Induction of 72-kDa heat shock protein does not produce second window of ischemic preconditioning in rat heart

Division of Cardiology, Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

	ABSTRACT

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Ischemic preconditioning (PC) induces delayed phase of protection, known as the second window of protection (SWOP). We investigated this phenomenon in rat and correlated it with the expression of 72-kDa heat shock protein (HSP 72). Rats were preconditioned with 1, 2, and 3 cycles of 5-min left anterior descending artery occlusions, each separated by a 10-min reperfusion (PC × 1, PC × 2 and PC × 3, respectively). Another group of rats was preconditioned with heat shock (HS) by raising temperature to 42°C for 15 min. Twenty-four hours later, rats were given sustained ischemia for 30 min and 90 min of reperfusion. Infarct sizes (%risk area) were 40.0 ± 7.5, 37.6 ± 5.6, and 47.6 ± 2.4 (mean ± SE) for PC × 1, PC × 2, and PC × 3 hearts, respectively, which were not different from the sham (49.9 ± 3.9, P > 0.05). In contrast, infarct size was reduced from 47.5 ± 3.8% in sham to 4.7 ± 2.3% (P < 0.01) 24 h after HS. Additionally, early PC significantly reduced infarct size from 47.5 ± 3.8% in controls to 6.0 ± 1.2 and 5.0 ± 1.1% with PC × 1 and PC × 3. Repeated PC cycles induced over a threefold increase in HSP 70 mRNA after 2 h compared with sham (P < 0.05). HSP 72, which increased 24 h after PC or HS, was not significantly different between the two PC stimuli. We conclude that PC does not induce SWOP in rat heart despite enhanced expression of HSP 72. In contrast, HS-induced delayed protection was associated with enhanced accumulation of HSP 72. It is possible that SWOP and HS have distinct mechanisms of protection that may not be exclusively related to HSP 72 expression.

ischemia; stress proteins; infarction

	INTRODUCTION

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RECENT STUDIES SUGGEST THAT repeated brief episodes of ischemia and reperfusion render the myocardium resistant to subsequent sustained ischemia and reperfusion (21). This phenomenon, known as ischemic preconditioning (PC) is an inherent capability of the myocardium to protect itself from ischemic damage. The initial infarct-limiting effect, called classical or early PC, is transient, subsiding within 2-3 h after the initial insult (22). Studies in rabbit and dog have shown that a significant effect of PC reappeared when sustained ischemia was initiated 24 h after repetitive short cycles of ischemia and reperfusion (17, 19). The phenomenon is referred to as second window of preconditioning (SWOP), or late PC. In addition to ischemia, SWOP can be produced by several other pathophysiological stressors, including heat shock (5, 15), free radicals (44), nontoxic derivative of endotoxin, monophosphoryl lipid A (11, 41, 42), and adenosine agonist (3). It has been shown that both PC and whole body heat shock elevate myocardial 72-kDa heat shock protein (HSP 72) to a similar extent and are associated with a significant reduction in infarct size after 24 h of recovery (19).

To date, SWOP has been shown to occur only in rabbits and dogs (17, 19, 40). However, the reproducibility of SWOP in rat has been controversial. Because of the apparent differences in the mechanisms of PC among species, it is unclear whether the phenomenon of SWOP is reproducible in other animals or even in humans. A preliminary report by Jagasia et al. (10) suggested the absence of SWOP in a rat model of myocardial infarction. In contrast, Yamashita et al. (38) recently showed the existence of SWOP in rat, which was suggested to be due to the differences in the protocols used in their study. However, it should be noted that these investigators used 20 min of sustained ischemia to induce myocardial infarction, which is in contrast to the conventional 30-min or higher duration of ischemia used by others (10, 16, 18, 27, 32). Therefore, considering the controversies surrounding this important field, we undertook this investigation to study SWOP in a rat model of myocardial infarction with 30 min of sustained ischemia followed by 90 min of reperfusion. This infarct model of rat heart has been shown to respond to early PC and delayed ischemic tolerance induced by heat shock (16, 18, 27). Our second objective was to see whether HSP-72, which is considered as the hallmark of ischemic or heat shock stress, was also induced and correlated with the protection.

	MATERIALS AND METHODS

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Animal Care

The care and use of animals in this study were conducted in accordance within the guidelines of the Committee on Animals of Virginia Commonwealth University and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Materials

Evans blue, mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), and triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemical (St. Louis, MO). All other chemicals used were of analytic reagent quality. The cDNA for HSP 70, which is a 2.3-kb BamH I/Hind III fragment, was obtained from American Type Culture Collection (Rockville, MD).

Experimental Groups and Protocol

Male Sprague-Dawley rats (200-250 g) were used in all the experiments. Four major protocols were designed, comprising 1) early "classical" PC, 2) SWOP after 24 h, 3) SWOP after 48 h, and 4) heat shock PC. Animals were assigned into one of 11 groups and subjected to 30 min of sustained left anterior descending artery (LAD) occlusion followed by 90 min of reperfusion. The animals used for mRNA or HSP 72 protein analyses were not subjected to sustained ischemia and reperfusion. The experimental protocol is illustrated in Fig. 1 and described as follows.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Diagrammatic representation of experimental protocol. PC, preconditioning; PC × 1, PC × 3, 1 and 3 cycles of preconditioning, respectively; SWOP, second window of opportunity; SW/1, SW/2, SW/3, SWOP plus 1, 2, and 3 cycles of PC, respectively; HS, heat shock.

Early (Acute) PC. All experimental animals in the following groups were subjected to sustained ischemia for 30 min and 90 min of reperfusion immediately following the PC protocols.

GROUP I. Group I (non-PC, n = 9) consisted of control rats receiving no PC. For the analysis of HSP 70 mRNA, three rats in this group were surgically operated and hearts were removed after 165 min. These animals were not subjected to sustained ischemia for 30 min.

GROUP II. In group II (PC × 1, n = 12), the animals received PC with one 5-min coronary artery occlusion followed by a 10-min reperfusion period. Six rats were used for HSP 70 mRNA analysis. These animals were allowed to reperfuse for the remaining experimental period of 150 min following the PC protocol.

GROUP III. In group III (PC × 3, n = 12), the animals received a PC protocol that consisted of three cycles of 5-min occlusion, each separated by a 10-min reperfusion. Six rats were used for HSP 70 mRNA analysis. The ventricular samples were harvested after reperfusing the hearts for the remaining experimental period, which in this case was 120 min.

SWOP: 24 h. The animals were subjected to sustained ischemia and reperfusion 24 h after sham operation or ischemic PC.

GROUP IV. In group IV (sham, n = 13), the animals were operated and the chest was closed. Five separate animals were used for analysis of HSP 72 before sustained ischemia-reperfusion.

GROUP V. In group V (n = 6), the animals were subjected to one cycle of PC with 5 min of LAD occlusion followed by 10 min of recovery period.

GROUP VI. In group VI (n = 6), PC was induced by two episodes of 5-min occlusion each followed by a 10-min reperfusion period.

GROUP VII. In group VII (n = 14), PC was induced by three cycles of 5 min of ischemia and 10 min of reperfusion. Five animals were killed for HSP 72 protein analysis before sustained ischemia-reperfusion.

SWOP: 48 h. These animals were subjected to sustained ischemia and reperfusion 48 h after sham operation or ischemic PC.

GROUP VIII. In group VIII (sham, n = 6), the animals were operated and the chest was opened and then closed without occlusion of the coronary artery.

GROUP IX. In group IX (n = 6), PC was induced by three cycles of 5 min of ischemia and 10 min of reperfusion.

Heat shock PC. The animals were subjected to sustained ischemia and reperfusion 24 h after anesthetization or whole body hyperthermia.

GROUP X. In group X (sham heat stress, n = 6), control animals were anesthetized without heat stress. Five animals were killed for HSP 72 protein analysis before sustained ischemia-reperfusion.

GROUP XI. In group XI (heat stress, n = 6), animals were subjected to heat stress by raising temperature to 42°C for 15 min. Five animals were used for HSP 72 protein analysis.

Surgical Procedures

Rats were anesthetized with 50 mg/kg ip pentobarbital sodium and, after tracheotomy, artificially ventilated with room air at a stroke volume of 12 ml/kg at a rate of 55 strokes/min. The animals were intubated orotracheally for SWOP protocols. Subsequent doses of pentobarbital sodium were administered during the experiment as needed to maintain surgical anesthesia. The animals were placed on a heating pad underneath a warming lamp to maintain body temperature at 37°C. The carotid artery was cannulated and connected to a 7D pressure transducer for monitoring arterial pressure throughout the experiment. Additionally, a branch of the jugular vein was cannulated for administering drugs. Electrocardiographic leads were attached to subcutaneous electrodes to monitor either limb leads I or II or lead III. For animals that did not require the recovery period of 24 h, airway as well as vascular access for monitoring purposes were carried out differently as described in Myocardial Infarction Protocol. The surgery was performed under sterile conditions. The pericardium was exposed through a left thoracotomy performed at the fourth intercostal space. The pericardium was opened and the heart exposed. A 5-0 silk suture with an atraumatic needle was then passed around the left coronary artery midway between the atrioventricular groove and the apex. The ends of the tie were then threaded through a small vinyl tube to form a snare. The coronary artery was occluded by pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat. Myocardial ischemia was confirmed visually in situ mainly by regional cyanosis, S-T segment elevation/depression, or T wave inversion on electrocardiogram and accompanying hypokinetic movement of the myocardium. Release of the snare for reperfusion was readily confirmed by hyperemia over the surface of the previously ischemic-cyanotic segment.

For animals assigned to groups I, II, and III, the experiment immediately proceeded to the infarction protocol after the PC protocol(s). However, for the animals that required a 24- or 48-h reperfusion before sustained ischemia (groups IV-IX), the following steps were carried out. After completion of the PC procedure, the loose suture was left in situ. The incision was then closed in layers and the chest evacuated of air. The animals were observed during recovery until fully conscious and then extubated. After surgery, the rats received intramuscular doses each of analgesia (buprenorphine, 0.02 mg/kg) and antibiotic (penicillin, 200,000 U/kg). They were then returned to their cage and allowed free access to food and water. In the sham-operated groups, the animals underwent the same surgical preparation with the heart exposed and a snare-suture occluder positioned. Without inducing ischemia, we closed the chest, and the rats were allowed to recover for 24 or 48 h before sustained ischemia.

Whole Body Hyperthermia

The animals were anesthetized with an intraperitoneal injection of 50 mg/kg of pentobarbital sodium and placed on a warming blanket. A rectal thermometer was inserted to monitor core body temperature while the rats were subjected to whole body hyperthermia at 42°C for 15 min. The rats were subsequently allowed to recover for 24 h at room temperature. Control rats were treated similarly without turning on the heating blanket.

Myocardial Infarction Protocol

Twenty-four hours after the PC protocol or sham surgery, rats were reanesthetized and, after tracheotomy, mechanically ventilated with room air. Body temperature was maintained at 37°C with the use of a heating pad. The carotid artery was cannulated and connected to a 7D pressure transducer for monitoring arterial pressure throughout the experiment. Additionally, the jugular vein was cannulated for intravenous access. The electrocardiogram was continuously recorded with standard limb leads. The thorax was reopened, and the heart was exposed to identify the coronary artery branch. For the animals in the control group, a ligature was positioned as described above. Otherwise, the previously placed in situ coronary artery was used again. To induce myocardial infarction, the coronary artery was occluded by manipulating the suture and snare as described above. The thoracic cavity was covered with a plastic film to minimize heat loss. After 30 min of ischemia, the ligature was released, and the heart was reperfused for 90 min. A successful occlusion was validated in situ by observing regional cyanosis, hypokinesis/dyskinesis of the relevant segment of the left ventricle (LV), a decrease in arterial pressure, and electrocardiogram changes indicative of injury (S-T segment elevation). Reperfusion was confirmed by a conspicuous reactive hyperemia and hemodynamic improvement in the blood pressure.

Infarct Size Assessment

At the end of the infarction protocol (90 min of reperfusion), the ligature around the coronary artery was retightened and ~1 ml of 10% Evans blue dye was injected as a bolus into the jugular vein until the eyes turned blue. The rat was killed immediately, and the heart was removed and frozen at 20°C in a freezer. The frozen heart was then cut from apex to base into about eight transverse slices of equal thickness. The area at risk was determined by negative staining with Evans blue. The slices were then incubated in 1% TTC solution in isotonic pH 7.4 phosphate buffer at 37°C for 20 min. Tetrazolium reacts with NADH in the presence of dehydrogenase enzymes, causing viable tissue to stain a deep red color. The slices were subsequently fixed in 10% Formalin solution. Red-stained viable tissue was easily distinguished from the infarcted pale unstained necrotic tissue. The areas of infarcted tissue, the risk zone, and the whole LV were determined by digital planimetry with computer morphometry using a Bioquant imaging software. The area for each region was averaged from slices. Infarct size was then expressed both as a percentage of the total LV and as a percentage of the ischemic risk area.

Measurement of HSP

After the protocols described above were completed, the animals were anesthetized, a median sternotomy was performed, and the heart was quickly removed. The LV was separated and kept frozen at 80°C until Northern or Western blot analysis was performed for measurement of HSP 70 mRNA or HSP 72 protein.

Northern Blot Analysis of HSP 70 mRNA

About 0.5 g of powdered frozen heart tissue was homogenized in 5 ml of guanidinium thiocyanate (4.0 M)-Tris (0.1 M) buffer containing 1% -mercaptoethanol. The homogenate (4 ml) was layered onto 5.7 M cesium chloride cushion and 0.01 M EDTA (pH 7.5) and centrifuged for 20 h at 20°C. After removal of the supernatant, the pellet was transferred to a microphage tube in 300 µl of 3 M sodium acetate (pH 5.2) and kept at 20°C overnight. The tube was then microcentrifuged for 30 min and the supernatant discarded. The remaining RNA pellet was washed with 70% ethanol, dried using a Speedvac (Savant), and suspended in 50 µl of water. The yield of RNA was assessed by measuring the optical density (OD) of the preparation at 260 nm (1 OD = 40 µg RNA/ml) to determine the concentration of RNA. The ratio between the readings at 260 and 280 nm (OD₂₆₀/OD₂₈₀) gave an estimate of the purity of the isolated RNA.

Twenty micrograms of RNA from each sample were subjected to electrophoresis on 1% agarose gel-2.2 M formaldehyde gel and then transferred to a Nytran membrane by capillary blot. The membranes were baked in an oven at 80°C for 2 h and then prehybridized at 42°C for 18 h in a solution containing 50% formamide, 6× SSC, 2× Denhardt's reagent, 0.1% SDS, and 100 µg/ml denatured salmon testis DNA. Hybridization was carried out at 42°C for 12-18 h with ~10⁷ counts per minute of denatured probe. With the use of a solution of 0.1% SDS-0.2× SSC, the membranes were washed for 30 min at room temperature followed by a wash at 55°C. The autoradiograph was established by exposing the filter for 24-48 h to X-ray film (Kodak XAR-2) at 70°C with an intensifying screen. Blots were reused by stripping and rehybridized with -actin cDNA probe for internal control of the quantity of total mRNA. In addition, the RNA load per lane was assessed by ethidium bromide staining of the original agarose gel. Autoradiograms were quantified by a scanning densitometer, and the strength of the message was presented as the ratio of expression of HSP-70 vs. -actin.

Western Blot Analysis of HSP 72

The LV tissue was homogenized in 0.1 M phosphate buffer containing 5% SDS, 1% mercaptoethanol, and 0.1 mM PMSF (protease inhibitor) for 1 min with a Polytron tissue homogenizer using PT10 probe and then strained through a 27-gauge needle followed by centrifugation at 14,000 g for 3 min. Protein concentration was measured using the Bio-Rad protein assay based on the Bradford dye-binding procedure with bovine serum albumin as the standard. The supernatant, representing cellular proteins, was divided into small aliquots and stored at 80°C until used. At the time of analysis, samples were thawed and recentrifuged and volumes were pipetted to allow loading of ~20 µg of total protein per lane on the slab gel. Proteins were separated by SDS-PAGE on 1-mm-thick, 12.5% acrylamide gel. After electrophoresis, the proteins on the gel were then transferred to Western polyvinylidine difluoride (PVDF) membranes (Schleicher & Schuell, Keene, NH) by electroelution. Protein transfer was confirmed by employing prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA). After transfer and blocking with nonfat dry milk, the PVDF membranes were incubated with a mouse monoclonal antibody cross-reacting to inducible isoform HSP 72 (Stressgen Biotechnologies, Victoria, BC, Canada) at a dilution of 1:2,000. The second antibody was a horseradish peroxidase-conjugated rabbit anti-mouse IgG and was used at 1:2,000 dilution. The membranes were developed using enhanced chemiluminescence (Amersham, Arlington Heights, IL) and exposed to X-ray film for the appropriate time.

Hemodynamic Measurements

Hemodynamic measurements included heart rate and systolic, diastolic, and mean arterial pressures. Rate-pressure product was calculated as the product of heart rate and systolic blood pressure. Measurements were made at the following time period of the protocol: baseline before 30-min coronary occlusion, end of 30-min ischemia, and at 15, 30, 60, and 90 min of reperfusion.

Statistical Analysis

All measurements of infarct size, area at risk, and hemodynamic data are expressed as group means ± SE. Changes in infarct size and hemodynamic variables were analyzed by ANOVA to determine the main effect of time, group, and time-by-group interaction. If the global tests showed major interactions, post hoc contrasts between different time points within the same group or between different groups were performed using Student's t-test. Statistical differences were considered significant for P values <0.05.

	RESULTS

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Expression of HSP 70 mRNA

As shown in Fig. 2A, there was no expression of HSP 70 mRNA in any of the three control hearts. Because HSP 70 mRNA was consistently absent in the control group, we used only three animals in this group. In contrast, the hearts subjected to PC cycles demonstrated some variability in the induction of HSP 70 mRNA, and, as a result, an additional three animals were included in these groups. Three hearts in the PC × 3 group demonstrated significant induction of HSP 70 mRNA. However, somewhat weaker signals were observed in the remaining three hearts. In PC × 1 hearts, the signal was intense in four of six hearts. One heart demonstrated a weak signal of HSP 70 mRNA, and another heart in this group showed complete lack of the signal. Normalization of HSP 70 mRNA with respect to the housekeeping gene -actin revealed an over threefold increase in the HSP 70 mRNA (P < 0.01) (Fig. 2B). No significant differences in the levels of HSP 70 mRNA between PC × 1 and PC × 3 groups were observed (P > 0.05).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   A: Northern blot analysis showing increase in heat shock protein (HSP) 70 mRNA after 1 or 3 cycles of ischemic PC in rat heart. Total cellular RNA was prepared from myocardial risk region, electrophoresed, transferred to Nytran membrane, and hybridized with [32P]HSP 70 probe. Blots were stripped and rehybridized with -actin cDNA probe. Each band represents HSP 70 mRNA from separate heart. B: transcript levels of HSP 70/-actin measured by densitometer scanning of autoradiogram bands. Results are means ± SE from 3 to 6 hearts. * P < 0.05 from control.

Infarct Size During Early PC

We first studied the reproducibility of early PC by subjecting the hearts to PC × 1 and PC × 3 protocols (Fig. 1). As shown in Fig. 3A, the areas at risk expressed as the percentage of LV were not significantly different among the three groups: control, 39.7 ± 2.3%; PC × 1, 35.0 ± 1.6%; and PC × 3, 38.5 ± 2.4% (means ± SE, P > 0.05). The infarct size expressed as percent area at risk was 47.5 ± 3.8% in the control hearts subjected to sustained ischemia and reperfusion. PC resulted in significant reduction of infarct size to 6.0 ± 1.2 and 5.0 ± 1.1% with PC × 1 and PC × 3, respectively (Fig. 3B). The infarct size was not significantly different between PC × 1 and PC × 3 groups (P > 0.05). A similar trend was observed when infarct size was expressed as percentage of LV (Fig. 3C).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Bar diagram showing risk area [expressed as % of left ventricle (LV)] and reduction in infarct size (expressed as %area at risk as well as %LV) following early PC with 1 or 3 cycles of 5 min of ischemia and 10 min of reperfusion. Significant reduction in infarct size in the ischemic reperfused heart was evident with 1 and 3 PC cycles. Each bar represents mean ± SE of 6-8 animals.

Infarct Size Following Delayed PC With Sublethal Ischemia or Heat Shock

Pilot experiments using PC × 3 stimulus (5 rats in each of the sham and preconditioned groups) failed to demonstrate reduction in infarct size 12 h after sustained ischemia (data not shown). We therefore proceeded to study the induction of delayed PC 24 and 48 h later. These time windows have already been demonstrated to reduce infarct size in the rabbit heart (2, 17, 19, 24). The areas at risk expressed as percentage of LV were also not significantly different in the four groups (Fig. 4A). Infarct size in the ischemic reperfused hearts was 49.9 ± 3.9% after 24 h of sham operation (group IV). In PC × 1, PC × 2, and PC × 3 groups, the infarct sizes were 40.0 ± 7.5, 37.6 ± 5.6, and 47.6 ± 2.4%, respectively (%risk area, means ± SE), which were not significantly different from each other or sham hearts (Fig. 4B, P > 0.05). A similar trend was observed when the infarct size was calculated in terms of total left ventricular area (Fig. 4C). Furthermore, animals subjected to PC × 3 cycles 48 h before the myocardial infarction protocol had infarct not significantly different compared with the corresponding sham (48.4 ± 4.0 in 48 h sham vs. 50.0 ± 4.2 in 48 h SWOP). The areas at risk in these groups were also identical (38.1 ± 4.0 in 48 h sham vs. 41.0 ± 4.1 in 48 h SWOP). In contrast, whole body hyperthermia 24 h before ischemia resulted in a significant reduction in infarct size from 47.5 ± 3.8% in the sham-anesthetized group to 4.7 ± 2.3% (% area at risk, Fig. 5). The areas at risk were also not different between the two groups.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Bar diagram showing risk area (expressed as % of LV) and infarct size (expressed as % of area at risk and %LV) in the ischemic-reperfused hearts 24 h after PC with 1-3 cycles of 5 min of ischemia and 10 min of reperfusion. See Experimental Groups and Protocol for experimental details. Each bar represents mean ± SE of 6-8 animals.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Bar diagram showing risk area (%LV) and infarct size (expressed as % of area at risk and %LV) in the ischemic-reperfused hearts 24 h after whole body hyperthermia. * P < 0.05 vs. controls.

Expression of Inducible HSP 72

Figure 6 is a representative blot showing expression of the inducible form of HSP 72 in the LV 24 h after ischemic and heat shock PC. Because HSP 70 mRNA was not significantly different between PC × 1 and PC × 3 groups, we chose to determine the expression of HSP 72 after 24 h using the PC × 3 protocol. As shown in Fig. 6A, there was a minimal or negligible amount of HSP 72 in the control hearts subjected to surgical operation. The expression of HSP 72 increased significantly in the SWOP group. However, the overall mean background levels of HSP 72 were high in the sham group (as indicated from density graph) because three of the five hearts demonstrated higher baseline levels of the protein (not shown). Heat shock also induced significantly higher expression of HSP 72 compared with the corresponding sham group. The baseline level of HSP 72 expression was significantly lower in sham compared with the heat-shocked group (Fig. 6B). The overall expression of HSP 72 between PC × 3 and heat-shocked rats was not significantly different (P > 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Western blot analysis showing expression of inducible form of HSP 72 in LV. Rats were subjected to similar protocol as shown in Fig. 1. A: 24 h SWOP, groups IV and V. B: heat shock PC, groups X and XI. Hearts for analysis of HSP 72 were not subjected to sustained period of ischemia and reperfusion. Hearts were excised and left ventricular tissue was dissected and utilized for preparing protein lysates. Proteins were separated by SDS-PAGE and electroeluted onto polyvinylidine difluoride membrane before probing with monoclonal antibody against human inducible HSP 72. Secondary antibody was a horseradish peroxidase-conjugated rabbit anti-mouse IgG.

Systemic Hemodynamics

Heart rate, mean arterial blood pressure, and rate-pressure product are shown in Table 1. In a majority of the experimental groups, no significant differences in the baseline levels of these parameters were observed between each group. With few indicated exceptions, the heart rate, mean arterial pressure, and rate-pressure product remained reasonably stable throughout the reperfusion period, although these parameters decreased gradually at most of the time points in all of the groups.

View this table: [in this window] [in a new window]   Table 1.   Hemodynamic data

	DISCUSSION

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Salient Findings

It is well established that PC immediately and markedly elevates cardiac tolerance against ischemia (21, 26). However, this tolerance wanes abruptly within 2 h (22). Recently it has been proposed that PC induces a slower form of tolerance resulting in the restoration of cardioprotection after the rapid PC effect has worn off. This slower form of protection was designated as SWOP (19). Because of the apparent differences in the mechanisms of ischemic PC among various species (18), the existence of SWOP in rats has been reported to be controversial (10, 38). Recent studies also proposed a role of HSP 72 in SWOP, because this protein was found to be elevated 24 h after ischemic PC and heat shock (19). In the present study, we investigated SWOP and compared it with the early ischemic PC and late PC induced by whole body heat shock in the rat heart. Our results show that early PC with PC × 1 and PC × 3 significantly reduced the infarct size. On the other hand, the PC × 1, PC × 2, and PC × 3 protocols failed to reduce infarct size in the ischemic hearts after 24 or 48 h. However, we observed a significant reduction in the infarct size in the heat-shocked hearts when compared with sham controls, which is similar to the previously published studies (27). These differences in the cardioprotective effects between the two types of stresses were not related to the changes in the hemodymanics in the experimental groups. The PC stress was clearly evident, as indicated by the rapid induction of HSP 70 mRNA within 3 h and increased expression of the inducible form of HSP 72 at 24 h compared with the nonpreconditioned sham hearts. Taken together, our data show that early ischemic PC or late PC with heat shock produces significant protection in the ischemic heart. However, ischemic PC failed to produce SWOP despite increased expression of inducible HSP 72. These results suggest that SWOP cannot be reproduced in the rat heart under experimental conditions that proved effective in inducing acute PC.

Controversies in SWOP

Marber et al. (19) showed a 45% reduction in infarct size in the preconditioned compared with sham hearts. A number of preliminary studies from our laboratory (2, 24) have also showed significant reduction in the infarct size in the rabbit, thus further corroborating the findings of Marber et al. (19). Similarly, Kuzuya et al. (17) were able to demonstrate ~62% reduction in infarct size 24 h after ischemic PC in dogs using four episodes of PC. On the other hand, Tanaka et al. (34) could not observe SWOP in the rabbit. Two additional reports failed to demonstrate SWOP against myocardial infarction in the pig (28, 33). Moreover, another preliminary study in rats also failed to produce SWOP (10). The latter study used two different PC protocols, i.e., 3 × 3 min or one 15 min followed by 45 min of sustained ischemia, compared with 1 × 5, 2 × 5, and 3 × 5 min PC cycles and 30 min of sustained ischemia used in the present investigation. Yamashita et al. (38) recently showed the existence of SWOP in rat heart. These investigators used a PC stimulus similar to the current investigation, i.e., 3 × 5 min. However, the duration of sustained ischemia was only 20 min, which is in contrast to the 30-min or higher ischemic period used by other investigators in the rat model of myocardial infarction (10, 16, 18, 27, 32). Curiously, the extent of myocardial infarction using 20-min ischemia was >50% in their sham-operated, ischemic-reperfused hearts, which is comparable to or even higher than reported by us (26, 27, 30) and other investigators using 30 min of ischemia in rats (29, 32, 42). Therefore, even if one argues that the ischemic injury might have been severe with 30 min of ischemia in our experimental protocol, the infarct size was not different in either of these studies with opposite results. The delayed PC effect has been demonstrated in the isolated rat myocytes with exogenously generated oxygen-derived free radicals (43), heat shock, and metabolic PC (23). However, the in vitro simulated biochemical PC and ischemic conditions are quite different and cannot explain the lack of in vivo protection with physiological stresses induced by repetitive cycles of ischemic PC. Marber et al. (19) and Kuzuya et al. (17) used four cycles of 5-min PC in their studies. Although we did not use four cycles of PC stimulus in our studies, a preliminary study reported that multiple PC cycles do not necessarily induce greater tolerance than one cycle in the rabbit heart (1). We therefore believe that PC stimulus with PC × 1 or PC × 3 was sufficient to induce the protective response as indicated by significant reduction in infarct during early PC. Moreover, the reduction in infarct size with PC × 1 or PC × 3 protocols was not significantly different, suggesting that additional PC stimulus may not have caused further protection during early PC.

Role of HSP

In the present investigation, PC induced an approximately threefold increase in HSP 70 mRNA with PC × 1 and PC × 3 protocols. PC × 3 also induced a significant increase in HSP 72 protein 24 h later compared with sham. It has been reported that a single 5-min coronary occlusion doubled the expression of HSP 70, whereas four cycles of 5 min of ischemia and 5 min of reperfusion resulted in a threefold increase in HSP 70 mRNA in the rabbit heart (14). Similarly, Das et al. (6) showed induction of several inducible genes for HSPs and antioxidant enzymes following four episodes of PC in the heart. In the present studies we did not find significant differences in the levels of HSP 70 mRNA between PC × 1 and PC × 3 groups. We do not know the reason for this discrepancy, although these differences could be due to the experimental models. We used an in situ model of regional ischemia as opposed to the globally ischemic isolated perfused rat heart used by Das and co-workers (6).

Expression of HSPs is associated with protection from a number of metabolic insults, including ischemia-reperfusion, although the evidence that this protection results directly from HSPs is controversial. Currie et al. (4) showed that exposure of rats to elevated temperature, with consequent cardiac HSP induction, resulted in an improved recovery of contractile function and reduced creatine kinase release after subsequent ischemia and reperfusion. Hutter et al. (9) suggested HSP 72 as the primary mediator of ischemic protection in the rat heart. These investigators pretreated rats with whole body hyperthermia at varying temperatures and subsequently subjected them to in vivo ischemia-reperfusion. Infarct size was highly correlated with the amount of HSP 72 induced. Marber et al. (19) suggested that ischemic and heat stress pretreatment increased HSP 72 to a similar extent and suggested that the protection occurred as a consequence of stress protein induction. Transgenic mice overexpressing HSP 70 have been shown to be tolerant to ischemia-reperfusion injury (20, 25). Although we observed a rapid rise in HSP 70 mRNA with its consequent translation into the protein 24 h later, we did not find its correlation with the myocardial protection following ischemic PC. Furthermore, a recent study from our laboratory demonstrated 80% of maximum HSP 72 and HSP 27 production in the rat heart by 4 h after whole body hyperthermia, whereas protection was not evident until after 12-24 h (27, 31). These data suggested that mere accumulation of HSPs was not sufficient to explain the ischemic protection after heat shock. The heat shock produced ~85% reduction in the infarct size after 30 min of sustained ischemia in the rat heart. This was significantly higher than the 64% reduction of infarct size in the rabbit heart reported previously (8).

Currie et al. (5) also reported that infarct size following ischemia-reperfusion was significantly reduced 24 h after heat shock; the protective effect was abolished by 40 h in vivo while HSP 72 was still present. Recently, another study from our laboratory by Xi et al. (37) failed to demonstrate significant recovery of postischemic ventricular function and reduction of myocardial infarct size following heat shock despite the enhanced expression of HSP 72. Joannidis et al. (13) also reported that whole body heat shock failed to prevent the ischemic injury of rat renal tubules either in vivo or in vitro, despite significant induction of HSPs. In the isolated cultured adult cardiac myocytes, both heat shock and metabolic PC were able to induce an identical delayed protective effect in terms of reduction in the release of cellular enzymes following simulated ischemia 24 h later (23). However, the amount of HSP 72 accumulated following metabolic PC was significantly lower compared with heat shock. Not only that, pharmacological agents such as monophosphoryl lipid A (41) as well as the adenosine A₁ receptor agonist 2-chloro-N⁶-cyclopentyl-adenosine induced delayed myocardial protection in the rabbit heart that was independent of the expression of HSP 72 (3).

Conclusions

The experimental approaches used in the early ischemic PC or delayed PC with heat shock produced significant reduction in infarct size after ischemia-reperfusion. The delayed PC with ischemia and heat shock were accompanied by increased synthesis of HSP 72. However, in contrast to the results reported in dogs (17) and rabbits (2, 19), ischemic PC under similar conditions failed to produce delayed protection after 24 or 48 h. We do not know the exact reason for this discrepancy. One plausible hypothesis is that rodents behave differently when it comes to delayed cardiac PC. In support of this argument, we and others have observed that rats and mice required at least a 10-fold higher concentration of the monophosphoryl lipid A for induction of pharmacological PC (12, 35) compared with rabbits and dogs (7, 40, 41). A second possibility is that the protective protein(s) responsible for causing delayed protection in the rabbit heart are probably not expressed in the rat heart. Additionally, SWOP and heat shock may have distinct mechanisms of protection that may not be exclusively related to enhanced HSP 72 expression. Further studies are required to clarify these issues.

	ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-51045 and HL-59469 to R. C. Kukreja. N. L. Bernardo was supported by a fellowship from the American Heart Association, Virginia affiliate. M. A. Nayeem and J. Chelliah were supported by a postdoctoral training fellowship from NHLBI Training Grant HL-07537.

	FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: R. C. Kukreja, Box 282, Division of Cardiology, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, VA 23298.

Received 26 June 1998; accepted in final form 9 September 1998.

	REFERENCES

Top Abstract Introduction Materials & Methods Results Discussion References

1.   Baxter, G. F., F. M. Goma, and D. M. Yellon. Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium. Basic Res. Cardiol. 92: 159-167, 1997[Medline].

2.   Bernardo, N. L., M. D'Angelo, P. V. Desai, J. E. Levasseur, and R. C. Kukreja. ATP-sensitive potassium (K_ATP) channel is involved in the second window of ischemic preconditioning in rabbit (Abstract). J. Mol. Cell. Cardiol. 29: A228, 1997.

3.   Bernardo, N. L., S. Okubo, M. A. Wood, and R. C. Kukreja. ATP-sensitive potassium channel blockers suppress monophasic action potential shortening and abolish delayed cardioprotection induced by 2-chloro-n-cyclopentyladenosine (CCPA) (Abstract). Circulation 96: I-257, 1997.

4.   Currie, R. W., B. M. Ross, and T. A. David. Induction of the heat shock response in rats modulates heart rate, creatine kinase and protein synthesis after a subsequent hyperthermic treatment. Cardiovasc. Res. 14: 87-93, 1990.

5.   Currie, R. W., R. M. Tanguay, and J. G. Kingma, Jr. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 87: 963-971, 1993[Abstract/Free Full Text].

6.   Das, D. K., R. M. Engelman, and Y. Kimura. Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia. Cardiovasc. Res. 27: 578-584, 1993[Abstract/Free Full Text].

7.   Elliott, G. T., M. L. Comerford, J. R. Smith, and L. Zhao. Myocardial ischemia/reperfusion protection using monophosphoryl lipid A is abrogated by the ATP-sensitive potassium channel blocker, glibenclamide. Cardiovasc. Res. 32: 1071-1080, 1996[Abstract/Free Full Text].

8.   Hoag, J. B., Y.-Z. Qian, M. A. Nayeem, M. D'Angelo, and R. C. Kukreja. ATP-sensitive potassium channel mediates delayed ischemic protection by heat stress in rabbit heart. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H861-H868, 1997[Abstract/Free Full Text].

9.   Hutter, M. W., R. E. Sievers, V. Barbosa, and C. L. Wolfe. Heat-shock protein induction in rat hearts: a direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 89: 355-360, 1994[Abstract/Free Full Text].

10.  Jagasia, D., J. M. Whiting, and P. H. McNully. Ischemic preconditioning fails to produce a second window of protection 24 hrs later in the rat (Abstract). Circulation 94, Suppl.: I-184, 1996.

11.   Janin, Y., Y.-Z. Qian, J. Hoag, G. T. Elliott, and R. C. Kukreja. Pharmacologic preconditioning with monophosphoryl lipid A is abolished by 5-hydroxydecanoate, a specific inhibitor of the K_ATP channel. J. Cardiovasc. Pharmacol. 32: 1163-1172, 1998.

12.   Jarrett, N. C., L. Xi, M. L. Hess, and R. C. Kukreja. Monophosphoryl lipid A induces delayed protection against ischemia/reperfusion injury in the mouse heart (Abstract). J. Mol. Cell. Cardiol. 30: A262, 1998.

13.   Joannidis, M., L. G. Cantley, K. Spokes, R. Medina, J. Pullman, S. Rosen, and F. H. Epstein. Induction of heat-shock proteins does not prevent renal tubular injury following ischemia. Kidney Int. 47: 1752-1759, 1995[Medline].

14.   Knowlton, A. A., P. Brecher, and C. S. Apstein. Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia. J. Clin. Invest. 87: 139-147, 1991.

15.   Kontos, M. C., J. S. Shipley, and R. C. Kukreja. Heat stress improves functional recovery and induces synthesis of 27- and 70-kDa heat shock proteins without preserving sarcoplasmic reticulum function in the ischemic rat heart. J. Mol. Cell. Cardiol. 28: 1885-1894, 1996[Medline].

16.  Kukreja, R. C., Y.-Z. Qian, and E. E. Flaherty. Protein kinase C is involved in heat stress-induced myocardial protection of rat heart. Circulation 94, Suppl.: I-423, 1996.

17.   Kuzuya, T., S. Hoshida, N. Yamashita, H. Fuji, H. Oe, M. Hori, T. Kamada, and M. Tada. Delayed effect of sublethal ischemia on the acquisition of tolerance to ischemia. Circ. Res. 72: 1293-1299, 1993[Abstract/Free Full Text].

18.   Li, Y., and R. A. Kloner. The cardioprotective effects of ischemic `preconditioning' are not mediated by adenosine receptors in rat hearts. Circulation 87: 1642-1648, 1993[Abstract/Free Full Text].

19.   Marber, M. S., D. S. Latchman, J. M. Walker, and D. M. Yellon. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation 88: 1264-1272, 1993[Abstract/Free Full Text].

20.   Marber, M. S., R. Metril, S.-H. Chi, R. Sayen, D. M. Yellon, and W. H. Dillmann. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 95: 1446-1456, 1995.

21.   Murry, C. E., R. B. Jennings, and K. A. Reimer. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124-1136, 1986[Abstract/Free Full Text].

22.   Murry, C. E., V. J. Richard, R. B. Jennings, and K. A. Reimer. Myocardial protection is lost before contractile function recovers from ischemic preconditioning. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H796-H804, 1991[Abstract/Free Full Text].

23.   Nayeem, M. A., M. L. Hess, Y.-Z. Qian, K. E. Loesser, and R. C. Kukreja. Delayed preconditioning of cultured adult rat cardiac myocytes: role of 70- and 90-kDa heat stress proteins. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H861-H868, 1997.

24.  Okubo, S., N. L. Bernardo, A. B. Jao, G. T. Elliott, R. C. Kukreja. Tyrosine phosphorylation is involved in second window of preconditioning in rabbit heart (Abstract). Circulation 96, Suppl.: I-313, 1997.

25.   Plumier, J.-C. L., B. M. Ross, R. W. Currie, C. E. Angelidis, H. Kazlaris, G. Kollias, and G. N. Pagoulatos. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J. Clin. Invest. 95: 1854-1860, 1995.

26.   Qian, Y.-Z., J. E. Levasseur, K.-I. Yoshida, and R. C. Kukreja. K_ATP channels in rat heart: blockade of ischemic and acetylcholine-mediated preconditioning by glibenclamide. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H23-H28, 1996[Abstract/Free Full Text].

27.   Qian, Y.-Z., J. S. Shipley, J. E. Levasseur, and R. C. Kukreja. Dissociation of the expresion of 72 and 27 kDa heat shock proteins with ischemic tolerance following heat shock in rat heart. J. Mol. Cell. Cardiol. 30: 1163-1172, 1998[Medline].

28.   Qiu, Y., X.-L. Tang, S. W. Park, J. V. Sun, A. Kalya, and R. Bolli. The early and late phases of ischemic preconditioning: a comparative analysis of their effects on infarct size, myocardial stunning, and arrhythmias in conscious pigs undergoing a 40 minute coronary occlusion. Circ. Res. 80: 730-742, 1997[Abstract/Free Full Text].

29.   Schultz, J. E. J., A. K. Hsu, and G. J. Gross. Ischemic preconditioning is mediated by a peripheral opioid receptor. J. Mol. Cell. Cardiol. 29: 1355-1362, 1997[Medline].

30.   Schultz, J. E. J., Y.-Z. Qian, G. J. Gross, and R. C. Kukreja. Specific ATP-sensitive potassium channel antagonist 5-hydroxydecanoate blocks ischemic preconditioning in the rat heart. J. Mol. Cell. Cardiol. 29: 1055-1060, 1997[Medline].

31.   Shipley, J. B., Y.-Z. Qian, J. E. Levasseur, and R. C. Kukreja. Expression of the stress proteins HSP-27 and HSP-72 in rat hearts does not correlate with ischemic tolerance after heat shock (Abstract). Circulation 92: I-654, 1995.

32.   Speechly-Dick, M. E., M. M. Mocannu, and D. M. Yellon. Protein kinase C. Its role in ischemic preconditioning in the rat. Circ. Res. 75: 586-590, 1994[Abstract/Free Full Text].

33.   Strasser, R., M. Arras, A. Vogt, A. Elsasser, E. R. Schwarts, M. Schlepper, and W. Schaper. Preconditioning of porcine myocardium: how much ischemia is required for induction? What is its duration? Is a renewal of effect possible? (Abstract). Circulation 90: I-109, 1994.

34.   Tanaka, M., H. Fujiwara, K. Yamasaki, M. Miyamae, R. Yokota, K. Hasegawa, T. Fujiwara, and S. Sasayama. Ischemic preconditioning elevates cardiac stress protein but does not limit infarct size 24 or 48 h later in rabbits. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H1476-H1482, 1994[Abstract/Free Full Text].

35.   Tosaki, A., N. Maulik, G. T. Elliott, R. M. Engelman, and D. K. Das. Preconditioning of rat heart with monophosphoryl lipid A: a role of nitric oxide. J. Pharmacol. Exp. Ther. 285: 1274-1279, 1998[Abstract/Free Full Text].

37.  Xi, L., J. Chelliah, M. A. Nayeem, J. E. Levasseur, and R. C. Kukreja. Whole body heat shock fails to protect mouse heart against ischemia-reperfusion injury in an isolated perfused heart model. J Mol Cell Cardiol. In press.

38.   Yamashita, N., S. Hoshida, N. Taniguchi, T. Kuzuya, and M. Hori. A "second window of protection" occurs 24 h after ischemic preconditioning in the rat heart. J. Mol. Cell. Cardiol. 30: 1181-1189, 1998[Medline].

39.   Yang, X. M., G. F. Baxter, R. J. Heads, D. M. Yellon, J. M. Downey, and M. V. Cohen. Infarct limitation of the second window of protection in a conscious rabbit model. Cardiovasc. Res. 31: 777-783, 1996[Medline].

40.   Yao, Z., J. A. Auchampach, G. M. Pieper, and G. J. Gross. Cardioprotective effects of monophosphoryl lipid A, a novel endotoxin analogue, in the dog. Cardiovasc. Res. 27: 832-838, 1993[Abstract/Free Full Text].

41.   Yoshida, K. I., M. M. Maaieh, J. B. Shipley, M. Doloresco, N. L. Bernardo, Y. Z. Qian, G. T. Elliott, and R. C. Kukreja. Monophosphoryl lipid A induces pharmacologic 'preconditioning' in rabbit hearts without concomitant expression of 70-kDa heat shock protein. Mol. Cell. Biochem. 159: 73-80, 1996[Medline].

42.   Ytrehus, K., Y. Liu, A. Tsuchida, T. Miura, G. S. Liu, X.-M. Yang, D. Herbert, M. V. Cohen, and J. M. Downey. Rat and rabbit heart infarction: effects of anesthesia, perfusate, risk zone, and method of infarct sizing. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2383-H2390, 1994[Abstract/Free Full Text].

43.   Zhou, X. B., X. L. Zhai, and M. Ashraf. Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes. Circulation 93: 1177-1184, 1996[Abstract/Free Full Text].