Activation of Mitochondrial ATP-Sensitive K⁺ Channel for Cardiac Protection Against Ischemic Injury Is Dependent on Protein Kinase C Activity

Heart Preparation

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).

Experimental Protocol

After equilibration, hearts were randomly divided into the following experimental groups.

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}

Morphological Examination

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}

Statistical Analysis

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⇓).

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Figure 1.

Effect of various interventions on LDH release in hearts subjected to I/R. LDH release is reduced in hearts preconditioned with Ca²⁺ and DE (80 μmol/L) compared with ischemic control hearts. 5-HD (100 μmol/L), CHE (6.4 μmol/L), CN (0.05 μmol/L), or VL (2.5 μmol/L), administered during the DE pretreatment period, completely blocked protection. 5-HD also blocked protection by PMA (100 nmol/L). Values are mean±SE; n=6 for each group. *P<0.05 vs ischemic control.

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Figure 2.

A, Effect of various interventions on ATP content in hearts at the end of the ischemic period. At the end of the ischemic period, ATP content was significantly preserved in Ca²⁺- and DE-preconditioned hearts compared with ischemic control hearts. Administration of 5-HD, CHE, CN, or VL during DE pretreatment did not augment ATP content, and its effect was in fact similar to that observed in ischemia only. Values are mean±SE; n=6 for each group. *P<0.05 vs ischemic control. B, Effect of various interventions on ATP content in hearts subjected to I/R. ATP content was markedly preserved in Ca²⁺- and DE-preconditioned hearts compared with ischemic control hearts. 5-HD, CHE, CN, or VL administered during the DE pretreatment period reduced ATP content. ATP content in hearts pretreated with 5-HD and PMA together was not different from that of ischemic controls. Similarly, VL alone did not reduce ischemic injury. Values are mean±SE; n=6 for each group. *P<0.05 vs ischemic control.

Role of CPC, MitoK_ATP Channel, and PKC in Cardiac Protection

Ca²⁺-Preconditioned Hearts

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⇑).

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Figure 3.

Effect of different interventions on the cell morphology. A, Light micrograph of normal (nonischemic) heart showing compact and well-preserved myofibers. Nuclei show uniform distribution of chromatin material (arrow). B, Light micrograph of ischemic control heart subjected to I/R showing extensive damage to the myofibers. Many myofibers are hypercontracted, forming contraction bands (arrow) and releasing intracellular contents (arrowhead). C, Ca²⁺ preconditioned heart subjected to I/R. The majority of cells are normal, with distinct nuclei. A few cells have undergone contraction band formation (arrow). D, Heart treated with 5-HD and CPC before I/R. Cellular damage is clearly observed. Intracellular vacuolization (arrow) and contraction bands (arrowhead) are observed. E, DE-pretreated hearts subjected to I/R. Overall, myofibers are well preserved; however, a few myofibers are hypercontracted (arrow). In PMA-pretreated hearts, myofibers are also preserved similarly to DE-pretreated hearts (data not shown). F, Heart pretreated with DE and 5-HD before I/R. Severe cellular damage is clearly observed. Intracellular vacuolization (arrowhead) and contraction bands (arrow) are observed. All magnifications, ×400.

To clarify the role of mitoK_ATP channel in CPC, a specific blocker, 5-HD, was infused before CPC pretreatment. 5-HD abolished the protective effects of CPC (Table 1⇑; Figures 1⇑, 2A⇑, 2B⇑, and 3D⇑).

MitoK_ATP 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 mitoK_ATP channel with 5-HD completely abolished the recovery of LVDP (Figure 4⇓) and CF (Table 1⇑).

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Figure 4.

Effect of various interventions on LVDP and end-diastolic pressure. A, LVDP is significantly increased in hearts preconditioned with DE (80 μmol/L) compared with ischemic control hearts. 5-HD and CHE administered during the DE pretreatment period completely blocked the recovery of LVDP. B, LVEDP is significantly reduced in DE-preconditioned hearts compared with ischemic control hearts. 5-HD and CHE given during the DE pretreatment period elevated LVEDP to the ischemic control level. Values are mean±SE; n=6 for each group.

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Figure 5.

Effect of the DE with or without washout on ATP and LDH release in hearts subjected to I/R. DE pretreatment with or without washout did not have significant effect on ATP content and LDH release. Values are mean±SE; n=6 hearts for each group. *P<0.05 vs ischemic control.

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.

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Figure 6.

Immunofluorescent staining of PKC isoforms. A, Negative control (no PKC antibody). B, PKC-α in control hearts. Diffuse cytoplasmic distribution of the α isoform is observed. Distribution of PKC-δ, -ε, -β₁, or others in control hearts is similar to that in Figure 6B⇓ (data not shown). C, PKC-α in a DE-treated heart. Immunofluorescence is observed in the sarcolemma (arrow). Confocal micrograph. D, PKC-ε in DE-treated heart. PKC-ε is prominently distributed in the intercalated disc (arrow). E, PKC-β₁ staining is observed in the nucleus (arrow). F, In DE-treated heart without TMRE loading, PKC-δ is positively localized in the mitochondrial sites between myofibrils (arrow), intercalated disc (arrowhead), and nucleus. Confocal micrograph. G, Heart pretreated with TMRE as visualized with confocal microscope. Mitochondria lying between myofibrils preferentially accumulated TMRE and have punctate appearance (arrow). H, Same field from the tissue as in panel G but double stained with PKC-δ antibody and TMRE to colocalize mitochondria and antibody staining. Pinkish red color represents immunostain for PKC-δ, which is seen over mitochondria (arrow), intercalated disc (arrowhead), and nucleus. No specific distribution of PKC-δ in hearts treated with DE and 5-HD, DE and CHE, or DE and VL is observed, which is similar to micrograph B (data not shown). All original magnifications ×400 except confocal imaging, which is 630×1.4.

Potential Role of Ca²⁺ in Modulating MitoK_ATP 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 mitoK_ATP 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 MitoK_ATP 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 mitoK_ATP 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 K_ATP 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 K_ATP channels after activation of PKC. Thus, our study strengthens the idea that activation of mitoK_ATP 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 mitoK_ATP channels. PKC inhibition with CHE and CN totally blocked protection by DE, which suggests a crucial role of PKC in the regulation of mitoK_ATP channels. The importance of PKC is further emphasized by the findings of Sato et al⁴⁹ that PKC primes the mitoK_ATP 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 mitoK_ATP 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 mitoK_ATP channels.^{1 5} The data in this study point out that DE stimulates both PKC and mitoK_ATP 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 mitoK_ATP 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 mitoK_ATP 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 mitoK_ATP 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 mitoK_ATP 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 mitoK_ATP channel, which is important for endogenous cardioprotection against ischemia. Therefore, the association of PKC with mitoK_ATP channel may be an important component of preconditioning. The pharmacological manipulation of PKC and the openers of mitoK_ATP channels could be exploited in the future for therapeutic purposes.