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Cardiovascular Research 1998 37(3):700-709; doi:10.1016/S0008-6363(97)00244-7
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes

Tetsuji Miuraa,*, Toshiro Miurab, Shuji Kawamurab, Mahiko Gotoa, Jun Sakamotoa, Akihito Tsuchidaa, Masunori Matsuzakib and Kazuaki Shimamotoa

aSecond Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060, Japan
bSecond Department of Internal Medicine, Yamaguchi University, School of Medicine, Ube, Japan

* Corresponding author. Tel.: +(81-11) 611 2111X3225; fax: (+81-11) 644 7958; e-mail: miura@samped.ac.jp

Received 21 March 1997; accepted 10 September 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: This study examined the possibility that the role of PKC and PC, and thus the response to PFC inhibitors, may differ depending on how many ischemic episodes are employed to precondition the heart. Methods: In the first series of experiments, myocardial infarct was induced by 30 min of coronary occlusion and 3 h of reperfusion in the rabbit. Infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Prior to the 30-min ischemia, rabbits were subjected to no PC, single PC (i.e., PC with an episode of 5 min ischemia/5 min reperfusion), and repetitive PC (2 cycles of 5 min ischemia/5 min reperfusion) with or without one of three treatments: polymyxin B (PolyB), staurosporine (Stauro), and 8-sulfophenylthephylline (SPT). In the second series of experiments, the rabbits received 5 min of coronary occlusion after repetitive PC with or without PolyB or Stauro treatment. Then, myocardial tissue in the ischemic region was sampled for assay of PKC activity. Untreated rabbits served as controls. Results: Single and repetitive PC limited %IS/AR to the same extent (%IS/AR=9.8±1.9 and 10.4±2.3, both p<0.05, vs. the control value of 44.5±3.4), and single PC was blocked by PolyB (%IS/AR=43.9±2.7) and Stauro (%IS/AR=31.5±3.2). Although the protocol of PolyB injection maintained the plasma PolyB level during sustained ischemia well above its Ki for PKC, this agent and also Stauro failed to abolish the protection by repetitive PC (%IS/AR=21.6±3.0 and 11.4±4.3, respectively). SPT, an adenosine receptor antagonist, not only blocked single PC (%IS/AR=44.4±4.4) but also attenuated protection by repetitive PC (%IS/AR=28.3±3.6). Infarct sizes in non-preconditioned hearts were not modified by PolyB, Stauro, or SPT. The ratio of membrane fraction PKC activity to cytosolic fraction PKC activity was elevated by repetitive PC plus 5 min ischemia, and this change in PKC was inhibited in hearts given PolyB and Stauro. Conclusions: In contrast to single PC, repetitive PC protects the heart against infarction even when PolyB and Stauro are administered to inhibit PKC during ischemic insult. This difference may be attributable to a PKC-independent mechanism, in which the adenosine receptor may be partly involved.

KEYWORDS Protein kinase C; Adenosine receptor; Myocardial infarction; Rabbit


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Exposing cardiomyocytes to a brief period of ischemia significantly enhances their tolerance against subsequent ischemic insult [1, 2]. Although this phenomenon, termed preconditioning, has been confirmed in virtually every animal model, its mechanism is still unclear. The involvement of adenosine A1 [2], bradykinin B2 [3, 4]and opioid [5]receptors in triggering the mechanism of preconditioning has been indicated, but signal pathways downstream to those receptors are controversial. Recently, Downey et al. [2, 4, 6]proposed a hypothesis that protein kinase C (PKC) activity in myocytes is up-regulated by A1, B2 and opioid receptors during preconditioning and that this kinase phosphorylates proteins during the early phase of ischemic insult, leading to enhancement of myocardial tolerance against lethal cell injury. This PKC hypothesis was supported by several studies showing that PKC inhibitors given between preconditioning and the onset of sustained ischemia rendered the heart refractory to preconditioning [4, 6–10]and that PKC activators administered prior to ischemia limited infarct size [6, 7]. On the other hand, there are studies arguing against the PKC hypotheses [11–13]. Przyklenk et al. [11]reported that PKC inhibitors failed to block infarct size limitation by preconditioning in the dog. In a study using pig hearts by Vogt et al. [12], preconditioning with 2 cycles of ischemia/reperfusion caused translocation of PKC, but PKC inhibitors did not alter the infarct size limitation by preconditioning. Most recently, Valhause et al. [13]also showed that preconditioning in the pig heart was not inhibited by staurosporine. The reason for these reported discrepant effects of PKC inhibitors remains unclear.

An important question is whether PKC activity is linearly related to infarct size reduction or whether there is a threshold of PKC activity for cardioprotection by preconditioning. However, it is a methodologically difficult question to answer, because earlier observations of alterations in PKC activity by preconditioning are contradictory [8, 11, 12, 14, 15], perhaps because of unidentified problems in PKC assay methods. Thus, to obtain an insight into the relationship between PKC activity and anti-infarct tolerance, the present study was designed to examine whether the effect of PKC inhibitors on the cardioprotection of preconditioning is the same and independent of preconditioning protocols as long as the protocols afford the same extent of protection. To test this possibility, we assessed the effects of polymyxin B and staurosporine on preconditioning with a single and two cycles of 5 min ischemia/5 min reperfusion, since both preconditioning protocols limit infarct size by approximately 80% in the rabbit [16]. The effect of 8-sulfophenyltheophylline (SPT), an adenosine receptor blocker, on single and repetitive preconditioning was also assessed to analyze the relationship between PKC and adenosine receptors. To confirm the sufficiency of polymyxin B for inhibiting PKC in vivo, the plasma level of this agent after administration was determined in a separate group of rabbits. In addition, the effect of repetitive preconditioning on tissue PKC during myocardial ischemia was assessed in rabbits treated with or without PKC inhibitors.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experiment 1. Effect of PKC inhibitors and SPT on infarct size-limiting effect of single and repetitive preconditioning
2.1.1 Surgical preparation
The present investigation conforms with the Guidelines of Sapporo Medical University on research animal use and the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985). A rabbit model of myocardial infarction was prepared as in our previous studies [16–19]. In brief, male Japanese White rabbits were anesthetized with intravenous pentobarbital (40 mg/kg), tracheotomized, and ventilated with a Harvard respirator with room air and oxygen supplement. An additional bolus (5–10 mg) of pentobarbital was given when necessary to maintain anesthesia at the level where the corneal reflex disappeared. The tidal volume was 15 min per stroke and the ventilation rate was approximately 30 strokes per min. The ventilation rate and oxygen supplement were adjusted to maintain arterial PO2 and pH in the ranges of 90–120 mmHg and 7.35–7.50, respectively. A catheter was inserted into the carotid artery for blood pressure monitoring, and another catheter was placed in the jugular vein for administration of drugs. Bipolar electrodes were placed across the chest to monitor precordial ECG. The chest was opened via left thoracotomy, and 4-0 silk thread was passed around the marginal branch of the left coronary artery. The silk thread was passed through a small vinyl tube to make a coronary snare for coronary occlusion. After surgical preparation was completed, 1000 units of heparin were injected intravenously. Body temperature was monitored by a rectal thermometer and maintained at 37.7–38.5°C by a heating lamp.

2.1.2 Experimental protocol
A total of 91 rabbits were used in the present experiment, and all rabbits underwent 30 min of coronary artery occlusion and 3 h of reperfusion. In the present protocols, ‘single preconditioning’ and ‘repetitive preconditioning’ consisted of a single episode of 5 min ischemia followed by 5 min reperfusion and 2 cycles of 5 min ischemia separated by 5 min reperfusion, respectively. As shown in Fig. 1, rabbits were divided into 12 groups to assess the effects of polymyxin B, staurosporine, and SPT on single and repetitive preconditioning. The Control group received no pretreatment, and the PCx1 group and PCx2 group were subjected to single and repetitive preconditioning, respectively. Earlier studies indicated that PKC activity during sustained ischemia but not during preconditioning ischemia is crucial for single preconditioning to be cardioprotective [6, 20]. Thus, the timings of administration of PKC inhibitors (polymyxin B and staurosporine) in the present study were selected to inhibit primarily PKC activity during sustained ischemia but also that during the second preconditioning ischemia. The PolyB group was treated with polymyxin B alone (25 mg/kg 15 min before ischemia), and the PolyB/PCx1 group received polymyxin B 1 min after single preconditioning. In the PolyB/PCx2 group, polymyxin B was injected 1 min after the first preconditioning ischemia of the repetitive preconditioning protocol. The duration of hypotension after staurosporine injection was shorter than that after polymyxin B, suggesting that PKC inhibitory action of the former inhibitor is shorter than the latter. Thus, staurosporine was administered in two boluses. The Stauro group was given 100 µg/kg of staurosporine (50 µg/kg i.v. at 15 min and 5 min before the sustained ischemia) alone. The Stauro/PCx1 group received 50 µg/kg of staurosporine 5 min before and also 1 min after single preconditioning. In the Stauro/PCx2 group, the heart was subjected to repetitive preconditioning, and 50 µg/kg of staurosporine was injected 1 min after both the first and the second preconditioning ischemia. The SPT group received 15 mg/kg of SPT alone, and the SPT/PCx1 and SPT/PCx2 groups were preconditioned with single and repetitive preconditioning, respectively, after SPT injection. SPT (15 mg/kg) was administered 20 min before the 30-min ischemia in the SPT group, and 10 min before preconditioning in the SPT/PCx1 and SPT/PCx2 groups.


Figure 1
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  		Fig. 1 Experimental protocol. Control group, one 30-min coronary occlusion; PCx1 group, preconditioning with 5 min ischemia/5 min reperfusion; PCx2 group, preconditioning with two cycles of 5 min ischemia/5 min reperfusion; PolyB group, polymyxin B bolus (25 mg/kg, i.v.) 15 min before the 30-min ischemia; PolyB/PCx1 group, polymyxin B bolus 1 min after single preconditioning; PolyB/PCx2, repetitive preconditioning plus polymyxin B bolus 1 min after the first preconditioning ischemia; Stauro group, staurosporine bolus (50 µg/kg, i.v.) at 15 min and 5 min before the 30-min ischemia; Stauro/PCx1, staurosporine bolus 5 min before and 1 min after single preconditioning; Stauro/PCx2, staurosporine bolus 1 min after the first and the second preconditioning; SPT, 8-sulfophenyltheophylline bolus (15 mg/kg, i.v.) 20 min before the 30-min ischemia; SPT/PCx1, 8-sulfophenyltheophylline bolus 10 min before single preconditioning; SPT/PCx2 group, 8-sulfophenyltheophylline bolus 10 min before repetitive preconditioning.

 
Although all of the 91 rabbits were not completely randomized, we randomly prepared untreated controls, preconditioning controls, and inhibitor-treated rabbits for assessing the effect of each class of inhibitors (i.e., SPT or PKC inhibitors).

2.1.3 Determination of infarct size and size of area at risk
After 3 h of reperfusion, the rabbits were heparinized with 2000 units of heparin and killed with a lethal dose of intravenous pentobarbital. The heart was quickly excised and processed for postmortem analysis. The excised heart was mounted onto a Langendorff apparatus and perfused with saline for approximately 10 s to wash out blood in the coronary vessels. The coronary branch was re-occluded by the coronary snare, and Monastral blue dye was injected into the perfusion line to negatively mark the region at risk. The heart was frozen and sliced into approximately 2-mm sections parallel to the atrio-ventricular groove. The heart sections were incubated in 100 mM phosphate buffer (pH=7.4) with 1% triphenyltetrazolium for 20 min to visualize infarcts [21]. The stained sections were placed in a glass press, which uniformly compressed the slices into a thickness of 2 mm. The press was overlaid with a clear acetate sheet, and the infarct and area at risk were traced on the acetate sheet. After enlarging by 200% using a Xerox copy machine, the heart traces were read by a Macintosh Quadra computer using a scanner (Hewlett Packard ScanJett IIcx), and the areas of infarct and risk zone were measured by NIH Image, an image analysis software package. The volumes of infarct and risk zone were calculated by multiplying their area by the thickness of the heart slice (i.e., 2 mm).

2.1.4 Exclusion criteria
Rabbits that developed irreversible ventricular fibrillation during ischemia or reperfusion as well as those that failed to maintain diastolic blood pressure above 40 mmHg during the reperfusion period were excluded. This diastolic pressure criterion is based on the results of our previous study [19], which suggested that hypotension below this level may cause myocardial hypoperfusion during reperfusion, resulting in extension of infarction.

2.2 Experiment 2. Determination of polymyxin B level after intravenous administration in the rabbit
Four rabbits were anesthetized and ventilated and catheters were placed in the jugular vein and carotid artery as in Experiment 1 except that thoracotomy was not performed. After blood pressure and heart rate were stabilized, 25 mg/kg of polymyxin B was administered intravenously. At 5 and 15 min after the polymyxin B injection, 1 ml of blood was sampled through the arterial catheter and centrifuged at 4°C to separate plasma, which was stored at –20°C until assay. Polymyxin B concentration in the plasma was determined by the officially accepted microbiological assay [22].

2.3 Experiment 3. Effect of repetitive preconditioning and PKC inhibitors on PKC activity during ischemic insult
2.3.1 Surgical preparation and experimental protocol
Rabbits were prepared as in Section 2.1, except that a catheter was place in the left atrium. Rabbits were divided into four groups: Control, PC, PolyB/PC, and Stauro/PC groups. The PC, PolyB/PC and Stauro/PC groups received the same pre-ischemic treatments as that of PCx2, PolyB/PCx2, and Stauro/PCx2 groups in Experiment 1 (see Fig. 1), respectively. After the pre-ischemic treatments, the coronary artery was occluded by the coronary snare in these preconditioned groups. Five min after the onset of ischemia, Monastral blue dye was injected through the catheter inserted into the left atrium, and the heart was excised and immediately soaked into ice-cold saline. Myocardial tissue in the ischemic region, which is not stained by the blue dye, was quickly sampled and frozen in liquid nitrogen. The Control group received neither preconditioning nor PKC inhibitors, and the heart was excised immediately after the coronary occlusion and blue dye injection. Tissue sampling and processing were performed in the same way as in the other three study groups.

2.3.2 PKC assay
Subcellular fractionation and PKC assay were performed as in a previous study [23]. In brief, the frozen heart samples were homogenized in 5 volumes of STE buffer with protease inhibitors (i.e., 320 mM sucrose, 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM β-mercaptoethanol, 20 µM leupeptin, 0.15 µM pepstatin A, 0.2 mM phenylmethanesulfonyl fluoride, 50 mM NaF, 1 mM orthovanadate). An equal volume of STE buffer was added to the homogenate, centrifuged at 1000xg for 10 min, and the supernatant was re-centrifuged at 100 000xg for 60 min. The 100 000xg pellet and the 100 000xg supernatant were referred to particulate and supernatant fractions, respectively. The supernatant fraction corresponds to the cystolic fraction and the particulate fraction to membrane vesicles and mitochondria [23]. The particulate fraction was then treated with 0.3% Triton X-100 before assay. The 1000xg pellet, which consisted of nuclei and myofibrils [23], was not used for PKC assay in the present study.

The PKC activity was determined by a Peg TagTM PKC assay kit (Promega, Madison, WI, USA). In this assay, 17.5 µl of buffer containing 20 mM Tris-HCl, pH 4, 10 mM MgCl2, 1 mM dithiothreitol, 1.5 mM CaCl2, 1 mM ATP, 20 µg/ml of phosphatidylserine, 0.08 mg/ml of specific fluorescent peptide for PKC (PLSRTLSVAAK), and the extract (14 µg protein from the supernatant fraction and 15 µg protein from the particulate fraction) were incubated for 30 min at 30°C. After incubation, the specimens were mixed with 50% glycerol, heated at 95°C for 10 min, and applied to agarose gel electrophoresis in buffer containing 20 mM Tris-HCl, pH 8.0 to separate the phosphorylated and unphosphorylated peptides. The fluorescent peptides were identified under UV light and the spots were quantified by densitometry. Specific activity was calculated from the density of the phosphorylated bands and expressed in arbitrary units.

2.3.3 Chemicals
Eight-sulfophenyltheophylline was purchased from Research Biochemical International, U.S.A. Polymyxin B and staurosporine were obtained from Sigma, U.S.A.

2.3.4 Statistical analysis
Alteration of hemodynamic parameters throughout the experiment and their inter-group differences were tested by two-way repeated measures analysis of variance (ANOVA). The heart weight, risk area size and infarct size were compared between the study groups by one-way ANOVA. When an overall difference was indicated by ANOVA, a multiple comparison was performed by the Student–Newman–Keul post-hoc test. These statistical analyses were performed by SigmaStatTM (Jandel Scientific Software, San Rafael, CA). Linear regression lines were calculated by the least squares method, and the differences in regression lines were tested by analysis of co-variance. The difference was considered significant at p<0.05. All data were presented as mean±SEM.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Experiment 1
3.1.1 Exclusion of rabbits
Of 91 rabbits used in the present experiments, three were excluded according to the exclusion criteria: one rabbit in the PCx2 group for intractable ventricular fibrillation and two rabbits according to the diastolic pressure criterion (one each in the Control and SPT/PCx1 groups). Thus, 88 rabbits contributed to the following analysis.

3.1.2 Hemodynamic data
Hemodynamic parameters are summarized in Table 1. Baseline heart rate and mean blood pressure were comparable in all study groups (p=NS). The time-course of the heart rate during ischemia and reperfusion did not significantly differ between the groups, though the heart rate was slightly reduced after injection of polymyxin B as in the previous studies [6, 18]. Mean arterial blood pressure decreased after ischemia/reperfusion, and the difference between the Control group and any other group did not reach statistical significance. Administration of polymyxin B and staurosporine reduced the mean blood pressure, though hypotension was more marked and long-lasting after polymyxin B injection.


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  		Table 1 Summary of hemodynamic data in Protocol 1

 
3.1.3 Infarct size data
Table 2 summarizes heart weight, the size of area at risk and infarct size. There was no significant difference in the heart weight and the size of area at risk, which is a baseline determinant of infarct size. Both single preconditioning (PCx1) and repetitive preconditioning (PCx2) limited infarct size to the same extent (%IS/AR=9.8±1.9 and 10.4±2.3, respectively) from the control value of 44.5±3.4. Similar limitation of infarct size by single and repetitive preconditioning was also shown by a scatterplot of infarct size against risk area size (Fig. 2): regression lines of the risk area size–infarct size relationship were shifted similarly downwards by single and repetitive preconditioning. Infarct size in non-preconditioned heart was not changed by polymyxin B and staurosporine, and hypotension by these agents did not correlate with infarct size. Both polymyxin B and staurosporine abolished infarct size limitation by single preconditioning (%IS/AR=43.9±2.7 in PolyB/PCx1 group, 31.5±3.2 in Stauro/PCx1 group), which is consistent with a report by Ytrehus et al. [6]. In contrast, cardioprotection by repetitive preconditioning was not blocked by polymyxin B or by staurosporine. The %IS/AR was 21.6±3.0 in the PolyB/PCx2 group and 11.4±4.3 in the Stauro/PCx2 group, both of which were significantly smaller than %IS/AR in the Control, and their drug controls. The difference between single and repetitive preconditioning in the response to PKC inhibitors was confirmed by analysis of the risk area size–infarct size relationship. As shown in Fig. 3Fig. 4, polymyxin B and staurosporine blocked the downward shift of the relationship by single preconditioning, but a shift of the regression line by repetitive preconditioning was observed in the groups treated with these PKC inhibitors.


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  		Table 2 Summary of infarct size data in Protocol 1

 

Figure 2
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  		Fig. 2 Effect of single and repetitive preconditioning on the risk area size–infarct size relationship. The slopes of the regression lines in the PCx1 and PCx2 groups (Y=0.029+0.061X, Y=–0.051+0.175X) were significantly smaller than that in the Control group (Y=–0.123+0.602X, r=0.862, p<0.05). The two regression lines in the PCx1 and PCx2 groups were essentially superimposable, indicating the same extent of infarct size-limiting effects of single and repetitive preconditioning.

 

Figure 3
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  		Fig. 3 Effect of polymyxin B on the risk area size–infarct size relationship in preconditioned and non-preconditioned hearts. The regression lines were similar in the Control, PolyB (Y=–0.254+0.825X, r=0.926, p<0.05) and PolyB/PCx1 (Y=–0.178+0.652X, r=0.931, p<0.05) groups. The regression line in the PolyB/PCx2 group (Y=–0.104+0.369X, r=0.805, p<0.05) was significantly shifted downward to the control regression line (p<0.05 for the y-intercept).

 

Figure 4
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  		Fig. 4 Effect of staurosporine on the risk area size–infarct size relationship in preconditioned and non-preconditioned hearts. There were no significant differences in the regression line between the Control, Stauro (Y=–0.088+0.493X, r=0.994, p<0.05) and Stauro/PCx1 (Y=–0.127+0.478X, r=0.963, p<0.05) groups. The slope of the regression line in the Stauro/PCx2 group (Y=–0.187+0.360X, r=0.554) was significantly smaller than those in the Control and Stauro groups.

 
While SPT alone did not significantly modify %IS/AR (57.3±4.2), this adenosine receptor blocker inhibited infarct size limitation by single and repetitive preconditioning (%IS/AR=44.4±4.4 and 28.3±3.6%, respectively). However, the inhibitory effect of SPT was less on repetitive preconditioning than on single preconditioning. The attenuation of preconditioning-induced cardioprotection by SPT was confirmed by analysis of the effect of SPT on the risk area size–infarct size relationship (Fig. 5). The regression line for the risk area size–infarct size relationship was not statistically different between the Control, SPT, and SPT/PCx1 groups. The slope of the regression line in the SPT/PCx2 group was significantly smaller than those in the Control and SPT groups but larger than that in the PCx2 group shown in Fig. 2. These findings indicate that SPT achieved a complete blockade of single preconditioning and a partial blockade of repetitive preconditioning.


Figure 5
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  		Fig. 5 Effect of 8-sulfophenyltheophylline on the risk area size–infarct size relationship in preconditioned and non-preconditioned hearts. There were no significant difference in the regression line between the Control, SPT (Y=–0.231+0.838X, r=0.895, p<0.05) and SPT/PCx1 (Y=0.138+0.259X, r=0.616, p=NS) groups. The slope of the regression line in the SPT/PCx2 (Y=–0.008+0.299X, r=0.758, p<0.05) was significantly smaller than those in the Control and SPT groups. However, the slope of the regression line in the SPT/PCx2 group was significantly larger than that in the PCx2 group (shown in Fig. 1), suggesting that SPT attenuated infarct size limitation by the PCx2 protocol.

 
3.2 Experiment 2
Baseline heart rate and mean blood pressure in Experiment 3 were 273±5 beats per min and 88±5 mmHg, respectively, and alteration in these parameters after polymyxin B injection in this series of experiments were similar to those in the PloyB group in Experiment 1. The plasma polymyxin B level was 97.8±12.7 µM at 5 min and 52.4±1.3 µM at 15 min after administration.

3.3 Experiment 3
PKC activity data are summarized in Table 3. Significant differences were not detected between PKC activity in the Control, PC, PolyB/PC, and Stauro/PC groups in either the particulate or supernatant fraction. However, the ratio of PKC activity in the particulate fraction to that in the supernatant fraction was 0.993±0.257 in the PC group, which was significantly higher than in the Control group (0.339±0.080) and Stauro group (0.272±0.043). The PKC activity ratio in the PolyB/PC group (0.702±0.135) was not significantly different from the values in the control and PC groups.


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  		Table 3 PKC activity in the myocardial tissue

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The main observation in the present study is that polymyxin B and staurosporine failed to block the infarct size-limiting effect of repetitive preconditioning, although the same doses of these two PKC inhibitors abolished the effect of single preconditioning as shown in earlier studies using rabbits [6, 9]. These results suggest that repetition of preconditioning makes the preconditioned myocardium resistant to PKC inhibitors and that the importance of PKC during ischemia for anti-infarct tolerance may depend on the number of preconditioning episodes. Another possible explanation is that PKC activity may be enhanced by repetition of preconditioning, which can overwhelm the inhibitory action of the PKC inhibitors. However, this explanation is argued against by two lines of evidence. Firstly, Experiment 2 strongly suggests that PKC activity in the heart was sufficiently blocked by polymyxin B during the second preconditioning and sustained ischemia in Experiment 1. At 15 min after injection of 25 mg/kg of polymyxin B, which corresponds to the onset of sustained myocardial ischemia in the infarct size experiments, the plasma level of this agent was 52.4±1.3 µM. Since the rabbit heart lacks significant native coronary collaterals [24], the polymyxin B level in the myocardium would have been maintained at approximately 50 µM until reperfusion. This level is well above Ki of polymyxin B for PKC in the heart (1.8 µM in the bovine myocardium) and blood vessels [25, 26], and previous studies using isolated rabbit hearts demonstrated that 50 µM of polymyxin B completely abolished infarct size limitation by single preconditioning and preconditioning mimetics (i.e., bradykinin and phenylephrine) in vitro [4, 6, 20].

Secondly, both polymyxin B and staurosporine inhibited the effect of repetitive preconditioning on the distribution of intracellular PKC during ischemia in Experiment 3. Since PKC activity in the present study was determined in the presence of phosphatidylserine and calcium, the value of PKC activity is probably close to its maximum and actually represents an estimate of PKC proteins. Thus, the failure of polymyxin B and staurosporine to suppress absolute PKC activity in the particulate fraction does not necessarily argue against successful inhibition of PKC activity by these agents in vivo. For the same reason, the fact that there was no significant difference in PKC activity in the particulate fraction between the Control and PC groups does not exclude the possibility that activity of this enzyme is upregulated by preconditioning in vivo. This is a limitation in the PKC assay we employed, as was the case in an earlier rabbit study [15]. Nevertheless, the ratio of PKC activity in the particulate fraction to that in the supernatant fraction (Table 3) is an index of the intracellular distribution of PKC, although this ratio is not equal to the percentage of translocated PKC. The increase in this PKC ratio in the preconditioned myocardium suggests translocation of PKC from cytosol to the membrane and possibly to nuclei, of which PKC activity (i.e., the activity in the 1000xg pellet) was not determined. Furthermore, the change in the particulate PKC-supernatant PKC ratio by preconditioning was attenuated by polymyxin B and completely abolished by staurosporine (Table 3). These results are consistent with a report that PKC inhibitors blocked ischemia-induced PKC translocation in isolated rat hearts [27], and confirm the efficacy of polymyxin B and staurosporine in the rabbit heart in vivo.

It is not clear how anti-infarct tolerance induced by repetitive preconditioning is preserved after treatment with PKC inhibitors. However, involvement of adenosine receptors in this cardioprotection is suggested by the result that SPT significantly attenuated the infarct size-limiting effect of repetitive preconditioning (Fig. 5). This finding is somewhat surprising, since previous studies, including ours [2, 18], indicate that PKC activation is probably a signal downstream to the adenosine (A1/A3) receptor as well as to bradykinin B2 and opioid receptors [14, 28]. However, if we assume that adenosine receptor activation induces both PKC-dependent and -independent protective mechanisms during repetitive preconditioning, it explains not only the attenuation of repetitive preconditioning by SPT but also why inhibitors of other PKC-linked receptors (i.e., bradykinin B2 and opioid receptors) abolish the protection by single preconditioning but not by repetitive preconditioning [4, 28].

It should be noted that SPT did not completely abolish the infarct size-limiting effect of repetitive preconditioning. This result cannot be attributable to a possible insufficiency in the dose of SPT, because repetition of brief ischemia suppresses elevation of the interstitial adenosine level during subsequent ischemia [29, 30]. In our series of experiments using rabbits [30], elevation of the adenosine level in the microdialysate from the myocardium during the second episode of 5 min ischemia was approximately 50% of its level during the first episode of 5 min ischemia. Thus, it is unlikely that adenosine produced during repetitive preconditioning ischemia overwhelmed the effect of SPT at the dose sufficient to make the heart refractory to single preconditioning. Accordingly, the partial blockade of repetitive preconditioning by SPT may suggest that the resistance of a repetitively preconditioned myocardium to PKC inhibitors cannot be explained by adenosine receptors alone.

The present study showed that a difference in the number of repetitions of preconditioning ischemia may be one of the reasons for the discrepancy in the literature concerning the effect of PKC inhibitors on preconditioning. However, differences in the preconditioning protocol cannot explain all of the contradictory findings. Successful inhibition of repetitive preconditioning by PKC inhibitors was observed in rat models of infarction [7, 10], while staurosporine failed to abolish infarct size limitation by single preconditioning in the swine [13]. However, it should be noted that the rat differs from other species in a number of features of preconditioning. In contrast with the rabbit [16]and dog hearts [31], the rat heart cannot be consistently protected against infarction by a single cycle of ischemia/reperfusion [32]. Furthermore, preconditioning in this species is rather resistant to glibenclamide [32, 33]and adenosine receptor blockers [32, 34], in contrast with the results in the dog [35, 36]and swine [37]. Thus, it appears that there are species differences in the response of PC to PKC inhibitors, although the subcellular mechanism of the species difference remains unclear.

However, neither differences in the protocol of preconditioning nor difference in animal species can explain the different results obtained in two studies using canine models of infarction. Activation of PKC by preconditioning and inhibition of the preconditioning effect by PKC inhibitors were reported by Kitakaze et al. [8]but not by Przyklenk et al. [11]. They used the same anesthetics, four cycles of ischemia/reperfusion to precondition the heart, and reasonably selective inhibitors of PKC. One methodological difference that may be responsible for the different results is the placement of an extracorporeal carotid–coronary bypass tube in the study by Kitakaze et al. [8], which required heparinization. The use of a large dose of heparin, which can inhibit PKC [38, 39], might have modified the effects of PKC inhibitors used in their preparation.

In conclusion, the present study demonstrated that the effect of PKC inhibitors on the infarct size-limiting effect of preconditioning differs depending on the number of preconditioning episodes employed. Protection by repetitive preconditioning was more resistant to PKC inhibitors than that afforded by single preconditioning. This may be attributable to a PKC-independent mechanism provoked by repetition of preconditioning, in which adenosine receptors may be partly involved.

Time for primary review 21 days.


   Acknowledgements
 
This study was supported in part by Grants-in-Aid for Research (Nos. 04670547 and 08670812) from the Ministry of Education, Science and Culture, Japan. Technical assistance for polymyxin B assay by Dr. K. Sekiguchi, Pfizer Drug Research Institute, Japan is gratefully acknowledged.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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