Introduction

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Preconditioning with metabolic inhibition (MIP), one of the consequences of myocardial ischemia, confers delayed cardioprotection (Nayeem et al., 1997). It has been shown that pretreatment with a -opioid receptor (OR) agonist, U50,488H (U50), also produces delayed cardioprotection, which mimics that of MIP, and that the protection of MIP and U50 pretreatment (UP) is attenuated and abolished, respectively, by blockade of the -OR with a selective antagonist, nor-BNI (Wu et al., 1999). The observations indicate that the delayed cardioprotection of MIP is mediated at least partly via -OR in the heart. In addition, the delayed cardioprotection of MIP or UP is abolished with blockade of PKC during preconditioning/pretreatment (Wu et al., 1999), indicating that PKC is a trigger of the delayed cardioprotection of both MIP and UP. The PKC isoform(s) involved is, however, not known.

PKC-, -, and - are the major isoforms expressed in adult rat cardiomyocytes (Rybin and Steinberg, 1994) and have been implicated in cardioprotection of ischemic preconditioning (IPC) in isolated rat heart (Mitchell et al., 1995; Yoshida et al., 1997; Kawamura et al., 1998). A recent study showed that PKC- mediates early cardioprotection of -opioid receptor stimulation (Fryer et al., 2001). On the other hand, it was found that delayed cardioprotection of administration of adrenaline in the rat and of rapid cardiac pacing in the dog against ischemia-induced arrhythmias was associated with translocation of PKC- from cytoplasm to membrane (Wilson et al., 1996). Furthermore, it was shown that IPC and nitric oxide donor-induced delayed cardioprotection against myocardial stunning was accompanied by translocation of PKC- from cytoplasm to membrane, and chelerythrine blocks both the delayed protection and translocation of the isoform in the conscious rabbit (Qiu et al., 1998; Ping et al., 1999). It is therefore likely that PKC- also mediates the delayed cardioprotection of MIP and UP.

The purpose of the present study was therefore to determine whether PKC- is involved as a trigger in delayed cardioprotection of MIP and UP against cell injury/death. We first determined the translocation of different PKC isoforms from cytosol to membrane fraction, an indication of the activation of PKC isoforms (Kraft and Erson, 1983), in isolated ventricular myocytes subjected to MIP or UP. This was correlated with the protective effects of MIP and UP against a delayed insult of lethal simulated ischemia (LSI), shown to cause myocardial injury and cell death (Zhou et al., 2001). We also compared the effects of MIP and UP with those of activation of PKC by a PKC activator. We determined the release of lactate dehydrogenase (LDH) and exclusion of trypan blue as indices of cell injury and death, respectively. The most important experiment was the use of a selective PKC- inhibitor, provided by Dr. D. Mochly-Rosen (Stanford University, Palo Alto, CA), as well as a selective PKC- inhibitor to determine the roles of PKC- and - in delayed protection of MIP and UP. The results showed that PKC- is a trigger of delayed cardioprotection of MIP and UP.

	Materials and Methods

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Experimental Protocol. Ventricular myocytes were isolated from the heart of male Sprague-Dawley rats (200-250 g) according to a procedure previously described (Dong et al., 1993). More than 70% of the cells were rod-shaped and impermeable to trypan blue. After the ventricular myocytes had been separated, they were allowed to stabilize for 30 min before the experiment started. We used a ventricular myocytes preparation and adopted a procedure described previously (Wu et al., 1999). As shown in Fig. 1, cells were subjected either to MIP, or activation of -OR or PKC. MIP was achieved by incubation of the myocytes for 30 min with a glucose-free Hepes buffer, pH 6.5, that contained 20 mM lactate and 10 mM 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis (Morris et al., 1996; Nayeem et al., 1997). Activation of -OR and PKC was achieved by incubation of the myocytes with normal Hepes buffer containing 10 µM U50, a selective agonist of -OR and 10 nM phorbol-12-myristate-13-acetate (PMA), a PKC activator, respectively. Cells were then suspended in 5 ml of Joklik's modified Eagle's medium (MEM) that contained 1 mM Ca²⁺, 0.2% bovine serum albumin, 0.01 µM insulin, 100 U/ml penicillin G, and 100 µg/ml streptomycin and incubated for 20 h in a CO₂ incubator (95% O₂, 5% CO₂) in culture dishes (Wu et al., 1999). They were then subjected to LSI (see "LSI" for details) for 2 h followed by reperfusion in MEM for another 2 h.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Experimental design: ventricular myocytes were isolated and stabilized for 30 min. They were then subjected to preconditioning with MIP or PMA or UP in the absence or presence of 1 µM nor-BNI or 5 µM chelerythrine (Che) or 5 µM rottlerin or 0.1 µM V1-2 or the negative control peptide of V1-2 for a period of 5 min before to 5 min after preconditioning/pretreatment. The cells were washed several times before they were incubated in a drug-free culture medium for 20 h and then subjected to LSI for 2 h followed by 2 h of normal incubation (reperfusion). Vehicle pretreatment (VP): cells were incubated with dimethyl sulfoxide; MIP: cells were subjected to a glucose-free Hepes buffer, pH 6.5, that contained 20 mM lactate and 10 mM 2-DOG, an inhibitor of glycolysis; UP (pretreatment with U50): cells were incubated in normal Hepes buffer containing 10 µM U50 for 30 min; and PMA (pretreatment with PMA): cells were incubated in normal Hepes buffer containing 10 nM PMA for 10 min.

LSI. Twenty hours after pretreatment, the ventricular myocytes were transferred to a glucose-free Hepes buffer supplemented with 10 mM 2-DOG, 0.75 mM sodium hydrosulfite, 12 mM KCl, and 20 mM lactate, pH 6.5, for 2 h in a CO₂ incubator as described previously (Esumi et al., 1991; Nayeem et al., 1997). The buffer contains high K⁺ and is acidic, which produces an environment that mimics myocardial ischemia and causes cell injury/death. Finally, the cells were transferred back to normal medium for further incubation for 2 h.

LDH Assay and Trypan Blue Exclusion. Ventricular myocytes were cultured in suspension (Zhou et al., 2001). LDH release and trypan blue exclusion were used as indices of cell injury (Morris et al., 1996; Nayeem et al., 1997) and viability, respectively. With the exception of one experiment, these two parameters were measured on the same culture in all experiments.

Sample Preparation and Western Blot Analysis of PKC. The ratio of membrane and cytosolic fractions was used to indicate PKC translocation. Myocardial samples were obtained at the end of treatment, i.e., 35 min after the start of MIP or U50 or PMA pretreatment (Fig. 1). The time chosen was adopted from previous studies (Qiu et al., 1998; Ping et al., 1999). A previous study has shown that the translocation of PKC-, -, and - occurred immediately after IPC and lasted for at least 10 min (Kawamura et al., 1998). Cytosolic and membrane fractions were prepared from myocytes according to methods described previously with minor modification (Rybin and Steinberg, 1994). Briefly, the cells were washed three times with normal Hepes buffer to remove dead cells and then harvested. Cells were immediately lysed in ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM -mercaptoethanol, 50 µg/ml leupeptin, 5 mM phenylmethylsulfonyl fluoride), homogenized by sonication, and centrifuged at 100,000g for 1 h at 4°C. The supernatant was removed (cytosolic fraction) and the pellet was resuspended in 0.3 ml of homogenization buffer containing 0.5% Triton X-100, shaken for 60 min at 4°C to elute the particulate proteins, and then centrifuged at 100,000g for 1 h at 4°C. The supernatant was removed as membrane fraction. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) based on the Bradford dye-binding procedure with bovine serum albumin as the standard.

Drugs and Chemicals. MEM, U50,488H, chelerythrine, PMA, type I collagenase, insulin, Hepes, bovine serum albumin, 2-DOG, lactic acid, sodium hydrosulfite, and LDH assay kit were purchased from Sigma; nor-BNI was purchased from Tocris Cookson (St. Louis, MO); and rottlerin from BIOMOL Research Laboratories (Plymouth Meeting, PA). All chemicals were dissolved in distilled water with the exception of PMA and rottlerin. PMA was dissolved in dimethyl sulfoxide at a final concentration <0.1%, at which no effect was observed. Rottlerin was dissolved in a 1:5 cocktail of ethanol/saline according to Fryer et al. (2001). The molecular weight marker, nitrocellulose membrane, and enhanced chemiluminescence kit were from Amersham Pharmacia Biotech UK, Ltd. (Little Chalfont, Buckinghamshire, UK). The peroxidase-conjugated goat antimouse IgG antibody was from DAKO (Copenhagen, Denmark). Antibodies of PKC isoforms (-, -, and -) were from Transduction Laboratories (Lexington, KY).

Statistical Analysis. All data are expressed as mean ± S.E.M. One-way analysis of variance was first carried out to test for any difference between the mean values within the same study. When a significant P value was obtained, comparisons between individual means of groups were performed by a two-tailed, unpaired Student's t test. A difference of P < 0.05 was considered significant.

	Results

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Subcellular Distribution and Translocation of PKC Isoforms after MIP or UP or Pretreatment with PMA. Figure 2 shows the representative Western blots indicating the distribution of PKC isoforms , , and  in the membrane and cytosolic fractions of ventricular myocytes at 35 min after the start of MIP or pretreatment with 10 µM U50 (UP) or with PMA. Both MIP (Fig. 2A) and UP (Fig. 2B) increased significantly the membrane/cytosol ratio of PKC- compared with that of the vehicle control, indicating the translocation of PKC- isoform from cytosolic to membrane fraction. No significant translocation of either PKC- or PKC- was observed (Fig. 2, A and B). On the other hand, PMA at 10 nM, shown to confer similar delayed cardioprotection as MIP or UP (Fig. 4A), increased the membrane/cytosol ratio of both PKC- and - (Fig. 2C).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effect of MIP (A) or UP (B) or PMA (C) on subcellular distribution of PKC isoforms in ventricular myocytes. Cytosolic and membrane fractions were prepared from myocytes 35 min after the start of preconditioning/pretreatment. Equal amounts of protein (20 µg/lane) from each sample were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting as described under Materials and Methods. Autoradiograms in the upper panels are representative of five separate experiments. The number at the right side of each tracing is the molecular mass of the marker protein. Lanes 1 and 3 correspond to cytosolic and membrane fractions, respectively, isolated from vehicle-treated cells, whereas lanes 2 and 4 correspond to cytosolic and membrane fractions, respectively, isolated from treated groups. Relative levels of PKC isoforms are assessed by densitometry (Bio-Rad Multi-Analyst/PC). Membrane/Cytosol ratios of immunoreactivity are calculated for each isoform as indices of PKC translocation. The data are expressed as mean ± S.E. (n = 5). *P < 0.05, **P < 0.01, versus VP. For experimental protocol, abbreviations, and concentrations of drugs used, refer to Fig. 1.

Effects of MIP or UP or Pretreatment with PMA on LDH Release, Percentage of Blue Cells, and Subcellular Distribution of PKC-. The LDH activity in the cultured medium and percentage of blue cells after LSI were significantly reduced by pretreatment with MIP or UP (Fig. 4, A and B) in agreement with our previous finding (Zhou et al., 2001). These were accompanied by an increased membrane/cytosol ratio of PKC- (Fig. 4, A and B, bottom). The effects of MIP and UP on LDH activity, percentage of blue cells, and translocation of PKC- were attenuated and abolished, respectively, by 1 µM nor-BNI (Fig. 3), a selective -OR antagonist, which itself had no effect (data not shown).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Effect of MIP or UP on LDH activity and blue cells induced by LSI (top) and subcellular distribution of PKC- (bottom) in the presence of nor-BNI. LDH activity in the culture medium and blue cells were measured at the end of experiment (Fig. 1.). Results are expressed as LDH released into the medium/total LDH activity and blue cells/total cells normalized to 100% for VP group (control). For PKC isoform expression, cytosolic and membrane fractions of PKC- were prepared from myocytes 35 min after the start of preconditioning/pretreatment. Equal amounts of protein (20 µg/lane) from each sample were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting as described under Materials and Methods. Autoradiograms are representative of five separate samples: lanes 1 to 3, cytosolic fraction; lanes 4 to 6, membrane fraction. Autoradiograms are representative of five separate samples. Relative levels of PKC isoforms are assessed by densitometry (Bio-Rad Multi-Analyst/PC). Membrane/cytosol ratios of immunoreactivity are calculated for each isoform as indices of PKC translocation. The data are expressed as mean ± S.E.M. (n = 5). *P, #P < 0.05 versus corresponding groups. For experimental protocol, abbreviations, and concentrations of drugs used, refer to Fig. 1.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of MIP or PMA (A) or UP (B) on LDH activity in culture medium and blue cells (top) induced by LSI and subcellular distribution of PKC- (bottom) in the presence of chelerythrine (Che). LDH activity in the culture medium and blue cells was measured at the end of experiment. Results are expressed as LDH released into the medium/total LDH activity and blue cells/total cells normalized to 100% for vehicle pretreatment group (control). For PKC isoform expression cytosolic and membrane fractions were prepared from myocytes 35 min after the start of preconditioning/pretreatment. Equal amounts of protein (20 µg/lane) from each sample were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting as described under Materials and Methods. Autoradiograms are representative of five separate samples. Relative levels of PKC isoforms are assessed by densitometry (Bio-Rad Multi-Analyst/PC). Membrane/cytosol ratios of immunoreactivity are calculated for each isoform as indices of PKC translocation. The data are expressed as mean ± S.E.M. (n = 5). *P, #P <0.05, **P, ##P < 0.01 versus corresponding groups. For experimental protocol, abbreviations, and concentrations of drugs used, refer to Fig. 1.

Effects of MIP or UP or Pretreatment with PMA on LDH Release, Percentage of Blue Cells, and Subcellular Distribution of PKC- in Presence of V1-2, a PKC- Inhibitor. To further delineate the role of PKC-, the effects of MIP and -OR stimulation were determined in the presence of a selective PKC- inhibitor, V1-2 peptide. The effects of U50 and MIP on LDH activity and percentage of blue cells (Fig. 5A) and translocation of PKC- (Fig. 5B) were significantly attenuated by V1-2 peptide, but not by the control peptide. V1-2 peptide and the control peptide themselves had no effect on LDH activity, percentage of blue cells (data not shown), and subcellular distribution of PKC- (Fig. 5B).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Effect of MIP or UP on LDH activity in culture medium and blue cells induced by LSI (A) and subcellular distribution of PKC- (B) in the presence of V1-2 peptide or the negative control peptide. The V1-2 and the control peptide both were conjugated to the cell-permeable Drosophila antennapedia carrier peptide and applied at 0.1 µM. Results are expressed as LDH released into the medium/total LDH activity and blue cells/total cells normalized to 100% for vehicle pretreatment group (control). For PKC isoform expression, cytosolic and membrane fractions were prepared from myocytes 35 min after the start of preconditioning/pretreatment. Equal amounts of protein (20 µg/lane) from each sample were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting as described under Materials and Methods. Autoradiograms are representative of six separate samples. Relative levels of PKC isoforms are assessed by densitometry (Bio-Rad Multi-Analyst/PC). Membrane/cytosol ratios of immunoreactivity are calculated for each isoform as indices of PKC translocation. The data are expressed as mean ± S.E.M. (n = 6). *P, #P < 0.05 versus corresponding groups. For experimental protocol, abbreviations, and concentrations of drugs used, refer to Fig. 1.

Effects of MIP or UP or Pretreatment with PMA on LDH Release and Percentage of Blue Cells in Presence of Rottlerin, a PKC- Inhibitor. In view of the fact that PKC- mediates early cardioprotection of -OR stimulation (Fryer et al., 2001), we determined whether this PKC isoform also mediated delayed cardioprotection of MIP or UP, making use of a specific PKC- inhibitor, rottlerin. Figure 6 shows that the effects of MIP or UP on LDH activity and percentage of blue cells were not affected by 5 µM rottlerin, in contrast to the attenuating effects of the PKC- inhibitor.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of MIP or UP on LDH activity in culture medium and blue cells induced by LSI in the absence or presence of rottlerin (5 µM). Results are expressed as LDH released into the medium/total LDH activity and blue cells/total cells normalized to 100% for vehicle pretreatment group (control). Data represent mean ± S.E.M. of six independent experiments in six different rats. *P < 0.05 versus corresponding groups.

	Discussion

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The aim of the present study was to test the hypothesis that PKC- is involved as a trigger in delayed cardioprotection of MIP and UP. The first piece of evidence in support of the hypothesis is the translocation of this isoform, but not the other two isoforms, PKC- or PKC-, from cytosolic to membrane fraction after MIP and UP, which was correlated with the delayed protective effects of both treatments against cell injury/death induced by LSI. In a subsequent and crucial experiment we found that blockade of PKC- with a selective antagonist, V1-2 peptide, before and during MIP or UP abolished the effects of both treatments on cardioprotection and translocation of PKC-. This is the first unequivocal evidence demonstrating that PKC- is activated by MIP/UP, leading to delayed cardioprotection against cell injury in the heart. In previous studies, PKC- has been shown to mediate delayed cardioprotection of preconditioning against both arrhythmias and myocardial stunning (Wilson et al., 1996; Qiu et al., 1998; Ping et al., 1999). It is likely that PKC- is the isoform that is involved in delayed cardioprotection of any kind of preconditioning.

A recent study has shown that PKC- mediates the early cardioprotection of -OR stimulation (Fryer et al., 2001). Although stimulation of -OR also confers delayed cardioprotection (Fryer et al., 1999), there is no evidence that PKC- is involved. In the present study we failed to find that blockade of PKC- with a selective PKC- inhibitor affected the delayed cardioprotection of either MIP or UP. Nor did we find that either of the two treatments induced translocation of the PKC isoform. The observations indicate that PKC- is not involved in delayed cardioprotection of MIP or UP. On the other hand, evidence from previous studies (Wilson et al., 1996; Qiu et al., 1998; Ping et al., 1999) and the present study indicates that PKC- mediates delayed cardioprotection of preconditioning. It has also been shown that cardioprotection of hypoxic preconditioning of 30 min against the insult of prolonged hypoxia in cultured rat ventricular myocytes is abolished by blockade of PKC- (Gray et al., 1997). In a subsequent study the same laboratory showed that the cardioprotection of preconditioning of rabbit ventricular myocyte with 10 min of anoxia against injuries induced by more prolonged anoxia (180 min) is also abolished by blockade of PKC-, but not other PKC-isoforms (Liu et al., 1999). The observations indicate that the PKC- also mediates immediate cardioprotection. It appears likely that PKC- may mediate early cardioprotection, whereas PKC- mediates both early and delayed cardioprotection, in the rat heart. However, the experimental design, preparations, and animals of the studies are different, which may affect the result. Further studies are needed before a conclusion can be reached.

It was found in the present study that administration of PKC- inhibitor before and during the MIP and UP could block delayed cardioprotection of MIP and UP, indicating that PKC- is required to put the heart into a preconditioned state. Similar observations were reported by Baxter et al. (1995) and Qiu et al. (1998). The role of PKC is, however, different in immediate cardioprotection of preconditioning. In a previous study, it was demonstrated that the infarct-sparing effect was blocked when the PKC inhibitor staurosporine was present during the second (sustained) ischemic insult, but not when it was administered during IPC (Yang et al., 1997). The observations indicate that the activity of PKC is required to mediate the protection but not to put the heart into a preconditioned state. The results suggest that PKC-mediated signaling events underlying early and late IPC may be different. Unlike early IPC, late IPC appears to involve changes in gene expression (Marber and Yellon, 1996) and would be the result of the activation of signal transduction pathways that control the synthesis of cardioprotective proteins.

In another study in our laboratory we showed that UP and MIP confer delayed protection against cell injury/death induced by LSI, which is correlated with increased expression of the inducible heat shock protein 70 (HSP 70). Blockade of the production of HSP 70 with a selective antisense abolishes the protective effects of MIP and UP (Zhou et al., 2001). The observations indicate that the inducible HSP 70 mediates the delayed cardioprotection of both MIP and UP. It has been shown that heat stress increases the production of HSP 72 accompanied by delayed cardioprotection and that the effects are inhibited by blockade of PKC with its inhibitor chelerythrine; however, the increased expression of the HSP is not affected by blockade of PKC with chelerythrine (Joyeux et al., 1997). The observations suggest that PKC may not directly affect the production of HSP. The relationship between PKC- and inducible HSP 70 in delayed cardioprotection of UP and MIP requires further study.

Findings from our previous (Wu et al., 1999) and present studies showed that the delayed cardioprotection of MIP is mediated via -OR and that the downstream signaling mechanism is PKC, which is in the phospholipase pathway. Because blockade of MIP with a selective -OR antagonist, nor-BNI, which completely blocks the delayed cardioprotection of -OR stimulation, only antagonized the protective effect of MIP, the protection conferred by MIP may also be mediated via other pathways. It has been shown that the delayed cardioprotection of IPC is abolished by genistein, an inhibitor of tyrosine kinase, in the rabbit, suggesting that tyrosine kinases are also involved in mediating the cardioprotection of preconditioning (Imagawa et al., 1997). It is likely that MIP activates different receptors, which in turn activate these two pathways. On the other hand, it is unlikely that UP also involves tyrosine kinases because the effects of -OR stimulation on the heart have been shown not to be affected at all upon blockade of tyrosine kinases with their inhibitors (Sheng et al., 1997).

The most important experiment of the present study was to determine the role of PKC- in mediating the delayed cardioprotective effects of MIP and UP. We made use of a selective PKC- inhibitor, V1-2 peptide, which is a small peptide of eight amino acids. It is derived from the first unique region of PKC- (amino acids 14-21), which duplicates portions of PKC- binding site contained in the first variable binding region of the PKC- molecule. It competes for binding of the PKC- to its receptor for activated C kinase, which are specific anchoring proteins within the particulate fraction (Mochly-Rosen et al., 1991; Ron et al., 1994), thus halting PKC- translocation and function (Johnson et al., 1996). We also used a control peptide, which is derived from PKC- V1 region and does not act as a PKC- inhibitor (Johnson et al., 1996). It has been shown that transient permeabilization of V1-2 peptide into cardiac myocytes inhibits translocation of PKC-, but not other PKC isoforms, induced by PMA (Johnson et al., 1996). Furthermore the PMA-mediated negative chronology, known to correlate closely with PKC- translocation (Johnson and Mochly-Rosen, 1995), is inhibited by PKC- in cardiac myocytes but not by the control peptide (Johnson et al., 1996).

In the present study we used Western blot analysis to determine the translocation of PKC isoforms from cytosol to membrane fraction, which is not the most accurate measure of PKC translocation, particularly if translocation within different organelles is to be determined. In this study we used cytosolic-to-membrane translocation as an experiment to obtain preliminary information on which isoforms may be activated after preconditioning. We found translocation of PKC- after MIP/UP, which suggests that this particular PKC isoform may mediate delayed cardioprotection of MIP/UP as a trigger. The most important experiment that followed is the blockade of PKC with a selective PKC- inhibitor, which provides definitive answer that this isoform indeed acted as a trigger of delayed cardioprotection of MIP/UP. For future study to determine translocation of PKC isoforms in different organelles, more sophisticated techniques such as immunofluorescence staining are needed.

In conclusion, the present study has provided evidence for the first time that the delayed cardioprotection of MIP and UP involves PKC- as a trigger. Further study is needed to delineate the relationship between PKC- and HSP 70, which has also been shown to mediate delayed cardioprotection of MIP and UP.