From the First Department of Medicine (N.Y., S.H., T.K., M.H.), the
Department of Pathophysiology (T.K.), and the Department of Biochemistry
(N.T.), Osaka University Medical School, Suita, Osaka, Japan.
Correspondence to Shiro Hoshida, MD, PhD, Cardiovascular Division, Osaka Rosai Hospital, 1179-3 Nagasone-cho, Sakai, Osaka 591, Japan.
Methods and Results—Hyperthermia was induced in
anesthetized rats by placement in a temperature-controlled
water bath. After the defined recovery interval(s) at room temperature,
ischemia was induced by occlusion of the left coronary
artery for 20 minutes, followed by reperfusion for 48 hours. The
exposure to hyperthermia led to a recovery interval– dependent,
biphasic reduction in the incidence of ventricular
fibrillation during ischemia and in the size of the myocardial
infarct as determined after 48 hours of reperfusion. The time course of
the late-phase (24- to 96-hour recovery interval) but not the
early-phase (0.5 hour) cardioprotection depended on the degree of
hyperthermia. The time course of the increase in myocardial manganese
superoxide dismutase (Mn-SOD) activity corresponded to that of the
cardioprotective effects, although an increase in the content of Mn-SOD
and of heat shock protein 72 corresponded only to the late-phase
effects. Administration of an antioxidant before hyperthermia abolished
the early- and late-phase cardioprotection and the increase in Mn-SOD
activity.
Conclusions—The activation of Mn-SOD mediated by free radical
production during hyperthermia is important in the acquisition
of early-phase and late-phase cardioprotection against
ischemia/reperfusion injury in rats.
Whole-body hyperthermia also induces tolerance of the heart to
ischemia/reperfusion injury 24 hours after
hyperthermia.2 10 Because free radicals are
produced by heat stress11 and transduce the heat
shock signal in mammalian cells,12 a redox
mechanism may be involved in the acquisition of late-phase tolerance
via the induction of rescue proteins, such as HSPs and
endogenous scavengers of reactive oxygen
species.13 14 However, heat stress induction of
early-phase tolerance of the heart to ischemia/reperfusion
injury and the role of free radicals in the acquisition of tolerance
have not yet been determined.
The purpose of this study was to determine the role of redox mechanisms
and the induction of rescue proteins in early-phase and late-phase
cardioprotection against ischemia/reperfusion injury by
whole-body hyperthermia. The relationship between the induction of
Mn-SOD or HSP72 by heat stress and the protective effects of heat
stress against incidence of ventricular fibrillation (VF)
during ischemia and extent of myocardial necrosis after
reperfusion were examined in a rat model of myocardial infarction.
To determine the involvement of reactive oxygen species during
hyperthermia in the acquisition of tolerance against
ischemia/reperfusion injury, the low-molecular-weight synthetic
antioxidant N-2-mercaptopropionyl glycine (MPG; 100 mg/kg
IP)16 was infused 10 minutes before whole-body
hyperthermia (42°C group) or normothermia. To facilitate the use of
MPG without causing acidosis, the highly acidic aqueous solution of MPG
was neutralized with NaOH to a pH of 7.3 to 7.4 before
administration.
Infarction Protocol
Arrhythmias were monitored by ECG. VF was defined according to
the criteria of the Lambeth Conventions.18 If VF
occurred during ischemia and did not resolve spontaneously
within 3 seconds, manual cardioversion was attempted by gentle
palpation of the nonischemic region of the heart. We excluded
from infarct-size analysis rats in which VF persisted for >6
seconds or in which cardioversion had to be performed >3 times.
Incidence of VF was evaluated as it occurred (yes/no).
Myocardial Tissue Sampling
Measurement of Activity and Content of Mn-SOD
Measurement of HSP72 Content
Materials
Statistics
Hemodynamic Data, Area at Risk, and Rectal
Temperature
The size of the area at risk expressed as a percentage of left
ventricular area did not differ significantly among the
groups (data not shown).
Incidence of VF
Size of Myocardial Infarct
Induction of Mn-SOD After Hyperthermia
Effect of MPG Treatment
MPG did not alter Mn-SOD activity at recovery intervals of 0.5 or 72
hours in sham-treated control rats (Figure 7
Induction of HSP72
The mechanism that underlies the cardioprotection observed at both the
early phase and the late phase after hyperthermia appeared to be
related to an increase in Mn-SOD activity. The time course of
cardioprotection coincided with that for the increase of Mn-SOD
activity after hyperthermia for various magnitudes of heat stress or
after treatment with an antioxidant. Brief, sublethal ischemic
or anoxic insults have been shown to increase Mn-SOD activity and to
induce cardioprotection or myocyte protection in a biphasic
manner.5 7 Other studies have also attempted to
measure Mn-SOD activity after sublethal ischemia and
reperfusion but have been less successful at establishing the
correlation of the level of Mn-SOD activity with the degree of
cardioprotection.23 24 Das et
al25 reported that mammalian hearts subjected to
heat shock increase expression of Mn-SOD mRNA. Heat shock also enhances
SOD activity in the pig heart.26 Mn-SOD is
directly associated with the protection of the myocyte against
hypoxia-reoxygenation
injury.6 19 However, the causal relation between
the hyperthermia-induced cardioprotection and the elevation of Mn-SOD
activity remains to be elucidated.
Whole-body hyperthermia significantly increased the late-phase levels
of HSP72. Treatment with a radical scavenger during hyperthermia
reduced the induction of HSP72. These data suggest that myocardial
HSP72 and Mn-SOD may be induced via a common pathway that involves the
production of free radicals during hyperthermia. However, HSP72
induction and hyperthermia-induced cardioprotection are not
correlated,14 15 although some reports have
demonstrated a positive correlation between heat-induced HSP72
expression and infarct size reduction.2 10 27 28
Data from in vitro experiments19 indicate that
Mn-SOD and HSP72 are induced in cardiac myocytes after hyperthermia,
but only the inhibition of Mn-SOD (by antisense
oligodeoxyribonucleotides) abolishes the late-phase
cardioprotective effect. Beckmann et al29 showed
that members of the HSP70 family bind transiently to nascent proteins
and act as intracellular chaperones, helping to stabilize these
proteins until they achieve their final conformation. Voos et
al30 suggested that the constitutively expressed
HSP70 in yeast cells may be an unfoldase that facilitates protein
transport through the membranes of the endoplasmic reticulum and
mitochondria. The induced HSP72 may have chaperon functions and help
refold partially denatured proteins after stress. Thus, induction of
HSP72 may promote the maturation of Mn-SOD. Overexpression of HSP70 in
rat hearts is reported to induce cardioprotection against
ischemia/reperfusion injury associated with increased levels of
myocardial Mn-SOD content.31 However, it remains
to be determined whether HSP72 and Mn-SOD are cooperative or
interacting factors in acquired ischemic tolerance.
Kingma et al14 reported that
endogenous catalase activity in myocardium is
increased after heat stress and could protect the heart against
ischemia-reperfusion injury. Although we did not examine
myocardial catalase activity in the present study, it is
conceivable that a heat shock–induced increase in
endogenous catalase activity in myocardial tissue could
facilitate or enhance the detoxification of the superoxide in
mitochondria and thus result in reduced tissue injury during the
process of ischemia-reperfusion.
Induction of Mn-SOD has been demonstrated in eukaryotes in conditions
that favor the production of free radicals such as
superoxide.32 The induction of Mn-SOD by tumor
necrosis factor-
We used the macroscopic TTC staining technique for determination of
necrotic tissue in this study, being fully aware of its documented
limitation. Previous studies in rabbit35 and
pig36 models have demonstrated that exogenous
treatment of hearts with Cu,Zn-SOD induced the artifactual SOD-mediated
preservation of the TTC reaction within 24 hours of reperfusion,
whereas that preservation disappeared 72 hours after
reperfusion.35 In our additional experiments,
however, there were no differences in the sizes of the infarcts between
48-hour-reperfusion and 72-hour-reperfusion models assessed by TTC
staining in the rats at 0.5 and 72 hours after 42°C whole-body
hyperthermia (data not shown). Moreover, the Mn-SOD activity in the
risk area of the myocardium decreased after reperfusion and
did not exceed the level of that in control rats without
ischemia-reperfusion (unpublished observations). Tanaka et
al37 reported that TTC macrochemistry provided a
reliable measurement of infarct size after 4 days or 4 hours of
reperfusion in a dog model, even if hearts were treated with SOD
exogenously. There may be differences in effects on measurement of
infarct size assessed by TTC staining between endogenous
and exogenous SOD, or between Cu,Zn-SOD and Mn-SOD.
Previous studies in rat and rabbit models2 10
indicate that heat stress at 42°C for 15 minutes induces maximal
cardioprotection 24 hours after whole-body hyperthermia. In the
present study, maximal late-phase protection was achieved 72 hours
after hyperthermia at 42°C for 15 minutes. The apparent discrepancy
may be explained by differences in the method used to induce
hyperthermia. Rats were placed in a temperature-controlled water bath,
rather than on a heating pad or blanket, to induce whole-body
hyperthermia in the present study.
Conclusions
Received November 14, 1997;
revision received April 24, 1998;
accepted May 4, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Whole-Body Hyperthermia Provides Biphasic Cardioprotection Against Ischemia/Reperfusion Injury in the Rat
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Hyperthermia
increases cardiac tolerance to ischemia/reperfusion injury 24
hours after the heat stress. Free radicals and redox mechanisms have
been implicated in such tolerance. However, the time course and its
relation to the induction of antioxidative enzymes in the protection
induced by whole-body hyperthermia against ischemia/reperfusion
injury are unknown.
Key Words: superoxide dismutase • proteins • free radicals • mercaptopropionyl glycine
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cardiac resistance to ischemia/reperfusion injury
is increased by exposure to such sublethal stress as a brief period of
ischemia or whole-body hyperthermia. Tolerance after exposure
to a brief period of ischemia (ischemic
preconditioning)1 is manifested in a biphasic
manner, both soon after and 24 hours after
preconditioning.2 3 Different mechanisms mediate
cardioprotection in the early phase and in the late phase. The de novo
synthesis of proteins is not involved in the early
phase,4 but the induction of
endogenous heat shock proteins
(HSPs)2 or antioxidative
enzymes5 6 7 such as manganese superoxide
dismutase (Mn-SOD) is important in the late phase. However, the
production of free radicals during ischemic
preconditioning is involved in the mechanism of tolerance in both the
early phase8 9 and the late
phase.7
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animals and Experimental Protocol
Male Wistar rats (300 to 350 g) were maintained on a
12-hour dark/light cycle, housed at 23±1.5°C (45±15% relative
humidity), and allowed access to water and rat chow ad libitum. After
the induction of light anesthesia with sodium pentobarbital
5 to 10 mg/kg IP, whole-body hyperthermia was induced by placing the
rats in a constant-temperature water bath as described
previously.15 During whole-body hyperthermia, the
animal was supported by a wire apparatus to prevent the
aspiration of water and to facilitate the measurement of rectal
temperature. Hyperthermia was maintained at 40.0±0.2°C for 5 minutes
(40°C group), 41±0.2°C for 10 minutes (41°C group), or
42±0.2°C for 15 minutes (42°C group) (Figure 1
). For hyperthermia at 42°C, 
5
minutes was required for the rat's core temperature to reach 42°C.
Rats in the sham-treated control group were placed in a water bath
maintained at 36.5±0.2°C for 20 minutes. Rats were allowed to
recover at room temperature for defined intervals (0.5, 3, 6, 12, 24,
36, 48, 60, 72, 84, 96, or 120 hours, n=10 to 12 each) before the
induction of myocardial infarction. Some rats received neither the
hyperthermic nor the normothermic water-bath treatment
(untreated controls).
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Figure 1. Experimental protocol. Anesthetized rats
were placed in water baths at indicated temperatures for indicated
intervals and allowed to recover for various intervals before
initiation of cardiac ischemia induced by ligation of left
coronary artery. Incidence of VF was determined during
ischemia. Animals were killed 48 hours after restoration of
cardiac perfusion. Antioxidant MPG 100 mg/kg IP was infused 10 minutes
before immersion in water bath.
At the end of the recovery interval, rats were
anesthetized with sodium pentobarbital 25 mg/kg IP, intubated,
and ventilated with a small-animal respirator (model SN-480-7-10,
Shinano Seisakusyo). The right femoral artery was cannulated with
polyethylene tubing for the continuous measurement of
arterial blood pressure with a pressure transducer
(TP-300T; Nihon Kohden). The heart rate, incidence of
arrhythmias, and ST-segment changes were monitored.
Hemodynamic variables were recorded
continuously (model WT-645G recorder; Nihon Kohden). The chest was
opened with a midline sternotomy. Silk thread (7-0) was passed
around the left coronary artery (LCA) 3 to 4 mm distal to
the LCA origin. After a 10-minute period of stabilization, measurement
of arterial pressure was initiated and the LCA was ligated.
After 20 minutes of coronary occlusion, the snare was released.
Reperfusion was indicated by a change in the color of the
ventricular surface. The silk thread around the
coronary artery was left in place. The surgical wounds were
repaired 60 minutes after reperfusion, and the rats were returned to
individual cages to recover. Aseptic surgical techniques were used
throughout. Benzylpenicillin 30 000 U/kg IM was injected as
prophylaxis against infection. Rats were reanesthetized with
sodium pentobarbital 25 mg/kg IP 48 hours after surgery and were
intubated and ventilated with a respirator. After the heart was exposed
and the LCA was reoccluded, Evans blue dye (2%) was injected via the
right femoral vein to estimate the area perfused by the occluded artery
(ischemic region). Rats were killed by an overdose of sodium
pentobarbital. The left ventricle was then cut into 6 pieces
perpendicular to the apex-base axis. These specimens were incubated
with 1% triphenyltetrazolium chloride
(TTC) at 37°C to stain the noninfarcted region. The ischemic,
infarcted, and nonischemic areas of tissue were separated with
scissors and weighed.15 17 The area at risk and
the size of the infarct were defined as the ratio of the mass of the
ischemic region to the left ventricular mass and
the ratio of the mass of the infarct region to the mass of the
ischemic region, respectively, expressed as a percentage.
To obtain tissue samples for measurement of enzyme content and
activity, rats were killed by an overdose of sodium pentobarbital. The
myocardial tissue was rinsed in PBS, and both atria and the right
ventricle were removed. Left ventricular myocardial samples
were rapidly frozen by immersion in liquid nitrogen and stored at
-80°C. To measure myocardial SOD activity and content, blood
remaining in the left and right coronary arteries was washed
out by retrograde infusion of PBS through the ascending aorta before
myocardial tissue was sampled.
Myocardial levels of Mn-SOD were determined in rats killed after
recovery intervals of 0.5, 3, 12, 24, 48, 72, 96, or 120 hours and in
control rats that did not receive water-bath treatment. SOD activity of
the myocardial samples was determined by the nitro blue tetrazolium
(NBT) method.5 6 Myocardium was
homogenized with 20 mmol/L PBS containing 1
mmol/L EDTA and centrifuged at 900g for 15 minutes.
The supernatant was sonicated and added to the reaction mixture of NBT
with xanthine–xanthine oxidase. SOD activity in the supernatant was
measured colorimetrically as inhibitory
activity against the formation of blue formazan by SOD in the reaction
mixture. To evaluate Mn-SOD activity, the assay was repeated in the
presence of potassium cyanide (1 mmol/L) to inhibit
copper,zinc–superoxide dismutase (Cu,Zn-SOD) activity. Cu,Zn-SOD
activity was determined by subtracting the activity of Mn-SOD from the
total SOD activity. Pentobarbital sodium had no effect on the SOD
activity of normal rat hearts in our preliminary study. Mn-SOD content
in rats of the 42°C group was measured by means of an ELISA as
reported previously.6 19 The activity and content
of Mn-SOD were expressed relative to the protein concentration in the
supernatant.
HSP72 content in heart tissue from sham-treated controls and
from rats exposed to whole-body hyperthermia at 42°C was evaluated
for recovery intervals of 0.5 and 72 hours. HSP72 content in the
supernatant of unsonicated heart tissue homogenate was
determined by means of Western blot
analysis.15 Protein samples were diluted
into a 1x Laemmli sample buffer solution. Equal total protein loads
(40 µg) were separated by SDS-PAGE on 1-mm-thick, 7.5%
polyacrylamide gels. The gels were run in duplicate, and 1 gel
was stained with Coomassie blue to determine equivalence of loading and
adequacy of sample preparation. The proteins were transferred onto a
nitrocellulose membrane by Western blotting. Membranes were washed in
PBS with 0.2% BSA to block nonspecific binding sites. The blocked
membranes were incubated at 4°C overnight in PBS containing mouse
monoclonal IgG cross-reactive to the inducible 72-kD heat-shock protein
(Stressgen) at 1:500 dilution and then incubated in PBS containing
horseradish peroxidase–conjugated rabbit anti-mouse IgG (Kirkegaard &
Perry Laboratories) at 1:5000 dilution at room temperature for 2
hours.
Chemicals were purchased from Sigma Immunochemicals and Wako
Fine Chemicals.
Data are expressed as mean±SEM. The significance of the
differences between shams and each hyperthermic group was assessed by
1-way ANOVA and the Fisher protected least significant difference post
hoc test. The significance of differences in the incidence of VF was
determined by 
2 testing with Yates'
correction. A value of P<0.05 was considered
statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Exclusion Due to VF and Death
A total of 24 rats that developed serious VF during occlusion (5
in the sham-treated control group, 4 in the 40°C group, 4 in the
41°C group, 8 in the 42°C group, 2 in the 42°C group pretreated
with MPG, and 1 in the sham-treated control group pretreated with MPG)
were excluded from the evaluation of myocardial infarct size. Eleven
rats died prematurely during the 48-hour reperfusion period (2 in the
sham-treated control group, 2 in the 40°C group, 3 in the 41°C
group, 2 in the 42°C group, 1 in the 42°C group pretreated with
MPG, and 1 in the sham-treated control group pretreated with MPG).
No significant differences were observed in the rate-pressure
product or in the rectal temperature during the infarct protocol
among the groups before ischemia, at the end of the
ischemic period, or 0.5 hour after reperfusion (data not
shown).
The sham-treated control animals that were placed in a water bath
at 36.5°C exhibited no significant differences in the incidence of VF
(yes/no) for recovery intervals of 0.5 to 72 hours. Prior exposure to
whole-body hyperthermia resulted in a time-dependent biphasic tolerance
against VF during myocardial ischemia (Figure 2). Hyperthermia at 40°C for 5 minutes
(Figure 2a) resulted in tolerance against VF (incidence of VF reduced
to 29% of control) in rats allowed to recover for 0.5 hour after
hyperthermia. Tolerance to VF decreased with increasing recovery
interval such that the incidence of VF reached the level for
sham-treated control animals without hyperthermia at a recovery
interval of 6 hours. However, tolerance to VF reappeared after 12 hours
of recovery. A 78% reduction in the incidence of VF was obtained 24
hours after hyperthermia. For recovery intervals >48 hours, the
incidence of VF in the treated group was not different from control. A
biphasic pattern of tolerance to VF during ischemia was also
observed in animals treated at 41°C and 42°C, but the time course
differed slightly (Figure 2b and 2c).
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Figure 2. Effects of hyperthermic treatment and recovery
interval on incidence of VF during ischemia in rats.
Arrhythmias were monitored by ECG during ischemia in
rats exposed to treatments as described in Figure 1 
. Open columns,
sham-treated control rats; solid columns, hyperthermia-treated rats.
Incidence of VF (yes/no) among sham-treated control groups was not
significantly different from incidence in untreated control rats (C).
Number of rats used is indicated in each column. Controls and shams are
shown in each panel for comparison. Incidence of VF was not evaluated
>60 hours after hyperthermia in 40°C group and >84 hours after
hyperthermia in 41°C group. *P<0.05 vs sham-treated
control rats by 2 analysis with Yates'
correction.
There were no significant differences in the size of the
myocardial infarct for recovery intervals of 0.5 to 72 hours among the
sham-treated control groups. The induction of whole-body hyperthermia
reduced the size of the ischemia/reperfusion–induced
myocardial infarct in a time-dependent, biphasic manner (Figure 3). Protection against ischemic
damage was evident in rats allowed a 0.5-hour recovery interval but was
no longer evident by 6 hours in all 3 groups. As the recovery interval
increased beyond 6 hours, protection from damage was again observed and
followed a time course similar to that for tolerance to VF (Figure 3).
Although not shown, the size of the infarct assessed by TTC staining
did not differ significantly from that assessed by histology in
additional rats with 48-hour reperfusion.
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Figure 3. Effects of hyperthermic treatment and recovery
interval on size of myocardial infarct in rats. Infarct size was
calculated as ratio of mass of infarct region to mass of
ischemic region in hearts from rats exposed to treatments as
described in Figure 1 . Open columns, sham-treated control rats; solid
columns, hyperthermia-treated rats. Infarct size among sham-treated
control groups was not significantly different from infarct size in
untreated control rats (c, 50±2%). Data are mean±SEM. Number of rats
is indicated in each column. Controls and shams are shown in each panel
for comparison. Infarct size was not evaluated >60 hours after
hyperthermia in 40°C group and >84 hours after hyperthermia in
41°C group. *P<0.05 vs sham-treated control rats by
ANOVA and Fisher protected least significant difference test.
In the myocardia from sham-treated controls, both Mn-SOD activity
(data not shown) and Mn-SOD content remained constant during the time
course of the experiment. The biphasic time course of changes in Mn-SOD
activity in hyperthermia-treated myocardium (Figure 4) was complementary to the time course
for protection against VF (Figure 2) and protection against
ischemic damage (Figure 3) for each group. Mn-SOD activity
increased at the 0.5-hour recovery interval but was not different from
control for recovery intervals of 3, 12, or 120 hours. Mn-SOD content
in rats of the 42°C hyperthermia group (Figure 5) did not change relative to control for
recovery intervals <24 hours. Thereafter, Mn-SOD content increased
significantly and reached 145% of sham-treated control values 72 hours
after hyperthermia at 42°C. The activity of the cytosolic isoform of
SOD (Cu,Zn-SOD) did not change after hyperthermia (data not shown).
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Figure 4. Effects of hyperthermia and recovery interval on
Mn-SOD activity in rat myocardium. Activity of Mn-SOD was
determined by NBT method in cardiac tissue homogenates from
rats exposed to treatments as described in Figure 1 . Untreated control
indicates value (identical in each panel) from rats receiving neither
hyperthermic nor normothermic water-bath treatment. There
was no significant change in values from sham-treated controls at any
point during recovery (data not shown), and values were not
significantly different from untreated control value. Each data point
represents mean±SEM of values from 
4 rats.
*P<0.05 vs control by ANOVA and the Fisher protected
least significant difference test.
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Figure 5. Effects of hyperthermia and recovery interval on
Mn-SOD content in rat myocardium. Mn-SOD content was
determined by ELISA in cardiac tissue homogenates from rats
exposed to treatments as described in Figure 1 . Untreated control
indicates value from rats that received neither hyperthermic nor
normothermic water-bath treatment. 
, Sham-treated
control group; 
, hyperthermia (42°C)–treated group. Each data
point represents mean±SEM of values from 4 rats.
*P<0.05 vs corresponding sham-treated control by ANOVA
and Fisher protected least significant difference test.
Because the half-time for elimination of MPG is 7 minutes in
vivo,16 MPG was considered to be effective as an
antioxidant only during hyperthermia. The infusion of MPG did not alter
the incidence of VF or the size of the myocardial infarct in
sham-treated control rats (Figure 6).
However, MPG completely abolished the protection against
ischemic damage and the occurrence of VF at both 0.5 and 72
hours after hyperthermia at 42°C.
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Figure 6. Effects of MPG on incidence of VF during
ischemia and on infarct size in rats. Arrhythmias were
monitored by ECG during ischemia, and infarct size was
calculated as ratio of mass of infarct region to mass of
ischemic region after reperfusion in rats exposed to treatments
as described in Figure 1 . Sham indicates sham-treated controls; MPG,
sham-treated controls receiving MPG 100 mg/kg IP; 42°C, rats from
42°C group; and MPG+42, rats receiving MPG before hyperthermic
treatment at 42°C. Number of rats used is indicated in each column.
*P<0.05 vs sham-treated controls by 2
analysis with Yates' correction (incidence of VF) and by ANOVA
(infarct size).). However, MPG completely abolished
both the early peak of Mn-SOD activity (0.5 hour after hyperthermia)
and the late-phase peak of Mn-SOD activity (72 hours after
hyperthermia) in the 42°C group (Figure 7). Moreover, MPG completely
abolished the increase of Mn-SOD content 72 hours after hyperthermia in
the 42°C group (data not shown).
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Figure 7. Effects of MPG and hyperthermic treatment on
Mn-SOD activity in rat myocardium. Activity of Mn-SOD was
determined by NBT method in cardiac tissue homogenates from
rats exposed to treatments as described in Figure 1 . Groups are defined
as in Figure 6. Data are mean±SEM values from at least 6 rats.
*P<0.05 vs sham-treated controls by ANOVA and Fisher
protected least significant difference test.
In sham-treated control rats, HSP72 levels did not increase after
recovery intervals of either 0.5 or 72 hours after
normothermic treatment (data not shown).
Representative results regarding HSP72 induction are
shown in Figure 8. HSP72 content was not
different from control 0.5 hour after 42°C hyperthermia. However,
HSP72 content was markedly increased at 72 hours after hyperthermia.
MPG did not alter the HSP72 levels after normothermic
treatment. However, MPG abolished the increased levels of HSP72
observed in myocardium at 72 hours after hyperthermia.
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Figure 8. Effect of MPG and hyperthermia on expression of
HSP72 in rat myocardium. HSP72 content was determined by
Western blot analysis of supernatants from unsonicated cardiac
tissue homogenates from rats exposed to treatments
described in Figure 1 . Representative results obtained
from indicated cardiac tissue are shown. Similar results were obtained
in experiments performed in at least 3 rats. Untreated control
indicates rats that received neither hyperthermic nor
normothermic water-bath treatment; MPG, sham-treated
controls receiving MPG 100 mg/kg IP before normothermic
treatment; 42°C, rats from 42°C group; and MPG+42, rats receiving
MPG before hyperthermic treatment at 42°C.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In ischemic preconditioning, cardioprotection at both the
early phase8 9 and the late
phase7 20 is mediated by the oxygen free radicals
that are produced during preconditioning. In particular, Baines et
al9 clearly demonstrated the role of free
radicals in classic preconditioning, albeit generated by
ischemia or the addition of hypoxanthine and xanthine oxidase
to the perfusate. The generation of superoxide by the
mitochondria of rat muscle increases in a temperature-dependent manner
from 37°C (muscle temperature at rest) to 45°C (muscle temperature
after exercise).11 Pretreatment with a radical
scavenger, MPG, abolished both the early-phase and late-phase
beneficial effects of hyperthermia on ischemia/reperfusion
injury, indicating that the oxygen free radicals produced during
hyperthermia were cardioprotective, not cardiotoxic. Numerous studies
have shown that small changes in thiol redox potential can exert
signaling functions that can be blocked by high levels of thiol
antioxidants. The transcription factors activating protein-1 and
nuclear factor (NF)-
B and the activity of several kinases and
phosphatases are subject to redox
regulation.21 22 The alteration of a redox state
in mammalian cells is also important in the heat shock signal
transduction.12 Previous studies have shown that
transient thiol oxidation results in activation of NF-B by tumor
necrosis factor-
and that N-acetylcysteine, a glutathione
precursor and antioxidant, blocks both the decrease in glutathione and
the activation of NF-B.22 In the present
study, MPG abolished the increase in levels of HSP72 observed in
myocardium at 72 hours after hyperthermia. MPG also
abolished the early-phase and late-phase peaks of Mn-SOD activity and
the late-phase induction of Mn-SOD. These data indicate that the thiol
redox potential and the generation of oxygen free radicals are
important in signal transduction after heat stress. was shown to be mediated by oxygen free
radicals.33 Oberley et al32
reported that the local x-irradiation of mouse heart, which is known to
produce oxygen free radicals, causes a large biphasic increase in
Mn-SOD activity; this increase was dose- and time-dependent. In the
present study, the late-phase increase in Mn-SOD activity shifted
to a later period as the magnitude of the prior heat stress increased,
although the reason for this shift is unclear. Also unclear is the
mechanism responsible for early-phase increased Mn-SOD activity in the
absence of an increase in Mn-SOD content. Modulation of early-phase
Mn-SOD activity by antioxidants may indicate direct action of reactive
oxygen species on the enzyme. In eukaryotic cells, Mn-SOD
is preformed and stored as a precursor.34 It is
also possible that precursors of Mn-SOD that have antigenicity but lack
enzyme activity are modulated by heat stress.
Whole-body hyperthermia protected against VF during
ischemia and limited the extent of myocardial infarction after
reperfusion both immediately and 24 to 72 hours after heat stress. The
time course of the protective effect is similar to that for increases
in myocardial Mn-SOD activity, although Mn-SOD content was increased
only in conjunction with late-phase protection. Activation of Mn-SOD
appears to be mediated through a common pathway that involves free
radical production during hyperthermia. It remains to be
determined whether elevation of Mn-SOD activity is causally related to
the cardioprotection induced by heat stress against
ischemia-reperfusion injury. Further studies should also be
undertaken to clarify other mechanisms of cytoprotection evoked by
hyperthermic preconditioning.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
B and AP-1. Proc
Natl Acad Sci U S A. 1994;91:1672–1676.
B and
transcription of human immunodeficiency virus. Proc Natl Acad Sci
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