Activation of Mitochondrial ATP-Sensitive K+ Channel for Cardiac Protection Against Ischemic Injury Is Dependent on Protein Kinase C Activity
Abstract—Protein kinase C (PKC) is involved in the second messenger signaling cascade during ischemic and Ca2+ preconditioning. Given that the pharmacological activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels also mimics preconditioning, the mechanisms linking PKC activation and mitoKATP channels remain to be established. We hypothesize that PKC activity is important for the opening of the mitoKATP channel. To examine this, a specific opener of the mitoKATP channel, diazoxide, was used in conjunction with subcellular distribution of PKC in a model of ischemia/reperfusion (I/R). Langendorff-perfused rat hearts were subjected to 40-minute ischemia followed by 30-minute reperfusion. Effects of activation of the mitoKATP channel and other interventions on functional, biochemical, and pathological changes in ischemic hearts were assessed. In hearts treated with diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly improved after I/R; lactate dehydrogenase release was also significantly decreased. The morphology was well preserved in diazoxide-treated hearts compared with nontreated ischemic control hearts. The salutary effects of diazoxide on the ischemic injury were similar to those of Ca2+ preconditioning. Administration of sodium 5-hydroxydecanoate, an effective blocker of the mitoKATP channel, or chelerythrine or calphostin C, an inhibitor of PKC, during diazoxide pretreatment or during continuous presence of diazoxide in the ischemic period, completely abolished the beneficial effects of the diazoxide on the I/R injury. Blockade of Ca2+ entry during diazoxide treatment by inhibiting the L-type Ca2+ channel with verapamil also completely reversed the beneficial effect of diazoxide during I/R. PKC-α was translocated to sarcolemma, whereas PKC-δ was translocated to the mitochondria and intercalated disc, and PKC-ε was translocated to the intercalated disc of the diazoxide-pretreated hearts. Colocalization studies for mitochondrial distribution with tetramethylrhodamine ethyl ester (TMRE) and PKC isoforms by immunoconfocal microscopy revealed that PKC-δ antibody specifically stained the mitochondria. ATP was significantly increased in the diazoxide-treated hearts. Moreover, the data suggest that activation and translocation of PKC to mitochondria appear to be important for the protection mediated by mitoKATP channel.
Ischemic preconditioning results in a drastic reduction in cellular injury in the ischemic myocardium. Protein kinase C (PKC) is an important component of the signal transduction pathways in the ischemic preconditioning–induced protection. One of the final effectors in this protection has been recently reported to be mitochondrial KATP (mitoKATP) channels.1 Previously, opening of sarcolemmal KATP channels has been shown to shorten the action potential causing depression of contractility.2 3 This was proposed as the mechanism of protection in ischemic myocardium.4 However, recent evidence contradicts this hypothesis, because the degree of action potential shortening can be divorced from the extent of protection and KATP channel openers are protective even in unstimulated cardiac myocytes,5 6 in which action potential abbreviation cannot be a factor.
Cardiac myocytes possess another type of KATP channel in the inner mitochondrial membrane.7 Opening of this channel would tend to dissipate the membrane potential established by the proton pump.8 Such dissipation accelerates electron transfer by the respiratory chain and leads to net oxidation of the mitochondrial matrix. Low concentration of diazoxide (DE) specifically activates mitoKATP channels without any effect on sarcolemmal KATP channels.9 Our recent study shows that stimulation of mitoKATP channels with DE is highly protective against the calcium overload injury and PKC activity is required for the effect of mitoKATP channel.10 11 Because inhibition of PKC can block the effect of mitoKATP channels, we tested the hypothesis that the activation and translocation of PKC are also required for the protection against lethal ischemia by mitoKATP channel. In this study, we investigated the role of the mitoKATP channel in cardiac protection and its dependence on various PKC isoforms. The results of this study show that the effect of mitoKATP channel is blocked by inhibition of PKC, the activation of which is indicated by both Ca2+-dependent and -independent pathways.
Materials and Methods
DE, chelerythrine (CHE), calphostin C (CN), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Verapamil (VL) hydrochloride was purchased from Research Biochemicals International. Sodium 5-hydroxydecanoate (5-HD) was purchased from ICN Pharmaceuticals, Inc. Tetramethylrhodamine ethyl ester (TMRE) was obtained from Molecular Probes. OCT compound (Tissue-Tek) was from Miles, Inc; rabbit polyclonal isoform-specific anti-PKC antibodies were purchased from Santa Cruz Biotechnology. The following antibodies were raised against isoform-unique peptide sequences within the carboxyl terminus: α, 651 to 672; β1, 656 to 671; β2, 657 to 673; γ, 679 to 697; δ, 657 to 673; ε, 722 to 736; η, 669 to 683; and ζ, 573 to 592. Indocarbocyanine-conjugated anti-rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories. DE was dissolved in DMSO before being added into the perfusion buffer. The final concentration of DMSO was <0.1%. Other reagents were dissolved in PBS. All of the agents except DE were directly administered through the aortic cannula at a rate of 0.2 mL/min by a Harvard infusion pump. DE was directly perfused through the aorta in a noncirculating Langendorff apparatus with Krebs-Henseleit (KH) buffer.
Hearts from male Sprague-Dawley rats were removed and retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with KH buffer, which consisted of (in mmol/L) NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.8, NaHCO3 25, and glucose 11. The buffer was saturated with 95% O2-5% CO2 (pH 7.4, 37°C) for 50 minutes. Hearts were perfused at a constant pressure of 80 mm Hg. A water-filled latex balloon-tipped catheter was inserted into the left ventricle through the left atrium and was adjusted to a left ventricular end-diastolic pressure (LVEDP) of 4 to 6 mm Hg during the initial equilibration. Thereafter, the balloon volume was not changed. The distal end of the catheter was connected to a Digi-Med Heart Performance Analyzer-τ (model 210, version 1.01, Micro-Med) via a pressure transducer (Case). Hearts were paced at 350 bpm except during ischemia. Pacing was reinitiated after 3 minutes of reperfusion in all groups. The index of myocardial function was determined as previously described.12 All hearts were perfused with the oxygenated KH buffer for a total of 95 minutes (37°C), consisting of a 25-minute preischemic period followed by 40 minutes of global ischemia and 30 minutes of reperfusion. The present study conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1985).
After equilibration, hearts were randomly divided into the following experimental groups.
Group 1: Normal Control and Vehicle Control
Hearts (n=6) were perfused for 95 minutes with KH buffer as a normal control for different experimental groups. Hearts were treated for 11 minutes with vehicle (0.04% DMSO, n=6), after which they were subjected to ischemia/reperfusion (I/R). The amount of DMSO used in vehicle control experiments did not affect any hemodynamic parameters, lactate dehydrogenase (LDH) release, ATP content, or cell morphology.1 12
Group 2: I/R
After equilibration, hearts (n=6) were subjected to ischemia for 40 minutes followed by reperfusion for 30 minutes.
Group 3A: Ca2+ Preconditioning (CPC)+I/R
This group was designed to test whether CPC mimics protection by opening of mitoKATP channel. Because Ca2+ plays a significant role in protection of CPC, DE may increase Ca2+ indirectly (see Discussion) and activate PKC in addition to activation of mitoKATP channel. Hearts (n=6) were perfused for 3 cycles of 1 minute each with Ca2+-free KH buffer followed by 5 minutes with Ca2+-containing KH buffer, and then the hearts were subjected to I/R as in group 2.
Group 3B: 5-HD+CPC+I/R
The rationale of this experiment is that Ca2+-mediated activation of PKC is important for the activation of mitoKATP channels resulting in attenuation of cellular injury. 5-HD, a blocker of mitoKATP channels, was used during CPC. Hearts (n=6) were perfused similarly to group 3A, except that 5-HD (100 μmol/L)1 was infused before CPC.
Group 4: Role of MitoKATP Channels in Cardiac Protection
A potent activator of the mitoKATP channel, DE was used before ischemia. DE opens the sarcolemmal KATP channel at a higher concentration (K1/2=855 μmoL), whereas it opens the mitoKATP channel at very low concentration (K1/2=0.4 μmoL).9 This group was designed to test the hypothesis that an activation of mitoKATP channels mimics the effect of CPC, which is dependent on stimulation of PKC. 5-HD, an inhibitor of the mitoKATP channels, was used to determine the effect of mitoKATP channels in the prevention of cell injury. 5-HD blocks both the mitoKATP channel1 and the action potential shortening.13
Group 4A: MitoKATP Channel Opener+I/R
Hearts were perfused with KH buffer containing DE for 6 minutes. After 5 minutes, washout hearts were subjected to I/R. A dose response of DE using 1, 20, 40, 80, and 100 μmol/L was studied (n=6 hearts for each subgroup). We found that 80 μmol/L DE had an optimal effect on the I/R injury. In some hearts (n=6), DE was present during the entire ischemic period.
Group 4B: Blockade of MitoKATP Channel+I/R
Hearts (n=6) were perfused similarly to those in group 4A, except 5-HD (100 μmol/L)1 14 was administered alone for 4 minutes and then with DE together for 6 minutes. After 5-minute washout, hearts were subjected to I/R.
Group 5: Influence of PKC on MitoKATP Channel
To determine whether DE activates mitoKATP channels via PKC signaling pathway, PKC activator, PMA, and 2 PKC inhibitors, CHE chloride and CN, were used in these studies.
Group 5A: CHE or CN+DE+I/R
Hearts (n=6) were perfused similarly to group 4A, except that they were pretreated with CHE chloride (6.4 μmol/L) or CN (0.05 μmol/L) for 4 minutes, followed by combined treatment with DE for 6 minutes. After 5-minute washout, hearts were subjected to I/R. The dose of CHE chloride or CN was chosen on the basis of previous reports.15 16 To determine whether CHE chloride alone attenuates or increases ischemic injury, hearts were treated for 10 minutes with CHE chloride (6.4 μmol/L, n=6), and after 5-minute washout, hearts were subjected to I/R.
Group 5B: PMA+I/R
Hearts were perfused with KH buffer containing PMA (100 nmol/L)11 for 6 minutes. After 5-minute washout, hearts (n=6) were subjected to I/R.
Group 5C: 5-HD+PMA+I/R
To test the hypothesis that a specific mitoKATP channel inhibitor, 5-HD, blocks the effect by direct activation of PKC by PMA on the ischemic injury, hearts were first pretreated with 5-HD (100 μmol/L) for 4 minutes and then together with PMA (100 nmol/L) for 6 minutes. After 5-minute washout, hearts were subjected to I/R (n=6).
Group 5D: Activation of MitoKATP Channel After PKC Blockade
The purpose of these experiments was to determine whether activation of mitoKATP channel after PKC blockade could protect the heart against ischemia. Hearts were perfused similarly to group 4A, except administration of DE was continued during the ischemic period (n=6).
Group 6: Calcium-PKC Link With MitoKATP Channel Opening
These experiments tested the hypothesis that activation of mitoKATP channels is facilitated by the PKC signaling pathway. The rationale behind these experiments is that DE may also indirectly influence the L-type Ca2+ channel and increase Ca2+ transient (see Discussion). The participation of Ca2+ in the signaling pathway was verified by blocking L-type Ca2+ channels with VL.
Group 6A: VL+DE+I/R
Hearts were first pretreated with VL (2.5 μmol/L) for 4 minutes and then together with DE for 6 minutes. After 5-minute washout, hearts were subjected to I/R (n=6).
Group 6B: VL Alone+I/R
To determine whether the concentration of VL used attenuates ischemic injury, hearts were treated for 10 minutes with VL (2.5 μmol/L, n=6); after 5-minute washout, hearts were subjected to I/R. The dose of VL was chosen on the basis of a previous report.15
Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in the coronary effluent by a coupled-enzyme spectrometric technique (DU Series 500 spectrophotometer, Beckman Instruments, Inc) using a Sigma assay kit as described.15 17 LDH was measured at 3, 5, 10, 20, and 30 minutes of reperfusion. The accumulated amount was obtained by integrating the area underneath the individual time-course curve for 30-minute reperfusion.
Measurement of Tissue ATP
After termination of the experiment, hearts were cut into transverse slices and were immediately frozen in liquid nitrogen until freeze-drying. A slice of left ventricle was processed for morphology and immunocytochemistry. Freeze-dried tissue (50 to 100 mg) was crushed in a precooled glass tube, and ATP was extracted with 5 mL of cold 6% trichloroacetic acid. The extract was analyzed for ATP by a spectrophotometric method.15
Subcellular Localization of PKC Isoforms by Immunocytochemistry
Subcellular localization of PKC isoforms after various interventions was performed by immunofluorescence staining and was compared with control hearts as previously described.10 15 Control and experimental specimens were harvested just before I/R. Left ventricular tissue was blotted and embedded in OCT compound, rapidly frozen in liquid nitrogen, and then stored at −70°C until use.15 Five-micrometer sections were fixed for 10 minutes in a 70% acetone-30% methanol mixture at –20°C, incubated in 10% normal goat serum in PBS for 30 minutes to block nonspecific binding, and incubated with primary antibodies (rabbit polyclonal antibodies against PKC isoforms PKC-α, PKC-β1, PKC-β2, PKC-γ, PKC-δ, PKC-ε, PKC-η, and PKC-ζ). Sections were examined and photographed with an Olympus BH-2 fluorescence microscope equipped with an automatic exposure control unit. Confocal images were also obtained with a Leitz DMRBE inverted research fluorescence microscope with a true confocal scanner (TCS 4D, Leica, Inc). Fluorescence was excited by the 568-nm line of krypton-argon laser, and the emission at 568 to 580 nm was recorded. For mitochondrial imaging, additional hearts were perfused with either 200 nmol/L TMRE alone or together with 80 μmol/L DE for 6 minutes. Specimens for microscopy were prepared in a manner similar to immunocytochemistry. TMRE is widely used as a structural marker for mitochondria and preferentially distributes into negatively charged cellular organelles, such as mitochondria.18 TMRE was excited with the 488-nm line of a krypton-argon laser and recorded at >530-nm with an FITC filter.5 19
Heart tissue taken from the left ventricular free wall at the end of experimental protocols was cut into 1.0-mm pieces that were fixed with 2.5% buffered glutaraldehyde. A semiquantitative estimate of cell damage was carried out on 1-μm-thick sections. Approximately 500 cells were analyzed in each heart by researchers without knowledge of the treatment, and 1 of 3 degrees of cell damage was assigned to each cell. Cell morphology was assessed according to the classification as previously described.15 17
All values are expressed as mean±SEM. Group comparisons were done by ANOVA (Statview 4.0.) All groups were analyzed simultaneously with a Bonferroni/Dunn test. A difference of P<0.05 was considered significant.
Ischemic Control Hearts
A significant decrease in functional recovery was observed in ischemic control hearts. At the end of reperfusion, LVEDP significantly increased, whereas left ventricular developed pressure (LVDP) and coronary flow (CF) were decreased (Table 1⇓). LDH leakage was significantly increased on reperfusion (Figure 1⇓), and ATP levels were reduced to 5.6±0.7 μmol/g dry weight as compared with the normal control value of 21.5±0.8 μmol/g dry weight (Figure 2⇓). The pathological changes were severe, and extensive necrosis was observed (Table 2⇓).
Role of CPC, MitoKATP Channel, and PKC in Cardiac Protection
A slight increase in LVDP and LVEDP was observed after 3 cycles of brief Ca2+ depletion and repletion. After I/R, a significant functional recovery was observed in these hearts (Table 1⇑). LDH release was significantly decreased, and ATP contents were markedly preserved both at the end of ischemia and at postischemic reperfusion (Figures 1⇑, 2A⇑, and 2B⇑). The cellular structure was extremely well preserved in hearts after CPC (Figure 3C⇓). A total of 56.2±4.3% cells were normal; 13.8±2.3% and 30.0±3.4% were mildly and severely damaged, respectively; and the degree of cell damage was much less than that in the ischemic control hearts (Table 2⇑).
MitoKATP Channel and Ischemic Changes
During and after DE treatment, the CF was significantly increased. A significant recovery of LVDP, HR, and CF was observed in DE-treated hearts as compared with ischemic control hearts (Table 1⇑, Figure 4⇓). LVEDP was significantly decreased in the treated hearts, and recovery of LVDP was amazing (Figure 4⇓). This level of protection is similar to that described in a recently published study on effects of DE on ischemia.1 A significant reduction in LDH release and a dramatic preservation of tissue ATP content both at the end of ischemia and at reperfusion were observed in contrast to ischemic control hearts (Figures 1⇑, 2A⇑, and 2B⇑). No significant differences were observed in ATP contents and LDH release in the DE-pretreated hearts with or without washout (Figure 5⇓). The cell structure was also well preserved in DE-pretreated hearts (Table 2⇑, Figure 3E⇑). The blockade of mitoKATP channel with 5-HD completely abolished the recovery of LVDP (Figure 4⇓) and CF (Table 1⇑).
PKC-Mediated Signaling Pathways in Cardiac Protection
PKC activation with PMA was itself protective. A significant functional recovery was observed after I/R (Table 1⇑). LDH release was significantly decreased and ATP contents were markedly preserved as compared with ischemic control hearts (Figures 1⇑, 2A⇑, and 2B⇑). The cell structure was also well preserved in PMA-pretreated hearts compared with ischemic control hearts (Table 2⇑). Morphological changes were similar to those of DE-pretreated hearts (data not shown).
To determine whether stimulation of mitoKATP channel is mediated via PKC signaling pathway, hearts were pretreated with CHE or CN before DE treatment. In these hearts, the contractile function was lost, and CF, LDH release, tissue ATP levels, and morphological damage were similar to those of the I/R group (Tables 1⇑ and 2⇑; Figures 1⇑, 2A⇑, 2B⇑, and 3⇑). CHE chloride alone did not have any effect on the cardiac function (Table 1⇑).
To further clarify the role of PKC in the regulation of mitoKATP channel, 5-HD was infused before the treatment with PMA. 5-HD abolished the protective effects induced by PKC activation (Table 1⇑; Figures 1⇑, 2A⇑, and 2B⇑). There were no significant differences in various parameters of cell injury between control ischemic hearts and 5-HD–pretreated hearts after PMA administration (Table 2⇑).
Because DE can indirectly increase cAMP,20 which leads to elevated [Ca2+]i transient in myocytes,21 22 we sought to determine whether a transient increase in [Ca2+]i by DE treatment activates PKC and its translocation to mitochondria. The effect of DE disappeared after pretreatment with VL, and functional recovery was significantly depressed (Table 1⇑). LDH release was significantly increased (16.7±1.5 U/g), and ATP content was also reduced to the level of I/R control hearts (Figures 1⇑, 2A⇑, and 2B⇑). VL alone did not provide any cardiac protection (Tables 1⇑ and 2⇑; Figures 1⇑, 2A⇑, and 2B⇑). The activation of mitoKATP channel with DE during blockade of PKC in the ischemic period did not reduce cell injury.
Immunohistochemical Distribution of PKC Isoforms After Various Interventions
Representative results from the immunohistochemical study are shown in Figure 6⇓. In the DE-pretreated hearts, PKC-α was distinctly localized in the cardiac cell sarcolemma, and PKC-δ was positively distributed in the mitochondria, which were confirmed with use of the membrane potential indicator TMRE. TMRE-treated tissue was also stained with PKC-δ antibody, which positively stained mitochondria, intercalated discs, and nuclei. PKC-ε was translocated to the intercalated disc in 90% of cells of DE-pretreated hearts. PKC-β1 staining was observed in the nuclear region. PKC-δ, -α, -ε, and -β1 showed diffuse and nonspecific staining in hearts pretreated with 5-HD, CHE, or VL, similar to results shown in Figure 6B⇓. PKC-β2, -γ, -η, and -ζ staining was less intense and weaker than by PKC-α, -δ, -ε and -β1 isoforms after DE treatment.
The major findings of this study are the following: (1) the mitoKATP channel appears to be an end effector of preconditioning against ischemia, and (2) the stimulation of the mitoKATP channel is dependent on PKC activation and translocation to mitochondria. In 1992, Gross et al23 showed that nicorandil, a KATP channel opener, was very effective against infarct size in dogs. Recent studies24 by the same group also showed cardioprotection in dogs treated with bimakalim, a KATP channel opener, which had minimal effect on action potential duration. Such dissociation with action potential shortening has also been shown in several other studies.24 25 These experimental results suggest that sarcolemmal KATP channels may not be the only effector of cardioprotection.
Cardiac myocytes possess KATP channels located in the inner mitochondrial membrane, which also respond to the openers of the sarcolemmal channels.7 26 Activation of any potassium-selective ion channels in the inner mitochondrial membrane would tend to dissipate the membrane potential established by the proton pump.8 DE has been reported to specifically activate mitoKATP channel with little effect on cardiac action potential duration.8 9 Thus, activation of the mitoKATP channel by DE may be necessary for higher ATP production to support increased workload in the heart. Mitochondria are known to play at least 2 roles essential for cell survival, ATP synthesis and maintenance of Ca2+ homeostasis. High-energy production is essential to support the action of ATP-dependent ion pumps, such as Na+/K+ and Ca2+ pumps27 to maintain membrane intactness and calcium ion gradients across various cell membranes, including the sarcolemma, sarcoplasmic reticulum, and mitochondria.28 It is likely that the enhancement of ATP production in the preconditioned heart maintains the Ca2+ homeostasis. We have shown that the effect of DE disappears after inhibition of the L-type Ca2+ channel and PKC, which suggests that DE is a trigger for the activation of both the mitoKATP channel and PKC.
Potential Role of Ca2+ in Modulating MitoKATP Channels
One of the main theories of the lethal cellular damage induced by I/R involves massive Ca2+ influx during reperfusion.28 However, recently it has been reported that brief ischemia evokes a transient elevation of [Ca2+]i.29 30 Smith et al31 have demonstrated that [Ca2+]i was increased 2- to 4-fold in preconditioning ischemia in isolated rat heart. We have previously demonstrated that the transient increase in [Ca2+]i is a trigger for the induction of CPC, which was strongly protective against ischemia and Ca2+ paradox.10 11 15 A Ca2+ transient also exists in the mitochondrial matrix.32 33 34 When rat myocytes were stimulated with a beta-adrenergic substance and paced at physiological frequency (4 Hz), Ca2+ concentration in the mitochondria exceeded that of cytosolic content, reaching a value of 0.6 μmol/L.35 This Ca2+ increase stimulates the activity of 3 enzymes (ie, pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) and increases the rate of ATP synthesis.36 37 38 Thus, [Ca2+]i increase could act as a second messenger within mitochondria.39 In addition, [Ca2+]i transient increase also activates Ca2+–independent PKC isoforms via phospholipase C pathway40 and causes their translocation to mitochondria.
The role of [Ca2+]i in mediating protection against lethal ischemia via PKC signaling pathway has been well established by our previous studies15 and by others.41 42 The mechanism by which DE may increase [Ca2+]i is not determined in this study. However, we have shown that by blocking the L-type Ca2+ channel, the effect of DE dissipates, which suggests the participation of Ca2+ in the signaling pathway leading to protection. In addition, DE is also a known inhibitor of phosphodiesterase,20 43 which degrades cAMP.21 Therefore, an increase in cAMP will influence L-type Ca2+ channel via protein kinases leading to an elevated Ca2+ transient in myocytes.22 This is in agreement with our study44 and work by Asimakis et al45 that isoproterenol elicits preconditioning effect against ischemia. Taken together, this study has shown that blockade of Ca2+ transient by VL in the DE-pretreated hearts prevented cardiac protection, which suggests that activation of the mitoKATP channel by DE may also involve Ca2+. Current studies in progress on isolated myocytes show a significant [Ca2+]i increase on introduction of DE as measured by the use of Fluo-3 (M.A., Y.W., unpublished observation, 1999).
Potential Role of PKC in Modulating MitoKATP Channel
The role of PKC in preconditioning has been well established by several investigations.41 46 The present study clearly demonstrates that [Ca2+]i plays an important role in activating various second messenger pathways including PKC. The blockade of [Ca2+]i transient by VL inhibited PKC activation and its effect on mitoKATP channels, which suggests the regulation of the latter channels by PKC. Similarly, DeWeille et al47 also demonstrated that stimulation of PKC by the phorbol ester phorbol myristate acetate activated the KATP channel. [Ca2+]i can also stimulate Ca2+-independent PKC isoforms via activation of phospholipase C, which forms inositol triphosphate and diacylglycerol (DAG). The latter 2 products stimulate Ca2+-independent isoforms of PKC.40 The role of DAG in the activation of PKC was further supported by our previous study,12 in which hearts were treated with 1-stearoyl-2-arachidonyl-glycerol, a precursor of DAG, causing significant protection. The protective effect, as expected, was abolished with CHE. Similarly, Van Winkle et al48 were able to block the protection in hearts pretreated with PMA after administration of 5-HD. It was suggested by their study that protection occurred with subsequent opening of KATP channels after activation of PKC. Thus, our study strengthens the idea that activation of mitoKATP channel after PKC inhibition is ineffective in reducing ischemic injury.
This study also strongly points out that PKC translocation to mitochondria was essential for the activation of mitoKATP channels. PKC inhibition with CHE and CN totally blocked protection by DE, which suggests a crucial role of PKC in the regulation of mitoKATP channels. The importance of PKC is further emphasized by the findings of Sato et al49 that PKC primes the mitoKATP channel to open earlier and more intensely during prolonged ischemia. On the other hand, Miura et al50 reported that PKC inhibition by CN was not effective in the reduction of infarction in rabbit hearts treated with DE. It is likely that these differences could arise as a result of different animal species and experimental protocols. However, PKC activation by adenosine A1 receptor leads to opening of mitoKATP channels, the effect of which can be blocked by PKC inhibition.50 This and other studies support the fact that various preconditioning stimuli induce myocardial protection against ischemia through mechanisms that appear to be mediated by PKC.
It is well established that DE selectively opens mitoKATP channels.1 5 The data in this study point out that DE stimulates both PKC and mitoKATP channels, thus conferring a greater protection as suggested by increased ATP preservation and improved cardiac function. Similar improvement in cardiac function has been recently reported by Garlid et al.1 Activation of mitoKATP channel retards calcium uptake and/or preserves ATP synthetic capability as reviewed by Buja.51 DE-pretreated hearts retained more ATP during ischemia, thus improving postischemic cardiac function and maintaining Ca2+ homeostasis as evidenced by less contraction band formation and Ca2+ accumulation by mitochondria. The loss of mitoKATP channel–mediated effects by PKC inhibition further reinforces the notion that the signal transduction cascades of a large number of receptors are coupled to PKC activation,42 46 which may phosphorylate different channels leading to preconditioning.41 The phosphorylating sites within the cell need further molecular characterization. Endogenous PKC exists in various isoforms with specific tissue distribution and sensitivity. Therefore, it is possible that different isoforms interact with mitoKATP channel after a specific response to PKC activation. The simultaneous visualization of mitochondria and staining with PKC-δ antibodies expand our existing knowledge of PKC isoform distribution in cardiac tissue and their significance in various signaling pathways. We used immunohistochemistry to determine subcellular localization of PKC isoforms. Pretreatment with DE clearly caused the translocation of the PKC-δ isoform to the mitochondrial sites, intercalated disc, and nucleus; PKC-α into the sarcolemma; PKC-ε into the intercalated disc; and PKC-β1 into the nucleus. Translocation of PKC-α to the sarcolemma was consistent with our previous study on CPC,52 which suggests that activation of PKC-α isoform by DE is Ca2+ dependent. Translocation of PKC-δ to the mitochondrial sites, intercalated disc, and nucleus may regulate intracellular processes not only by direct activation of mitoKATP channels but also by phosphorylation of numerous rate-limiting enzymes or gene transcription.40 41 Translocation of PKC-ε to the intercalated disc in DE-treated hearts may facilitate intercellular communication through cell junctions located in the intercalated disc of myocytes.
Potential Importance of Our Findings
The present study indicates the potential of [Ca2+]i and PKC in the regulation of the mitoKATP channel, which is important for endogenous cardioprotection against ischemia. Therefore, the association of PKC with mitoKATP channel may be an important component of preconditioning. The pharmacological manipulation of PKC and the openers of mitoKATP channels could be exploited in the future for therapeutic purposes.
This work was supported in part by NIH Grants HL 23597 and HL 55678.
- Received February 16, 1999.
- Accepted July 26, 1999.
- © 1999 American Heart Association, Inc.
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