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Am J Physiol Heart Circ Physiol 283: H440-H447, 2002. First published March 28, 2002; doi:10.1152/ajpheart.00434.2001
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Vol. 283, Issue 1, H440-H447, July 2002

Opening of mitochondrial KATP channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

Yoshito Ohnuma, Tetsuji Miura, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Akihito Tsuchida, and Kazuaki Shimamoto

Second Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined whether the mitochondrial ATP-sensitive K channel (KATP) is an effector downstream of protein kinase C-epsilon (PKC-epsilon ) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 ± 6.8% of the left ventricle to 20.3 ± 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 ± 4% of the total to 60 ± 4%, whereas no translocation was observed for PKC-delta and PKC-alpha . In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial KATP blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial KATP opener, infused 10 min before ischemia limited infarct size to 5.2 ± 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial KATP opening by PKC, the present results suggest that mitochondrial KATP-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.

mitochondria; protein kinase C; infarct size


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PHENOMENON of a transient sublethal episode of ischemia affording the myocardium a marked tolerance to subsequent lethal ischemia is termed ischemic preconditioning (PC), and its mechanism has been a subject of intensive investigation over the past decade (6, 20, 21). Involvement of Gi/Gq-coupled receptors as triggers in the PC mechanism has been established (6, 20), and studies to characterize the signal transduction downstream of these receptors have been carried out in a number of laboratories. Furthermore, a substantial amount of evidence indicates that protein kinase C (PKC) activation (15, 17, 19, 24, 40, 42) and opening of mitochondrial ATP-sensitive potassium (KATP) channels (1, 3, 8, 10, 11, 36, 41) are crucial steps in the mechanism of PC. However, the interrelationship between these two has not been fully elucidated. Initially, it was proposed that PKC, which is activated by G protein-coupled receptors, induces opening of the mitochondrial KATP channel, and this hypothesis is supported by two lines of evidence. First, a PKC-activating phorbol ester increased the mitochondrial KATP channel activity, which was monitored by flavoprotein oxidation, in isolated cardiomyocytes (30). Second, we found that infarct size limitation by a selective mitochondrial KATP channel opener (diazoxide) was abolished by a selective inhibitor of the mitochondrial KATP channel (5-hydroxydecanoate, 5-HD) but not by a PKC inhibitor (calphostin C), although both of these inhibitors completely blocked cardioprotection afforded by preischemic activation of a PKC-coupled receptor, adenosine A1 receptor (18).

However, recent studies have suggested that the mitochondrial KATP channel also plays a role in triggering the PC mechanism by producing free radicals (7, 25, 34). Pain et al. (25) reported that transient infusion of a mitochondrial KATP channel opener (diazoxide) mimicked the infarct size-limiting effect of PC, even with a 30-min washout period between the diazoxide infusion and onset of ischemic insult. This effect of diazoxide was abolished by coinfusion of free radical scavengers, suggesting that diazoxide contributes to PC by generation of free radicals. Furthermore, Forbes et al. (7) showed that free radical generation, which was monitored by dichlorofluorescin, was significantly increased by diazoxide in isolated cardiomyocytes. However, whether the mitochondrial KATP channel opening alone is responsible for free radical generation by PC remains unclear. Also, it has not been determined whether opening of the mitochondrial KATP channel is necessary for PKC activation by PC, although pharmacological evidence suggests that the role of free radicals in PC is induction of PKC activation (2).

Accordingly, the present study was aimed to clarify the relationship between activation of PKC and opening of mitochondrial KATP channels in the mechanism of cardioprotection by PC in a rabbit model of myocardial infarction. PKC isoforms that contribute to PC are species dependent, being the alpha -isoform in the dog (14), delta - and epsilon -isoforms in the rat (12, 17, 38), and the epsilon -isoform in the rabbit (15, 28). Therefore, we primarily focused on PKC-epsilon and examined 1) whether both infarct size limitation and translocation of PKC-epsilon afforded by PC are modified by a blockade of the mitochondrial KATP channel, and 2) whether cardioprotection by mitochondrial KATP channel opening is accompanied by translocation of PKC-epsilon in rabbit hearts. Additionally, the effects of PC and a mitochondrial KATP channel opener on PKC-alpha and PKC-delta in this species were compared with those on PKC-epsilon .


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted in accordance with The Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and was permitted by the Animal Use Committee of Sapporo Medical University.

Experiment 1: Studies of Myocardial Infarction

Surgical preparation and perfusion of isolated hearts. Male rabbits (Japanese White), weighing 2.2~2.9 kg, were anesthetized with intravenous pentobarbital sodium (30 mg/kg), tracheostomized, and ventilated with a Harvard respirator (model 683; Harvard Apparatus) using room air and oxygen supplement. After a left thoracotomy, the heart was quickly excised, mounted onto a Langendorff apparatus with a water jacket, and perfused with nonrecirculating modified Krebs-Henseleit buffer (in mM: 118.5 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 24.8 NaHCO3, 2.5 CaCl2, and 10 glucose) at a constant pressure of 75 mmHg. The buffer was gassed with 95% O2-5% CO2, resulting in pH of 7.4-7.5, and the temperature of the perfusate was maintained at 38°C. A fluid-filled latex balloon with a polyethylene-160 tube was inserted into the left ventricle and was connected to an SCK-580 transducer. Baseline left ventricular end-diastolic pressure was adjusted to <5 mmHg. Coronary flow was measured by timed collection of perfusate dripping from the heart. The heart was excluded from the study if the left ventricular systolic pressure was <70 mmHg or if arrhythmias persisted after a 20-min stabilization period.

Experimental protocols. After stabilization, all hearts underwent 30 min of global ischemia and 2 h of reperfusion. Global ischemia was achieved by complete interruption of coronary perfusion. Before global ischemia, each heart was subjected to one of the following six treatments: no pretreatment (control group); two cycles of 5-min global ischemia-5- min reperfusion (PC group); infusion of 100 µM 5-HD, a selective mitochondrial KATP channel blocker, for 5 min before ischemia (5-HD group); a combination of PC and 5-HD infusion (PC + 5-HD group); infusion of 100 µM diazoxide, a selective mitochondrial KATP channel opener, for 10 min before ischemia (diazoxide group); and infusion of 200 nM calphostin C, a PKC blocker, for 15 min and 100 µM diazoxide for 10 min before ischemia (calphostin C + diazoxide group).

We selected 100 µM as the dose of diazoxide for the following two reasons. First, cardioprotection by diazoxide was dose dependent, and 100 µM gave the maximal protection (9). Second, this dose of diazoxide has been demonstrated to indeed open the mitochondrial KATP channel in rabbit cardiomyocytes (30). The dose of calphostin C (i.e., 200 nM) is fourfold higher than its IC50 to inhibit PKC activity, and we confirmed that this dose was sufficient to inhibit PKC translocation by PC in rabbit hearts (unpublished observation). We did not set up a calphostin C control group, because our previous study (18) demonstrated that 200 nM calphostin C alone did not modify infarct size in the same rabbit model of myocardial infarction as that used in the present study.

Measurement of infarct size. Infarct size was measured as previously reported (13, 18). In brief, after 2 h of reperfusion, hearts were excised, frozen, and cut into 2-mm-thick sections from apex to base. The uppermost slices, which include valves, were not used for infarct size analysis. Infarcts in the heart slices were visualized by staining with 1% triphenyltetrazolium. The sizes of the infarct and the left ventricle were measured by computer-assisted planimetry. Their volumes were obtained by multiplying each area by 2 mm; i.e., the thickness of the heart slice.

Experiment 2: Western Blotting for PKC

Tissue sampling and sample preparation. protocol 1 . This protocol was designed to examine translocation of PKC from the cytosol to the membrane compartments by PC and diazoxide. Rabbit hearts were isolated and divided into four treatment groups as in experiment 1: control, PC, PC + 5-HD, and diazoxide groups. After measurement of basal hemodynamics, left ventricular biopsy samples (0.5~1.0 g) were taken from the hearts in each study group at three time points: after stabilization, immediately before the onset of global ischemia, and at 10 min after ischemia. Samples at these time points were quickly taken from the apical 1/4, 2/4, and 3/4 of the left ventricle, respectively, using sharp ophthalmology scissors. Immediately after sampling was performed, the tissues were frozen in liquid nitrogen and stored at -70°C until biochemical analysis. Tissue sample preparation was performed as previously reported (19). In brief, frozen heart samples were homogenized in cold buffer containing 50 mM Tris · HCl (pH 7.4), 5 mM EDTA, 10 mM EGTA, 50 mM NaF, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.3% beta -mercaptoethanol. The homogenate was centrifuged at 1,000 g for 10 min, and then the supernatant was centrifuged at 100,000 g for 60 min. The 100,000-g supernatant was designated to the cytosolic fraction, and the 100,000-g pellet was treated with 0.3% Triton X-100 and centrifuged at 10,000 g for 10 min to obtain supernatant (particulate fraction). The 1,000-g pellet, which consisted of nuclei and myofibrils, was not used in the present study. Protein concentration was determined using a Bio-Rad Protein Assay Kit (Bio-Rad; Hercules, CA).

PROTOCOL 2 . In this protocol, tissue samples were processed to selectively isolate mitochondrial fractions. Isolated rabbit hearts were perfused and pretreated, and then biopsy samples were taken after stabilization, immediately before the onset of global ischemia, and at 10 min after ischemia as in protocol 1. The mitochondrial fraction, the sarcolemma plus sarcoplasmic reticulum (SL/SR) fraction, and the cytosolic fraction of the samples were separated by the method of Chen et al. (5) with slight modification. In brief, ventricles were immediately minced in ice-cold buffer containing (in mM), 225 mannitol, 75 sucrose, 1 EGTA, 20 HEPES-KOH (pH 7.4), and a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals; Mannheim, Germany). The minced samples were homogenized for 5 s at maximal power output using a Polytron PT-MR3100 (Kinematica; Littau, Switzerland) equipped with a rotor knife of 7 mm in diameter. The homogenate was centrifuged at 1,000 g for 10 min, and then the supernatant was centrifuged at 10,000 g for 15 min. The 10,000-g mitochondrial pellet was washed twice and resuspended in MSE buffer. The postmitochondrial supernatant was centrifuged at 100,000 g for 60 min to obtain the cytosolic fraction (supernatant) and SL/SR fraction (pellet). Protein concentration was determined using a BCA Protein Assay Kit (Pierce; Rockford, IL).

PKC and cytochrome c Western immunoblotting analysis. Samples in protocol 1 were electrophoresed on a 12.5% polyacrylamide gel and then electroblotted onto polyvinylilidine difluoride membranes (Millipore, Bedford, MA). The blots were blocked with 5% nonfat dry milk in buffer containing 100 mM NaCl, 10 mM Tris · HCl (pH 7.4), and 0.1% Tween 20 for 1 h. The blots were then incubated with 1,000-fold diluted antibody against PKC-epsilon , PKC-alpha , or PKC-delta (Transduction Laboratories; Lexington, KY). These PKC isoforms were then visualized using an ECL Western blotting detection kit (Amersham; Buckinghamshire, UK) and quantified by using SigmaGel, gel analysis software (SPSS; Chicago, IL). Samples in protocol 2 were electrophoresed on 12.5% polyacrylamide gel for PKC and also on 15/25% polyacrylamide gel for cytochrome c detection. Subsequent processes for blotting and analysis were the same as those for protocol 1 samples. For cytochrome c detection, 2,000-fold diluted antibody against cytochrome c (Santa Cruz; Santa Cruz, CA) was used.

Chemicals. Diazoxide, 5-HD, and calphostin C were obtained from Sigma (St. Louis, MO). The other reagents used for preparing heart-perfusing buffer and tissue-homogenizing buffer were purchased from Katayama Chemical (Osaka, Japan).

Statistics. All data are presented as means ± SE. Differences in body weight, heart weight, and infarct size were examined by one-way analysis of variance (ANOVA) combined with the Student-Newman-Keuls post hoc test. Differences in hemodynamics in any given group were compared by two-way repeated-measures ANOVA. An ANOVA with repeated measures was used to analyze the subcellular distribution of PKC isoforms. SigmaStat (SPSS) was used to perform the statistical analysis. The difference was considered significant if the P value was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exclusion of Hearts

Ninety hearts were used in the present experiments. Five were excluded according to the exclusion criteria (i.e., 2 hearts that showed arrhythmias and 3 that failed to develop LV pressure >70 mmHg).

Experiment 1

Hemodynamic data. The hemodynamic parameters are summarized in Table 1. There were no significant differences in baseline heart rate, left ventricular developed pressure (LVDP), and coronary flow among the study groups. PC with two cycles of 5-min global ischemia-5-min reperfusion reduced heart rate and LVDP and tended to increase coronary flow before the 30-min global ischemia. Administration of diazoxide had little effect on heart rate and LVDP but significantly increased coronary flow. LVDP and coronary flow after reperfusion were significantly decreased in all groups, but recovery of LVDP was better in the diazoxide-treated hearts than that in the controls.

                              
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  		Table 1.   Hemodynamic parameters in isolated hearts (experiment 1)

Infarct size data. As shown in Table 2, body weight, heart weight, and left ventricular volume were comparable among the study groups. Infarct size as a percentage of the left ventricle (%IS/LV) in each heart is presented in Fig. 1. PC significantly reduced %IS/LV from 50.3 ± 6.8% of the control value to 20.3 ± 3.7%. This infarct size-limiting effect of PC was completely blocked by 5-HD, although 5-HD alone did not modify %IS/LV. Diazoxide significantly reduced %IS/LV to 5.2 ± 1.4%. This cardioprotective effect of diazoxide was not inhibited by pretreatment of hearts with calphostin C (%IS/LV = 7.0 ± 1.7%).

                              
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  		Table 2.   Infarct size data in experiment 1 



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  		Fig. 1.   Infarct size normalized as a percentage of the left ventricle in the study groups. Open circles represent individual data, and means ± SE are also shown for each group. Control, control group; PC, preconditioning group; 5-HD, 5-hydroxydecanoate group, Diaz, diazoxide group; Cal C, calphostin C. *P < 0.05 vs. Control.

Experiment 2

Protocol 1. There were no significant differences in heart rate, LVDP, and coronary flow among the groups before left ventricular biopsies were performed (data not shown). The percentage of particulate fraction in total (i.e., particulate fraction/cytosolic fraction plus particulate fraction; %PF) of PKC-epsilon under the baseline condition was 53 ± 3%, and it was increased to 66 ± 2% after 10 min of ischemia (P < 0.05; Fig. 2A). Because there was no significant change in %PF after the time control period in the control group (base vs. treatment in Fig. 2A), it is unlikely that our sampling method alone substantially affected PKC distribution. PC significantly increased the %PF of PKC-epsilon from 51 ± 4% at baseline to 60 ± 4%, and it was further increased to 71 ± 3% after 10 min of ischemia (both P < 0.05 vs. baseline; Fig. 2B). Infusion of 5-HD did not inhibit the increase in %PF of PKC-epsilon after PC and after 10 min of ischemia (55 ± 4% and 69 ± 2%, respectively, both P < 0.05 vs. 45 ± 6% at baseline; Fig. 2C). Diazoxide neither increased the %PF of PKC-epsilon by itself nor accelerated the increase in the %PF of PKC-epsilon after ischemia (Fig. 2D). In contrast to the alteration of PKC-epsilon , the %PF of PKC-alpha and %PF of PKC-delta were not increased either by PC, diazoxide, or ischemia (Fig. 3, A-D).


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  		Fig. 2.   Translocation of protein kinase C (PKC)-epsilon from the cytosol to the particulate fraction by preconditioning and sustained ischemia. Representative Western blot and summarized data are presented for each treatment group. Lanes 1-3, PKC-epsilon in cytosol; lanes 4-6, PKC-epsilon in particulate fractions. Lanes 1 and 4 are samples under baseline conditions (Base); lanes 2 and 5 are samples taken after treatment or time-matched controls (Tx); lanes 3 and 6 are samples taken 10 min into the sustained ischemia (I-10). Lane 7 is a positive control of PKC-epsilon obtained from rat brain lysate. A: control group; B: PC group; C: PC + 5-HD group; D: diazoxide group. *P < 0.05 vs. Base, dagger P < 0.05 vs. Tx. Summarized data are means ± SE (n = 5 in each group).



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  		Fig. 3.   Absence of significant translocation of PKC-alpha and PKC-delta after preconditioning and diazoxide infusion. Lanes 1-3, PKC in the cytosol; lanes 4-6 are PKC in the particulate fractions. Lanes 1 and 4 are samples under baseline conditions (Base); lanes 2 and 5 are samples taken after treatment (Tx); lanes 3 and 6 are samples taken 10 min into the sustained ischemia (I-10). Lane 7 is a positive control of PKC-alpha (A and B) or PKC-delta (C and D). A: PKC-alpha in the PC group; B: PKC-alpha in the diazoxide group; C: PKC-delta in the PC group; D: PKC-delta in the diazoxide group. Summarized data are means ± SE (n = 3 or 5 in each group).

Protocol 2. Cytochrome c was detected almost exclusively in the mitochondrial fractions, confirming proper separation of the mitochondria. Under the baseline condition, PKC-epsilon level in the mitochondria fraction (per gram protein) was 3% of the total. This level was 52% and 45% in the cytosolic and SR/SL fractions, respectively. Accordingly, to clearly present changes in the mitochondrial PKC-epsilon , the levels of mitochondrial PKC-epsilon after PC and drug treatments were presented as percentages of the baseline value (Fig. 4). PC increased the mitochondrial PKC-epsilon level by ~50% and tended to further increase the level after 10 min into the index ischemia. This time course of PKC-epsilon level in the mitochondria after PC and subsequent ischemia was not modified by administration of 5-HD. Diazoxide did not modify the mitochondrial PKC-epsilon level before and after 10 min ischemia. Alterations in PKC-epsilon level in the SL/SR fraction after PC and drug treatment were essentially the same as those in the particulate fractions in protocol 1 (data not shown).


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  		Fig. 4.   Time course of PKC-epsilon in the mitochondrial fraction in protocol 2. PKC-epsilon in the mitochondria increased after PC, but no further change was observed after 10 min into sustained ischemia. Administration of 5-HD did not modify PC-induced elevation of the PKC-epsilon level, and diazoxide did not mimic the effect of PC. Base, baseline; Tx, treatment; I-10, 10 min into the sustained ischemia. A: control group; B: PC group; C: PC + 5-HD group; D: diazoxide group. * P < 0.05 vs. Base. n = 3~7.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that 5-HD administered after PC ischemia completely inhibited infarct size limitation by PC without having any effect on PKC-epsilon translocation from the cytosol to the membrane fractions, including mitochondria. Furthermore, direct opening of the mitochondrial KATP channel by diazoxide limited infarct size, whereas this mitochondrial KATP channel opener neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. The cardioprotection afforded by diazoxide was not abolished by a PKC inhibitor, calphostin C.

These results indicate that translocation of PKC-epsilon is not sufficient but that activation of the mitochondrial KATP channel before ischemia is necessary for cardioprotection afforded by PC. Together with earlier findings regarding PKC-induced mitochondrial KATP channel opening (30), the present findings suggest that activation of the mitochondrial KATP channel occurs downstream from the PKC-epsilon translocation in the mechanism of PC against infarction.

Brief ischemia simultaneously activates several PKC isoforms, and different PKC isoforms have been suggested to be responsible for PC effects in various animal species (14, 17, 26-28, 31). However, two lines of evidence strongly support the notion that PKC-epsilon is the isoform relevant to PC in rabbit cardiomyocytes (15, 28). In this species, only epsilon - and eta -isoforms of PKC are activated by repetitive brief ischemia, and tolerance of the cardiomyocytes to ischemic injury was found to be correlated with translocation of PKC-epsilon but not with that of PKC-eta (28). Furthermore, a peptide selectively antagonizing PKC-epsilon receptor protein inhibited PC in isolated rabbit cardiomyocytes, whereas such an effect on PC was not detected for peptides that antagonize receptor proteins for other PKC isoforms (15). In the present experiments, the particulate fraction of PKC-epsilon was significantly increased after two cycles of 5 min of ischemia and 5 min of reperfusion and further translocation of PKC-epsilon was observed after 10 min of subsequent ischemia (Fig. 2B). This cumulative response to repetitive ischemia was similar to that in a study by Ping et al. (28), in which translocation of PKC-epsilon exhibited a dose-dependent pattern in response to the number of ischemia-reperfusion cycles. In contrast, neither PKC-alpha nor PKC-delta translocated either after PC or after 10 min of sustained ischemia (Fig. 3). These findings are consistent with earlier observations (28) and confirm that a pattern of PC-induced translocation of PKC isoforms in rabbit hearts differs from the pattern in rat hearts, in which both PKC-delta and PKC-epsilon translocates to the membrane fractions after PC (12, 17).

To examine whether PKC-epsilon directly translocates to the mitochondria after PC, we assessed the PKC level in the mitochondrial fraction in protocol 2. Consistent with the results of a recent preliminary study in the mouse (4), PKC-epsilon was detected in the cardiac mitochondria from the rabbits, although its level was substantially lower than those in the cytosol and the other membranes. PKC-epsilon in the mitochondrial fraction was significantly increased after PC, and the effects of 5-HD and diazoxide on the mitochondrial PKC-epsilon (Fig. 4) were also similar to those on PKC-epsilon in the SL/SR fraction (data not shown) and in the nonseparated particulate fractions (Fig. 2). Therefore, these results do not allow us to specify the site of PKC-epsilon translocation, which is crucial for transduction of PC-related signals. However, the findings in PKC-epsilon in the mitochondria (Fig. 4) and in the total membrane fractions (Fig. 2) indicate that both the inhibitory effect of 5-HD on PC protection and the diazoxide-induced cardioprotection (Fig. 1) are independent of translocation PKC-epsilon to the mitochondria and other membrane compartments.

There is pharmacological evidence indicating that PKC induces activation of the mitochondrial KATP channel (30). However, it is still not clear how PKC-epsilon translocation induces opening of the mitochondrial KATP channel. It is possible that PKC-epsilon translocated to the mitochondria directly regulates the mitochondrial KATP channel activity. However, we cannot exclude the possibility that PKC-epsilon translocated to the sarcolemma induces a cascade of protein kinase activation, which also contributes to facilitated opening of the mitochondrial KATP channel. Mitogen-activated protein kinases (MAPKs) (26, 29) and Src and Lck tyrosine kinases (27) are suggested to be kinases subsequently activated by PKC-epsilon . Interestingly, a p38-MAPK blocker, SB-203580, abrogated PC-induced infarct size limitation (16, 23, 29). Furthermore, anisomycin, which is an activator of p38-MAPK, mimicked PC effects on infarct size (3, 22), and this protective effect was completely inhibited by 5-HD in a study by Baines et al. (3). Therefore, p38-MAPK might be responsible for transmission of signals from PKC-epsilon to the mitochondrial KATP channel.

The results of the present study strongly support the notion that the mitochondrial KATP channel is an effector downstream of PKC in the mechanism of PC in the rabbit heart. However, a series of studies by Wang et al. (37-39) suggests that PKC activity is important for mitochondrial KATP channel opening to enhance anti-ischemic tolerance in rat hearts. In their studies (37-39), PKC inhibitors and PKC downregulation prevented both PKC-delta translocation and cardioprotection that were induced by diazoxide pretreatment in isolated rat hearts. In contrast, in rabbit hearts, neither PKC-epsilon nor PKC-delta was translocated after diazoxide infusion (Figs. 2 and 3), and PKC inhibitors (calphostin C and chelerythrime) failed to abolish the infarct size-limiting effect of diazoxide (18, 25) (Fig. 1). These discrepancies cannot be readily explained but may be due to species differences in the PC mechanism. In fact, species differences in the PC mechanism have been suggested with respect to the roles of adenosine receptors (6), PKC isoforms (12, 14, 17, 28), and the relationship between PKC and tyrosine kinase (33, 35, 42).

In the present study, we cannot totally exclude the possibility that the mitochondrial KATP channel activity during repetitive PC contributed to translocation of PKC-epsilon . Because 5-HD was infused from the end of the second PC ischemia to the onset of sustained ischemia, activity of the mitochondrial KATP channel was undisturbed during the first PC ischemia-reperfusion and the second PC ischemia. However, it is unlikely that the mitochondrial KATP channel activity during that period of the PC protocol played a major role in induction of PKC-epsilon translocation. First, a single cycle of PC did not afford significant infarct size limitation in the present model of infarction (unpublished observation). Second, translocation of PKC-epsilon was not provoked by a relatively high dose of diazoxide (Figs. 2D and 4), although the same dose of diazoxide afforded larger cardioprotection than that afforded by PC (Fig. 1). In the present study, cardioprotection was greater in diazoxide-treated hearts than in the preconditioned hearts (i.e., infarct size limitation by 90% vs. 60%), as was found in our recent study using the same isolated rabbit heart preparation (32). This difference may be explained by the possible difference in the extents of the mitochondrial KATP channel opening, because diazoxide-induced protection is dose dependent (9) and is completely abolished by 5-HD (3, 18, 32) as is cardioprotection afforded by PC (1, 6, 8).

Although PC increased PKC-epsilon in the membrane compartments before the onset of ischemia, the PKC-epsilon levels 10 min after the onset of ischemia in nonpreconditioned and preconditioned hearts were comparable (Fig. 2, A vs. B). These findings indicate the importance of the PKC-epsilon level at the time of ischemia onset and do not necessarily argue against our proposal that PKC-epsilon opens the mitochondrial KATP channels in PC. Studies on the critical timing of PKC activation and mitochondrial KATP channel opening for cardioprotection suggest that their interaction in PC may be during the very early phase of index ischemia. Yang et al. (40) demonstrated that PKC activity during the first 10 min of index ischemia is crucial for PC to be protective. Our recent study (34) using diazoxide showed that the mitochondrial KATP channel needs to be activated during the early phase of sustained ischemia to protect the cardiomyocytes from ischemic necrosis. Taken together, the results suggest that the level of PKC-epsilon translocation at the onset of ischemia is important in the PC mechanism because it enables enhanced and earlier opening of the mitochondrial KATP channels during ischemic insult.

In the present experiments, as in earlier studies (1, 8), 5-HD infused after PC eliminated anti-infarct tolerance in the preconditioned heart, indicating the importance of mitochondrial KATP channel activity during ischemia in PC-induced protection. However, in a recent study by Pain et al. (25), 5-HD administered after PC did not abolish the PC effect on infarct size. We do not have a clear explanation for this contradiction, but there are two possible reasons. First, the levels of mitochondrial KATP channel activation by PC may be different in these experiments, resulting in different responses to similar doses of 5-HD. Second, in addition to the mitochondrial KATP channel, there may be an effector of PC [such as cytoskeletal proteins (3)], and its contribution may have been predominant, masking the role of the mitochondrial KATP channel under the experimental conditions employed by Pain et al. (25). Nevertheless, the present findings (Fig. 2) suggest that anti-infarct tolerance afforded by mitochondrial KATP channel activation does not depend on PKC-epsilon translocation before and after ischemic insult in cardiomyocytes.


    ACKNOWLEDGEMENTS

This study was supported in part by a grant-in-aid for Scientific Research 13670731 (to T. Miura) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: Tetsuji Miura, Second Dept. of Internal Medicine, Sapporo Medical Univ. School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan (E-mail: miura{at}sapmed.ac.jp).

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. Section 1734 solely to indicate this fact.

First published March 28, 2002;10.1152/ajpheart.00434.2001

Received 22 May 2001; accepted in final form 26 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Auchampach, JA, Grover GJ, and Gross GJ. Blockade of ischaemic preconditioning in dogs by the novel ATP-dependent potassium channel antagonist sodium 5-hydroxydecanoate. Cardiovasc Res 26: 1054-1062, 1992[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 283(1):H440-H447
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