Table 2, Figures 2,3).
Changes in myocardial energy Compared with the NS
group, there was more ATP, phosphocreatine and glycogen
content in the reserpine group, but the difference was not
significant (P>0.05). These values were significantly lower
in the NE group compared to the other 2 groups
(P<0.05, Table 3).
Changes in coronary flow There was no significant
difference in coronary flow among the NS, reserpine,and NE
groups before ischemia, but after 120 min ischemia,
coronary flow was significantly lower in the NE group compared
with the other 2 groups (between which the difference was
not significant) (Table 4).
Discussion
Short-term myocardial hibernation model Myocardial
hibernation is a term that was first used by
Rahimtoola[1] to describe a postulated condition of chronic sustained
abnormal contraction because of chronic underperfusion in
patients with coronary heart disease, in whom revascularization
or an improved oxygen supply-demand relationship could
cause recovery of regional function. Initially, the concept of
myocardial hibernation was based on clinical observations
only, but this phenomenon has indeed been observed in
experimental studies in vitro and in
vivo. To describe this phenomenon, Ross
introduced the term
"perfusion-contraction
matching"[11]. With reference to sustained
perfusion-contraction matching, as demonstrated in experimental
studies over several hours, Ross defined "short-term
hibernation" and distinguished it from
"chronic hibernation", that is, a hypothetical condition of
chronic perfusion-contraction matching. The
existence and adaptive nature of
short-term hibernation has been accepted, but
the existence of perfusion-contraction matching over prolonged
periods of time (chronic hibernation) has been questioned, and chronic
hibernation has been suggested to be a result of repetitive
or cumulative myocardial
stunning[3,12,13].
In our perfusion pressure-controlled isolated rat heart
model, low-flow ischemia induced sustained hypo-contraction, that is, sustained perfusion-contraction match
ing during which there was no morphological or biological
evidence of myocardial injury: the PRP recovered to control
levels after reperfusion, and hearts retained their
responsiveness to dobutamine. These observations accord with
those for short-term hibernating
myocardium[5,14]. Our experiments were performed in isolated buffer-perfused rat
hearts, a model that allows the function of myocardial NE
during the development of short-term hibernation to be
studied, and the degree and composition of coronary flow,
heart rate, temperature, and myocardial function to be to
precisely controlled in the absence of systemic
neuroendocrine changes.
Role of myocardial NE in the development of
short-term hibernation The observed reduction in myocardial
contractile function during hibernation is not regarded as the
consequence of a sustained energetic deficit, but instead as a
regulatory event that serves to avoid an energetic deficit
and to maintain myocardial integrity and
viability[5,14]. In this active adaptation, activation of ATP-dependent
potassium channels and increases in the concentration of
interstitial adenosine are not involved in the development of
short-term myocardial hibernation. In a study by Heusch
et al it was suggested that a decrease in transient calcium and
calcium responsiveness might contribute to the development
of short-term hibernation[15]. Whereas the density of
myocardial b adrenoceptors has been shown to be reduced in
chronic animal models of myocardial
hibernation[17] or stunning, b adrenoceptor density and affinity was unaltered
in short-term hibernating myocardium after 90 min of
moderate ischemia[16], which is consistent with our present results.
In our short-term hibernation model in isolated rat heart, there
was no significant difference between the NS group and
reserpine group with respect to contraction function,
coronary flow, ATP content, phosphocreatine content, glycogen
content, myocardial ultrastructure, LDH leakage, apoptosis
of myocytes according to TUNEL, or the amount Bax/Bcl-2
products, suggesting that myocardial NE may not
contribute to the development of short-term hibernation, and that
there might not be significant myocardial NE release during
short-term hibernation, because significant NE release may
injure myocytes[6,17,18].
Effect of exogenous NE on short-term hibernation
Although short-term hibernation has been mimicked both
in vitro and in vivo, there are many significant differences
between them. Among these differences is the in
vitro absence of neural or hormonal factors that have an important
role in the regulation of cardiac performance and metabolic
processes. NE is the main transmitter released from
sympathetic nerve terminals and is an important component of
blood. During myocardial infarction, the concentration of
NE increases not only in the damaged myocardium but also
in the circulating system because of increased peripheral
releases from the pain, anxiety and reflex activation of the
sympathetic nerve system. The excessive activation of the
sympathetic nerve system is an important factor in
accelerating ventricular remodeling and delayed heart failure. NE
can increase cardiac performance, but it may also increase
oxygen cost and apoptosis or injury to myocytes, decrease
cardiac performance, injure the adaptation mechanism, and
destroy the development of hibernation. In the present
study, NE induced significant increases in cardiac
performance despite producing no significant change in coronary
flow during normal perfusion, but it induced significant
decreases both in cardiac performance and coronary flow after
120 min ischemia, suggesting that the concentration of NE
that induces increases in cardiac performance during normal
perfusion induces decreases in cardiac performance during
hibernation. ATP, phosphocreatine, and glycogen content
was significantly lower in the NE group than in the NS group,
suggesting that the balance of energy metabolism and
perfusion-contraction matching was destroyed by high
concentrations of NE.
Myocyte loss may occur in 2 distinct ways in the
myocardium: apoptosis and necrosis. In our experiments,
the proportion of apoptotic myocytes as observed by TUNEL
was significantly greater in the NE group relative to the NS
group, suggesting that a high concentration of NE might
induce apoptosis of myocytes during hibernation, which
could partially explain why NE induced the decrease in
cardiac performance during hibernation. The
bcl-2 proto-oncogene diagfamily is critical in the control of apoptosis. It
has been proposed that cell viability after an apoptotic
stimulus may depend on the ratio of Bcl-2 to
Bax[19]. The mean absorbance of Bcl-2 immunopositive expression was
significantly lower in the NE group than in the NS group, and the
mean absorbance of Bax products was significantly higher
in the NE group relative to the NS group, which indicates
that NE increases the expression of Bax and decreases the
expression of Bcl-2 during hibernation. The Bcl-2/Bax ratio
became smaller, suggesting that the Bcl-2/Bax ratio may have
an important role in the apoptosis of myocytes induced by
NE during hibernation. It has been reported that NE induced
the apoptosis of myocytes mainly by activating the
b1 recep-tor[20], and mitogen-activated protein kinase and the reactive
oxygen species mechanism may also have a
role[21,22]. In the present study, NE not only induced the apoptosis of
myocytes, but also induced injury to the myocardial cell
membrane and caused changes in the ultrastructure of the
myocardium, suggesting that NE may also induce necrosis
in myocardium during hibernation. Another important
reason why NE induces apoptosis and necrosis is that the
decrease in coronary flow resulted in a decrease in energy
supply to the myocardium, which might induce surviving
myocytes to undergo apoptosis or
necrosis[23].
The present study indicated that NE induced
progressive decreases in coronary flow and cardiac performance,
which might result from increases in apoptosis and necrosis,
suggesting that NE may be an important factor in the
deterioration of myocardial structure and function during
hibernation, and that anti-adrenergic treatment may be
helpful in the development and sustainment of short-term
hibernation. However myocardial NE may not contribute to
the development of short-term hibernation.
References
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