INTRODUCTION

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APOPTOTIC CELL DEATH is characterized morphologically by chromatin condensation and biochemically by fragmentation of internucleosomal DNA to multiples of 180-200 bp fragments (28). Recently, Gottlieb et al. (8) showed that apoptotic cell death as well as necrosis occurs in myocytes subjected to ischemia-reperfusion insult in rabbit hearts. We and others (22, 24) have previously reported that a transient preceding ischemia termed ischemic preconditioning reduced both myocardial infarct size and the incidence of apoptosis. However, the underlying mechanism by which ischemic preconditioning reduces apoptosis induced by ischemia-reperfusion has remained to be clarified.

Ischemic preconditioning is a phenomenon where a series of brief periods of myocardial ischemia-reperfusion increase myocardial tolerance to the subsequent prolonged ischemia. Since this phenomenon was first reported in 1986 by Murry et al. (20), the mechanism of ischemic preconditioning has been intensively studied, and it is believed that the activation of protein kinase C (PKC) plays a pivotal role in mediating ischemic preconditioning (10, 15, 31). However, the role of PKC in the mechanism of ischemic preconditioning attenuating apoptosis remains unknown.

Thus the purpose of the present study was to clarify the role of PKC in the mechanism of reducing apoptosis by ischemic preconditioning.

	METHODS

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Animal preparation. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23), and the protocol was approved by the Animal Research Committee of Yamaguchi University School of Medicine. Male Sprague-Dawley rats weighing 280-330 g were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). Additional anesthesia was supplied during the experiment if necessary. Rats were intubated and ventilated with a respirator using a mixture of 100% oxygen and room air (tidal volume, 1.2 ml/100 g body wt; respiratory rate, 65-70 breaths/min). Arterial blood gases were monitored, and arterial PO₂, PCO₂, and pH were maintained within the physiological range. Left thoracotomy was performed in the fourth intercostal space, and the heart was exposed. A 6-0 silk thread was passed around the left anterior descending coronary artery using a tapered needle. Both ends of the thread were passed through a 1-cm length polyethylene tube (outer diameter 1.5 mm) to occlude and reperfuse the coronary artery. Left ventricular pressure was measured by a 2-F. catheter-tip manometer (model SPC-320, Millar Instruments) inserted via the right carotid artery. Myocardial ischemia was confirmed by the bulging of the relevant segment of the left ventricle, the increase of the left ventricular end-diastolic pressure, and the S-T segment elevation in the electrocardiogram. The body temperature was measured by an electric thermistor and maintained at 37 ± 0.5°C with a heating pad placed under the rat.

Heart rate, peak and end-diastolic left ventricular pressures, the first derivative of the left ventricular pressure (dP/dt), and the time constant of isovolumic left ventricular pressure decay calculated by a monoexponential model without asymptote were measured (5).

Reagents. Calphostin C (Kyowa Medex, Tokyo, Japan) is a specific inhibitor of PKC (13). It was dissolved in DMSO at a concentration of 2 mg/ml, and the solution was diluted with sterilized water to the concentration of 0.1 mg/ml before use.

Experimental protocol. Rats were randomly assigned to one of the following four groups (Fig. 1). In all groups rats were subjected to 30 min of coronary occlusion followed by a 6-h reperfusion period. In the vehicle group (control; n = 7), 0.3 ml of 5% DMSO was administered intravenously 5 min before the 30-min coronary occlusion. In the calphostin C group (Cal; n = 7), calphostin C (0.1 mg/kg) was administered intravenously 5 min before the 30-min coronary occlusion. In the ischemic preconditioning group (IP; n = 7), three cycles of 3 min of ischemia followed by 3 min of reperfusion was performed as ischemic preconditioning before the 30-min coronary occlusion. In the ischemic preconditioning + calphostin C group (IP + Cal; n = 7), calphostin C (0.1 mg/kg) was administered 5 min before the ischemic preconditioning followed by the 30 min of ischemia. After the 6-h reperfusion period, rats were euthanized with an overdose of the anesthetic.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Experimental protocol showing a 30-min coronary occlusion followed by a 6-h reperfusion period in all groups (n = no. of animals). Three cycles of 3-min ischemia-reperfusion were performed before the 30-min ischemia as ischemic preconditioning (IP). Cal, calphostin C.

Definition of area at risk and area of infarction. After the rats were euthanized, the left anterior descending coronary artery was reoccluded and the heart was excised. Monastral blue dye (1.5%, 1 ml) was injected into the ascending aorta to delineate the risk area as a perfusion defect. The heart was then sliced into 1-mm thick sections, and the slices were incubated in 1% triphenyltetrazolium chloride (TTC) at 37°C for 20 min. Viable myocardium is stained in red by TTC, whereas infarcted tissue remains unstained (27). The slices were imaged in color at four times the magnification by a charge-coupled digital camera (FV-10, Fuji Photo Film, Japan), and stored on a Macintosh computer for image analysis. The area stained by the blue dye (perfused area), the unstained area (area at risk; AR), and the area of infarcted (AI) and noninfarcted myocardium were defined using an image analysis software (NIH Image). The AR normalized by the left ventricular area (AR/LV), and the AI normalized by the AR (AI/AR) were calculated. The heart slices were fixed with 10% Formalin and embedded in paraffin for subsequent in situ nick end labeling (TUNEL).

In situ nick end labeling. The TUNEL protocol was based on the previously published procedure (6). It depends on the preferential labeling by terminal deoxynucleotidyl transferase of 3'-OH ends of DNA. In brief, 4-µm thick sections of the heart were deparaffinized by a xylene wash and a descending ethanol series (100%, 95%, 70%, and 0%) and then washed with phosphate-buffered saline. The sections were subsequently incubated with 20 µg/ml proteinase K for 15 min at room temperature, and endogenous peroxidase was inactivated by the treatment with 3% hydrogen peroxide for 5 min. Equilibration buffer (42 µl) was then added to the sections and incubated with terminal deoxynucleotidyl transferase (ApopTag, Oncor) for 1.5 h at 37°C. After end labeling was completed, the sections were incubated with anti-digoxygenin peroxidase for 30 min at room temperature. For the color development, 3% aminoethylcarbazole was applied to the section for 3 min at room temperature. Finally, the sections were counterstained with hematoxylin. After the procedures, 1,000 nuclei in the subendo-, mid-, and subepicardial regions in the risk area were examined by microscopy at a magnification of ×200, and the number of TUNEL-positive myocyte nuclei was counted and expressed as a percentage of the total number of myocyte nuclei in each region (%AP).

Agarose gel electrophoresis of DNA. In the separate series of experiments, the same protocol (n = 5, each) was carried out for the detection of DNA ladder. Transmural myocardial samples from ischemic and nonischemic areas were isolated, frozen in liquid nitrogen, and stored at 80°C. The DNA was extracted from 70-80 mg of nonrisk and risk area of the left ventricular myocardium using a nucleic acid extraction kit (IsoQuick, Microprobe). The concentration of DNA in each sample was measured by spectrophotometry (260 nm). A same amount (10 µg) of extracted DNA was loaded on a 2.0% agarose gel containing ethidium bromide in 0.04 mol/l Tris, 0.04 mol/l borate acid, 2 mmol/l EDTA, pH 8.0 (TBE), and electrophoresed on a flatbed gel apparatus (Mupid-3, ADVANCE, Japan) at 100 V. The gel was photographed under transmitted ultraviolet light.

Statistical analysis. All values are expressed as means ± SE. The hemodynamic changes were assessed by a two-way ANOVA with repeated measure, and Fisher's test was used for multiple comparisons when a significant F value was obtained. Intergroup differences in AR/LV, AI/AR, and the percentage of TUNEL-positive myocyte nuclei were analyzed by one-way ANOVA, and Fisher's test was used for multiple comparisons. Differences at P < 0.05 were considered significant.

	RESULTS

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Hemodynamic responses and arrhythmias. There were no significant differences in body weight, heart rate, peak and end-diastolic left ventricular pressures, LV +dP/dt, and the time constant between the groups before and during the 30-min coronary occlusion and at 10 min after the reperfusion (Table 1). All rats of control, Cal, and IP + Cal groups showed arrhythmias such as ventricular tachycardia during the 30 min of ischemia, especially during the initial 5-10 min of ischemia, whereas the incidence of ventricular tachycardia was markedly reduced in the IP group.

Myocardial infarct size. Figures 2 and 3 show results of AR/LV and AI/AR. AR/LV did not differ significantly among the groups (51 ± 2% in the control group, 59 ± 3% in the Cal group, 47 ± 4% in the IP group, 48 ± 3% in the IP + Cal group, NS). However, AI/AR was significantly smaller in the IP group (26 ± 2%) than in the other three groups (60 ± 2% in the control group, 63 ± 2% in the Cal group, 51 ± 3% in the IP + Cal group, P < 0.01 vs. IP, respectively).

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Representative samples stained by monastral blue and triphenyltetrazolium chloride (TTC). Nonrisk areas are stained blue by monastral blue. Viable myocardium is stained red by TTC. A: control group; B, Cal group; C, IP group; D, IP + Cal group.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Bar graphs represent area at risk (AR) by the whole left ventricular area (LV) (AR/LV, A) and area of infarction (AI) by the AR (AI/AR, B). AR/LV in all groups are identical. AI/AR in IP group was sinificantly smaller than in other groups. Error bar shows ± SE. * P < 0.01 between groups.

Count of myocytes with TUNEL-positive nuclei. TUNEL-positive myocyte nuclei were counted in the histological sections. Figure 4 shows TUNEL-positive nuclei at a magnification of ×200. We counted myocyte nuclei stained by hematoxylin or TUNEL. The TUNEL-positive nuclei were observed only in the risk area and were mainly located in the subendocardial region.

View larger version (180K): [in this window] [in a new window]   Fig. 4.   Paraffin sections stained by TUNEL (ApopTag kit) and hematoxylin are presented (×200 magnification). A: risk area in control group; B: risk area in Cal group; C: risk area in IP group; D: risk area in Cal + IP group. Myocyte nuclei stained by TUNEL in red indicate apoptotic cells (arrowheads). TUNEL-positive nuclei are mainly observed in subendocardial region. E: DNase I-treated positive control. F: nonrisk area in control group. No TUNEL-positive nuclei are observed.

Figure 5 shows %AP in the subendocardial region. The %AP was reduced significantly by ischemic preconditioning (13 ± 2% in the IP group vs. 44 ± 3% in the control group, P < 0.01), and the effect of ischemic preconditioning was abolished by the pretreatment of calphostin C. 

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Percentage of TUNEL-positive nuclei (%AP) in risk area. Bar graphs represent effect of IP and calphostin C on %AP in risk area. IP significantly reduced TUNEL-positive myocyte nuclei in subendocardial region, and this effect was abolished by calphostin C. * P < 0.01 between groups. Error bar shows ± SE.

DNA laddering in agarose gel electrophoresis. A series of DNA fragments with a size range in multiples of 180-200 bp of a so-called "ladder pattern" indicates apoptotic internucleosomal DNA fragmentation. The ladder pattern was observed in the myocardium from the risk area of control, Cal, and IP + Cal groups but was less in the IP group compared with other groups (Fig. 6). No ladder pattern was observed in the myocardium from the nonrisk area of all groups.

View larger version (102K): [in this window] [in a new window]   Fig. 6.   Electrophoretic pattern of DNA fragments extracted from myocardium of control, Cal, IP, and IP + Cal groups. Lanes are as follows: lane 1, risk area of control group; lane 2, nonrisk area of control group; lane 3, risk area of Cal group; lane 4, risk area of IP group; lane 5, risk area of IP + Cal group; M, molecular size markers. DNA ladder pattern was seen in risk area of all groups but less apparent in IP group (lane 4).

	DISCUSSION

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The present study demonstrates that ischemic preconditioning reduced both myocardial infarct size and myocyte apoptosis caused by the ischemia-reperfusion insult. This effect of ischemic preconditioning was abolished by a selective PKC inhibitor calphostin C, indicating a crucial role of PKC in the mechanism of ischemic preconditioning.

Mechanism of ischemic preconditioning. Ischemic preconditioning exerts a strong cardioprotective effect against myocardial ischemic insult. This phenomenon has been confirmed in many animal models (14, 16, 18, 20, 23, 25), including human hearts (4, 21, 30). Whereas the mechanism of ischemic preconditioning has not been fully understood, several mediators such as adenosine (17), bradykinin (7), norepinephrine (1), and free radicals (26) have been reported. These mediators initiate intracellular signaling pathways by acting on G protein-linked receptors. Then phospholipase C activated by G proteins produces diacylglycerol, which activates cytosolic PKC (31). The possible end-effectors to induce myocardial protection such as ATP-sensitive potassium channels (10, 29) and the vacuolar proton ATPase may be phosphorylated and activated by PKC (9).

Apoptosis in ischemia-reperfusion insult. Gottlieb et al. (8) reported that the chromatin condensation and nucleosomal ladders were observed in the rabbit myocytes subjected to 30 min of ischemia followed by 4 h of reperfusion, and they indicated that the ischemia-reperfusion injury caused necrosis and apoptosis. Necrosis is characterized by the swelling of mitochondria, loss of plasma membrane integrity, and the random digestion of DNA (3). In contrast, apoptosis is characterized morphologically by a chromatin condensation and biochemically by internucleosomal DNA fragmentation (28). The fragmentation of genomic DNA into nucleosome-sized fragments by the endonucleolytic cleavage is a biochemical hallmark of apoptosis (2).

The present study domonstrates that the myocardial infarct size defined by TTC staining was significantly attenuated by ischemic preconditioning together with a significant reduction of apoptosis. This result is supported by previous studies done by Gottlieb et al. (9) and Piot et al. (22).

PKC and incidence of apoptosis. Calphostin C is a specific inhibitor of PKC acting on its regulatory domain (13). We and others (12, 16) have already demonstrated that the ischemic preconditioning is mediated by PKC, especially by the translocation of PKC- and -, and the effect was blocked by calphostin C or chelerythrine. The present study demonstrated that the pretreatment of calphostin C abolished the effect of ischemic preconditioning on both infarct size and the incidence of apoptosis. This suggests the pivotal role of PKC in mediating a cardioprotective effect of ischemic preconditioning not only on infarct size but also on the incidence of apoptosis. Whereas the underlying mechanism of reducing apoptosis by ischemic preconditioning is not fully understood, the attenuation of acidosis induced by vacuolar proton ATPase, which is activated by PKC, may be involved (9). The other possible role of PKC activation in inhibiting apoptosis may be related to the phosphorylation of anti-apoptotic proteins such as Bcl-2 and Bcl-x, because the anti-apoptotic effect of these proteins may depend on its phosphorylation (11, 19).

Further studies are needed to clarify the mechanism of PKC-dependent modulation of apoptosis in ischemia-reperfusion model.

In conclusion ischemic preconditioning reduces apoptosis induced by ischemia-reperfusion insult, and the effect may be mediated by PKC activation during ischemic preconditioning.