INTRODUCTION

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RECENTLY opioid receptor-mediated cardioprotection against myocardial ischemia has been demonstrated and is the subject of increasing interest. Paradis et al. (20) demonstrated increased preproenkephalin mRNA and enkephalins in the rat heart after myocardial infarction. Therefore, the ventricle may release enkephalins capable of stimulating the -opioid receptor to induce a cardioprotective signal transduction cascade within the cardiac myocyte. Additionally, Weil et al. (35) demonstrated that preproenkephalin mRNA levels were four times higher in the left versus right ventricle and suggested that the ventricle may be an endocrine organ that supplies the body with enkephalins. Indeed, Howells et al. (9) demonstrated higher levels of preproenkephalin mRNA in the ventricular myocardium than any other tissue of the rat.

Stimulation of opioid receptors has been shown to be cardioprotective against myocardial infarction and may be a trigger for ischemic preconditioning (IPC) in several species (27). Cardioprotection has been demonstrated with morphine in intact rat hearts (24), isolated rabbit hearts (16), and isolated chick cardiomyocytes (12). Morphine is primarily a µ-opioid receptor agonist; however, its cardioprotective effect does not appear to be the result of activation of the µ-opioid receptor. Indeed, Traynor and Elliott (32) suggested that the µ-opioid receptor can cross-talk with the -opioid receptor. In addition, Liang and Gross (12) demonstrated that morphine-induced cardioprotection of chick cardiac ventricular myocytes could be abolished by the selective ₁-opioid receptor antagonist 7-benzylidenenaltrexone (BNTX). Similarly, both acute (25) and delayed (4) cardioprotection in the rat heart has been attributed to stimulation of the ₁-opioid receptor.

Cardioprotection via the stimulation of ₁-opioid receptors has been shown to signal via a pertussis toxin-sensitive mechanism, implicating the involvement of a G_i/o protein (26). Additionally, morphine has been shown in an isolated rabbit heart to reduce infarct size (IS) via a chelerythrine-sensitive mechanism (16). This is in agreement with data demonstrating the involvement of protein kinase C (PKC) in IPC-induced cardioprotection (6, 21, 28, 30, 37). Finally, it has been suggested that activation of the ATP-sensitive potassium (K_ATP) channel may mediate opioid-induced cardioprotection in the rat (4, 26).

Because PKC is not a single entity but rather a family of related isoenzymes comprising at least nine different members with differences in requirements for activity, subcellular localization, and substrate specificity, it is important to determine which PKC isoform(s) mediates cardioprotection induced by opioid treatment. The PKC family of serine/threonine kinases can be divided into three distinct groups: conventional (, ₁, ₂, and ), novel (, , and ), and atypical (). These isoforms of PKC are expressed in a tissue-specific manner, and the translocation of specific PKC isoforms has been implicated in ₁-adrenergic preconditioning (17), Ca²⁺-induced preconditioning (18), and IPC (13, 11). The present study sought to examine the role of PKC in cardioprotection against myocardial ischemia induced by the selective -opioid agonists TAN-67 and D-Ala²-D-Leu⁵-enkephalin (DADLE) in the rat heart. We also examined the localization of specific PKC isoforms to multiple sites within the cell during opioid treatment in the presence or absence of the ₁-opioid receptor and PKC inhibitor BNTX and chelerythrine, respectively. Recently, a selective inhibitor of PKC- has been made available. Thus we were able to further investigate the effect of PKC- inhibition on cardioprotection and subcellular PKC localization. The results suggest that PKC is an integral component in the signal transduction cascade mediating opioid-induced cardioprotection and demonstrate that specific PKC isoforms may be translocated to distinct cellular loci after -opioid receptor stimulation. Finally, these studies suggest that PKC- is a key mediator of infarct size reduction after ₁-opioid receptor stimulation.

	METHODS

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This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.

General surgical preparation. The preparation of rats for ischemia-reperfusion studies were performed as previously described (4-6). Briefly, male Wistar rats weighing 350-450 g were anesthetized via Inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated and connected to a rodent ventilator (model CIV-101, Columbus Instruments; Columbus, OH). Rats were ventilated at 60-65 breaths/min. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5-10 mmH₂O. Arterial pH, PCO₂, and PO₂ were monitored by a blood gas system (AVL 995 pH/Blood Gas Analyzer) and maintained within a normal physiological range (pH, 7.35-7.45; PCO₂, 25-40 mmHg; and PO₂, 80-110 mmHg).

Drugs and materials. Inactin (thiobutabarbital sodium) and chelerythrine were purchased from Research Biochemical International and dissolved in distilled H₂O. 2,3,5-Triphenyltetrazolium chloride and GF 109203X were purchased from Sigma. GF 109203X was dissolved in a 1:10 cocktail of DMSO-distilled H₂O. TAN-67 and BNTX were kindly synthesized and furnished by Dr. Hiroshi Nagase of Toray Industries (Kanagawa, Japan) and dissolved in saline and a 1:10 cocktail of polyethylene glycol 400-distilled H₂O, respectively. Rottlerin was purchased from BioMol and dissolved in a 1:5 cocktail of ethanol-saline. Optimum cutting tissue (OCT) compound was purchased from Miles Laboratories. Rabbit polyclonal isoform-specific anti-PKC antibodies were purchased from Santa Cruz Biotechnology. Indocarbocyanine-conjugated anti-rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories.

Study groups and experimental protocols. The effect of opioid treatment on the rat myocardium and the regulation of this process by PKC was assessed in an in vivo rat model of ischemia-reperfusion injury previously utilized in our laboratory. Rats were divided among 11 study groups (Fig. 1). All animals were subjected to 30 min of ischemia and 2 h of reperfusion (control). The effects of opioids were assessed on the heart by administering the ₁/₂-opioid receptor agonist DADLE (1 or 2 mg/kg) or via administration of the ₁-selective opioid receptor agonist TAN-67 (10 mg/kg) 15 min before the ischemic period. This dose of TAN-67 has previously been shown to induce cardioprotection via stimulation of the ₁-opioid receptor because the ₁-opioid receptor antagonist BNTX administered before TAN-67 could abolish cardioprotection (26). The effect of PKC inhibition was examined in the absence or presence of DADLE (1 mg/kg) and TAN-67 via administration of the PKC antagonist chelerythrine (5 mg/kg) 5 min before the ischemic period. We (5) have previously shown that chelerythrine produces total blockade of one cycle of IPC when administered at this dose and time point in rats. Additionally, we examined the role of PKC in TAN-67-induced cardioprotection with another PKC inhibitor GF 109203X (0.05 mg/kg) administered 5 min before the ischemic period. Finally, we employed the PKC- selective inhibitor rottlerin (0.3 mg/kg) to determine the involvement of PKC- in opioid-induced cardioprotection administered 25 min before ischemia.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Protocol bar for infarct size determination in animals treated in the presence or absence of an opioid [TAN-67 or D-Ala2-D-Leu5-enkephalin (DADLE)]. Control animals were subjected to 30 min of ischemia and 120 min of reperfusion (A). A bolus of DADLE or an infusion of TAN-67 was given 15 min before ischemia (B). Chelerythrine or GF 109203X was administered 5 min before ischemia in the presence (C) or absence (D) of TAN-67 or DADLE. Rottlerin was administered 25 min before ischemia in the presence (E) or absence (F) of TAN-67.

Determination of infarct size. On completion of the above protocols, the coronary artery was reoccluded, and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to stain the nonoccluded area of the LV. The heart was excised, and the LV was removed from the remaining tissue and cut into six cross-sectional pieces. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% triphenyltetrazolium chloride stain in 100 mM of phosphate buffer at 37°C. Tissues were stored in vials of 10% formaldehyde overnight, and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments). IS and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).

Immunofluorescent staining of PKC isoforms. Subcellular localization of PKC isoforms after various interventions were performed by immunofluoresence staining and compared with control hearts as previously described (33, 34). Control and experimental specimens were harvested immediately before ischemia. LV tissue was embedded in OCT compound, rapidly frozen in liquid nitrogen, and stored at 70°C until use. Transverse cryosections (5 µm) were prepared with a cryostat (Jung Friocut 2800E, Leica) and collected on poly-L-lysine-coated slides. Sections were fixed for 10 min in a 70% acetone-30% methanol mixture at 20°C, rinsed in PBS, and incubated in 10% normal goat serum in PBS for 30 min to block nonspecific binding. Primary antibodies (rabbit polyclonal antibodies against PKC-, -₁, -₂, -, -, -, -, and -) were diluted with PBS containing 0.1% BSA. Sections were then incubated for 1 h at room temperature with diluted primary antibodies and subsequently washed three times in PBS. Sections were then incubated for 45 min with indocarbocyanine-conjugated goat anti-rabbit IgG followed by washing once with 0.1% Triton X-100 in PBS and twice with PBS. Nuclear staining was achieved with bis-benzamid (10 mg/ml in PBS) for 30 s and washed with PBS three times. Sections were examined and photographed with a microscope equipped with fluorescence optics (BH-2 with a PM-CBSP camera, Olympus). Confocal images were also obtained with a Leitz DME fluroescence microscope with a TCS 4D confocal scanning attachment (Leica). Fluorescence was excited by the 568-nm line of a krypton laser, and the emission at 568-580 nm was recorded.

Exclusion criteria. A total of 98 rats successfully completed the above protocols. Rats were excluded from data analysis if they exhibited severe hypotension (<30 systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range.

Statistical analysis of data. All values are expressed as means ± SE. Analysis of variance (ANOVA) with Newman-Keuls post hoc test was used to determine whether any significant differences existed among groups for hemodynamics, LV weight, IS, and AAR. Significant differences were determined at P < 0.05.

	RESULTS

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Hemodynamics. The hemodynamic parameters measured are shown in Table 1. The HR for baseline, 15 min of ischemia, and 120 min of reperfusion in control animals were 371 ± 9, 372 ± 12, and 411 ± 29 beats/min, respectively. The mean blood pressures for baseline, 15 min of ischemia, and 120 min of reperfusion in control animals were 86 ± 4, 79 ± 8, and 67 ± 9 mmHg, respectively. The rate pressure products for baseline, 15 min of ischemia, and 120 min of reperfusion in control animals were 39 ± 2, 35 ± 2, and 38 ± 6 mmHg · min¹ · 1,000¹, respectively. No significant differences were seen between control and treatment groups for rate pressure products. The HR at baseline was significantly increased in the chelerythrine control group and decreased in the group administered rottlerin or administered DADLE (1 mg/kg) in the presence of chelerythrine. The HR at 15 min of ischemia was also lower in rottlerin-treated animals, and the HR at 120 min of reperfusion was significantly less in the groups administered rottlerin or DADLE (1 or 2 mg/kg) before the control protocol. Mean blood pressures were significantly elevated only in rottlerin-treated animals during baseline and 120 min of reperfusion. All other HR and mean blood pressure measurements were not significantly different from control.

Infarct size after various interventions. LV weight and AAR expressed as a percentage of the LV (AAR/LV) were not significantly different in any of the groups (data not shown). IS/AAR (in %) for animals untreated or treated with opioids in the presence or absence of chelerythrine are shown in Figs. 2 and 3. IS/AAR in control animals averaged 59.7 ± 1.6. ₁/₂-Opioid receptor stimulation with DADLE (1 or 2 mg/kg) reduced IS/AAR (36.9 ± 3.9 and 36.7 ± 4.7%, respectively) versus control. Similarly, the ₁-selective opioid receptor agonist TAN-67 (10 mg/kg) reduced IS/AAR (29.6 ± 3.3%) versus control. Chelerythrine, administered in the presence of 1 mg/kg DADLE or 10 mg/kg TAN-67, completely abolished cardioprotection (61.8 ± 3.2 and 55.4 ± 4.0, respectively). Similarly, GF 109203X completely abolished TAN-67-induced cardioprotection (53.3 ± 2.5). However, chelerythine and GF 109203X did not affect IS/AAR versus control in nonopioid-treated animals (57.6 ± 5.7 and 54.6 ± 6.6%, respectively). The PKC--selective inhibitor rottlerin abolished TAN-67-induced cardioprotection (48.9 ± 4.8); however, it did not alter IS/AAR in nonopioid-treated animals (55.0 ± 5.6%).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Infarct size expressed as a percentage of the area at risk in rats. Control animals were subjected to 30 min of ischemia and 2 h of reperfusion. TAN-67 and DADLE induced cardioprotection. The protein kinase C (PKC) inhibitors chelerythrine (Che) and GF 109203X were administered in the absence or presence of TAN-67 and/or DADLE and abolished opioid-induced cardioprotection. *P < 0.05 vs. control.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Infarct size expressed as a percentage of the area at risk in rats. Control animals were subjected to 30 min of ischemia and 2 h of reperfusion. TAN-67 induced cardioprotection that was abolished by the PKC inhibitor rottlerin. However, rottlerin did not affect infarct size in non-TAN-67-treated animals. *P < 0.05 vs. control.

Immunohistochemical distribution of PKC isoforms after various interventions. Representative results from the immunohistochemical study are shown in Figs. 4 and 5. In the TAN-67-pretreated hearts, PKC- was distinctly localized in the sarcolemmal membrane, and PKC-₁ positively stained the nucleus. PKC- and - were translocated to the mitochondria and mitochondria/intercalated disks in TAN-67-pretreated hearts, respectively. Staining for PKC-₂, -, -, and - was less intense and more diffuse than staining produced by PKC-, -₁, -, and - isoforms after TAN-67 treatment. A diffuse and nonspecific staining for PKC-, -₁, -, and - were observed in hearts pretreated with saline, chelerythrine, BNTX, or TAN-67 in the presence of chelerythrine or BNTX. In TAN-67-treated animals in the presence of rottlerin, PKC-, -₁, and - maintained their distinct cellular translocation; however, the translocation of PKC- was abolished.

View larger version (112K): [in this window] [in a new window]   Fig. 4.   Immunofluorescent staining of PKC isoforms in TAN-67-pretreated hearts. A: negative control (no PKC antibody). B: PKC- in saline-treated control hearts. Diffuse cytoplasmic distribution of the -isoform was observed. PKC-1, -, -, and other isoforms in control hearts were similar to that of B (not shown). C: PKC- in a TAN-67-treated heart. Immunofluorescence is observed in the sarcolemma (arrow). D: PKC-1 in TAN-67-treated heart. PKC-1 staining was observed in the nuclear region (arrow). E: PKC- in a TAN-67-treated heart. PKC is positively localized in the mitochondrial sites between myofibrils (arrow). Inset: confocal image from the same section showing mitochondrial zones (arrow) F: PKC- in a TAN-67-treated heart. PKC- is prominently distributed in the intercalated disk (arrowhead) and mitochondrial sites (arrow). Inset: confocal image from the same section showing mitochondrial zones (arrow) with beaded appearance. G: heart treated with TAN-67 and 7-benzylidenenaltrexone (BNTX) and stained for PKC-. A diffuse and nonspecific immunofluorescence is observed. PKC-, -1, and - are similar to that of G in the presence of BNTX (not shown). H: heart treated with TAN-67 and chelerythrine and stained for PKC-. No specific localization is observed. PKC-, -1, and - are similar to that of H in the presence of chelerythrine (not shown). All original magnifications: ×400.

View larger version (126K): [in this window] [in a new window]   Fig. 5.   Immunofluorescent staining of PKC-, -1, -, and - in TAN-67-pretreated hearts in the presence of the PKC- antagonist rottlerin. Rottlerin alone did not induce any PKC translocation (not shown). A: immunofluorescence of PKC- is observed in the sarcolemma (arrow). B: PKC-1 staining was observed in the nuclear region (arrow). C: PKC- translocation to the mitochondria was abolished by rottlerin. Diffuse cytoplasmic distribution of the -isoform was observed. D: PKC- is prominently distributed in the intercalated disk (arrowhead) and mitochondrial sites (arrow). All original magnifications: ×400.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We demonstrated PKC-dependent cardioprotection to prolonged ischemia after stimulation of the ₁- or ₁/₂-opioid receptors in the intact blood-perfused rat heart. Infusion of either the ₁/₂-opioid agonist DADLE or the ₁-selective opioid receptor agonist TAN-67 induced marked cardioprotection versus control animals subjected to ischemia and reperfusion. Additionally, we could completely abolish TAN-67- or DADLE-induced cardioprotection with the PKC inhibitor chelerythrine and the recently characterized potent and selective inhibitor of PKC (31) GF 109203X. Additionally, we demonstrated that specific PKC isoforms are activated by opioid treatment and demonstrate the importance of PKC- with the PKC- antagonist rottlerin in infarct size reduction.

Rottlerin has been previously shown to inhibit PKC- with an IC₅₀ value of 3-6 µM (7). However, the IC₅₀ for inhibition of PKC-, -, and - and PKC-, -, and - are 30-42 and 80-100 µM, respectively. Furthermore, our immunohistochemical data suggest that the dose we used, 0.3 mg/kg, is selective for PKC- versus PKC-, -₁, and -.

Translocation of PKC from the cytosolic to particulate compartments is a commonly used index of PKC activation (19, 33). We utilized immunohistochemistry to determine the subcellular localization of specific PKC isoforms. The use of isoform-specific anti-PKC antibodies allows the assessment of both isoform-selective activation and compartmentalization. TAN-67 induced the translocation of PKC- to the sarcolemma, PKC-₁ to the nucleus, PKC- to the mitochondria, and PKC- to the mitochondria and intercalated disk. Our data indicate that -opioid agonists are capable of stimulating the translocation of both Ca²⁺-dependent (PKC- and -₁) and Ca²⁺-independent (PKC- and -) isoforms. This is in agreement with Miyawaki and Ashraf (18), who reported that high-Ca²⁺ preconditioning can induce translocation of both Ca²⁺-dependent and -independent PKC isoforms.

This is the first report of specific PKC isoform translocation via opioid receptor stimulation and is in agreement with the observations of Kawamura et al. (11) and Miyawaki et al. (19), who demonstrated that IPC induces the translocation of PKC- and - in the isolated rat heart. The most notable finding of this investigation is the establishment of PKC- as an integral component of opioid-initiated IS reduction from ischemia. Indeed, Inagaki et al. (10) demonstrated that the benzothiazepine derivative JTV519 confers cardioprotection against Ca²⁺ overload-induced myocardial injury via specific activation of PKC- in the rat myocardium, and Mitchell et al. (17) showed that PKC- translocation mediates cardioprotection during ₁-adrenergic or classical IPC. Although we have demonstrated that isoform-specific PKC translocation may be important in cardioprotection, it is possible that activation of these kinases may effect downstream signaling pathways that do not necessarily exert their protective effects at the area of PKC translocation.

Ping et al. (21) demonstrated that IPC induces the translocation of PKC- in the conscious rabbit heart that correlates with cardioprotection. However, they also demonstrated that PKC- was activated via IPC. These discrepancies may be explained by species differences in the rat versus the rabbit, which have been previously reported (1), or more likely may be explained by the use of different experimental protocols to induce cardioprotection (opioid receptor stimulation versus IPC).

The involvement of PKC as a mediator of IPC was first proposed in 1994 by Ytrehus et al. (37). They reported that the PKC inhibitors staurosporine or polymyxin B could effectively abolish IPC-induced cardioprotection in rabbits. In the same year, Liu et al. (15) reported that colchicine-induced disruption of cytoskeletal microtubules (14, 22), which may be involved in PKC translocation within the cell, could abolish IPC-induced cardioprotection in the rabbit. Since these initial observations, PKC has been shown to be an integral component of IPC in the rat (6, 28).

Miyawaki et al. (18, 19) reported that PKC- and - are translocated to the cell membrane during IPC and high-Ca²⁺ preconditioning. They also demonstrated PKC- translocation to the intercalated disk and suggested that PKC- may modulate myocardial function through cell-to-cell interactions (18). In the same light, Doble et al. (2) suggested that PKC- stimulation by fibroblast growth factor-2 may interact with and phosphorylate connexin43, a critical component of gap junctions that may affect intercellular communication. Although this investigation does not eliminate the importance of PKC- in IS reduction, it appears that PKC- activation is not essential for opiates to confer cardioprotection.

Delayed cardioprotection has also been demonstrated on -opiate receptor stimulation (4). We demonstrated cardioprotection that was maximal 48 h after an intraperitoneal injection of TAN-67 could be abolished by the ₁-opioid receptor antagonist BNTX and the mitochondrial selective K_ATP channel inhibitor 5-hydroxydecanoic acid. It has also been suggested that delayed cardioprotection produced by metabolic inhibition in rat ventricular myocytes involves opioid receptor stimulation (36). Additionally, this delayed cardioprotection induced by metabolic inhibition could be inhibited with chelerythrine (36). This suggests that delayed cardioprotection induced by opioids may also be a PKC-dependent event. In the present study, we demonstrated localization of PKC-₁ to the nucleus and propose that this isoform may be an important component of the signal transduction cascade leading to delayed cardioprotection because delayed cardioprotection is thought to be dependent on nuclear transcription and translation. Gutstein et al. (8) reported that - or µ-opioid receptors transiently introduced into COS cells revealed potent stimulation of extracellular signal-regulated kinase (ERK) on receptor activation. Additionally, Schonwasser et al. (23) demonstrated a link between the activation of specific PKC isoforms and the ERK/mitogen-actived protein kinase cascade. They found that all PKC isoforms examined (, ₁, , , and ) had the capacity to activate ERK, which may be important in gene regulation critical to the development of delayed cardioprotection.

The activation of PKC by opioids, specifically PKC-, may induce cardioprotection via stimulation of the K_ATP channel. PKC activation has been shown to induce activation of the K_ATP channel in rabbit ventricular myocytes via patch-clamp studies by Light et al. (13). Activation of the K_ATP channel in the mitochondria may induce potassium flux into the mitochondria. This potassium flux may limit calcium overload within the mitochondria via depolarization and limited calcium entry via the calcium uniporter. We propose that PKC- translocation to the mitochondria, leading to K_ATP channel activation within the inner membrane, is important in opioid-induced cardioprotection because we (3) recently demonstrated that TAN-67-induced cardioprotection could be abolished with inhibitors of the mitochondrial, but not sarcolemmal, K_ATP channel.

Cardioprotection as a result of opioid receptor stimulation in the heart has clinical implications. Tomai et al. (29) demonstrated that the adaptation to ischemia observed in humans after repeated balloon inflations during coronary angioplasty can by abolished by the opioid receptor antagonist naloxone. This was evidenced by the observation that in naloxone-treated patients, the mean S-T segment shift at the end of the second balloon inflation was similar to that at the end of the first inflation, whereas in placebo-treated patients, the S-T segment shift at the end of the second inflation was markedly reduced. Additionally, naloxone-treated patients manifested a greater severity and shorter time to onset of cardiac pain versus placebo-treated patients.

In conclusion, ₁-opioid receptor stimulation protects the myocardium from a prolonged ischemic insult and induces the translocation of specific PKC isoforms (, ₁, , and ). However, these PKC isoforms are not translocated to the same cellular locus on opiate stimulation. PKC- immunofluoresence was observed in the sarcolemma. PKC-₁ was localized within the nucleus, PKC- was positively localized in the mitochondria lying between the myofibers, and PKC- was predominately localized within the intercalated disk and mitochondrial sites. With the use of the selective PKC- inhibitor rottlerin, we demonstrated that PKC- is a necessary component of opioid-induced IS reduction. These data indicate that if PKC is stimulating the K_ATP channel to induce cardioprotection, it is likely that PKC-, which would be expected to preferentially activate the sarcolemmal K_ATP channel, or PKC- and -, which would be expected to preferentially activate the mitochondrial K_ATP channel, are involved. Additionally, translocation of PKC-₁ may be important in gene regulation involved in delayed cardioprotection from opioids.