Role of Protein Kinase C in Mitochondrial K ATP Channel Mediated Protection Against Ca 2 Overload Injury in Rat Myocardium

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1 Role of Protein Kinase C in Mitochondrial K ATP Channel Mediated Protection Against Ca 2 Overload Injury in Rat Myocardium Yigang Wang, Muhammad Ashraf Abstract Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoK ATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoK ATP channel diazoxide attenuates the lethal injury associated with Ca 2 overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoK ATP channels; whether diazoxide mimics the effects of Ca 2 preconditioning; and whether diazoxide reduces Ca 2 paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca 2 PD (10 minutes of Ca 2 depletion followed by 10 minutes of Ca 2 repletion). The effects of the MitoK ATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca 2 PD were assessed. In hearts treated with 80 mol/l diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca 2 PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxidetreated hearts compared with nontreated Ca 2 PD hearts. The salutary effects of diazoxide on the Ca 2 PD injury were similar to those in hearts that underwent Ca 2 preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca 2 PD. The addition of sodium 5-hydroxydecanoate, a specific MitoK ATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca 2 PD. Blockade of Ca 2 entry during diazoxide treatment by inhibiting L-type Ca 2 channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca 2 PD. PKC- was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC- and PKC- were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoK ATP channel is mediated by PKC-mediated signaling pathway. (Circ Res. 1999;84: ) Key Words: K channel Ca 2 paradox Ca 2 preconditioning protein kinase C diazoxide Massive cell damage occurs in the heart within seconds when it is perfused with perfusate devoid of Ca 2 followed by perfusion with a solution that contains Ca 2. This phenomenon has been called the Ca 2 paradox (Ca 2 PD). The common features of the Ca 2 PD are Ca 2 overload, sarcolemmal destruction, necrosis of the cell, depletion of high-energy phosphates, and loss of intracellular contents. 1,2 Mechanisms by which Ca 2 PD induces cell injury include altered Ca 2 channels, damaged cell membranes, disrupted intercalated disks, impaired Na -Ca 2 exchange, disruptive mechanical forces, and oxidative stress. 3 6 To understand these mechanisms, we plan to use the reverse strategy of testing interventions to discover cellular pathways that lead to reduction of Ca 2 PD injury. A mild stress induced by brief Ca 2 depletion and repletion, called Ca 2 preconditioning, 2 has been shown to protect the myocardium from Ca 2 PD injury or subsequent sustained ischemia/reperfusion. 7 Protein kinase C (PKC) was shown to be a major component in the attenuation of cell injury caused by Ca 2 preconditioning (CPC). Recent evidence suggests that the MitoK ATP channel is mainly involved in preconditioning against ischemia/reperfusion. 8,9 Garlid and his colleagues 10 reported that the K ATP channel from cardiac mitochondria is 2000 times more sensitive to diazoxide than the sarcolemmal channel, which indicates that 2 distinct channel subtypes coexist within the myocyte. Liu and colleagues 9 also reported that diazoxide targets mitochondrial but not sarcolemmal K ATP channels. We have previously shown that CPC elicits its protection through PKC mediated signaling pathways. 7 Therefore, it appears that the effects of both PKC and the MitoK ATP channel are interlinked. Accordingly, we hypothesize that diazoxide opens both MitoK ATP channels and activates PKC. The results of this study show that the participation of PKC is essential in MitoK ATP channel mediated cardiac protection. Received October 14, 1998; accepted March 4, From the Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Ohio. Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Ave, Cincinnati, OH American Heart Association, Inc. Circulation Research is available at

2 Wang and Ashraf May 28, Materials and Methods Materials Diazoxide, phorbol 12-myristate 13-acetate (PMA), and chelerythrine chloride were purchased from Sigma Chemical Co. Verapamil hydrochloride was purchased from Research Biochemicals International. Sodium 5-hydroxydecanoate (5-HD) was purchased from ICN Pharmaceuticals, Inc. OCT compound was from Miles Laboratories; Rabbit polyclonal isoform specific anti-pkc antibodies were purchased from Santa Cruz Biotechnology Inc. These antibodies were raised against isoform-unique peptide sequences within the carboxy terminus (, 651 to 672; I, 656 to 671; II, 657 to 673;, 679 to 697;, 657 to 673;, 722 to 736;, 669 to 683; and, 573 to 592). Indocarbocyanine-conjugated anti-rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. PMA and diazoxide were 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 the agents except diazoxide and PMA were directly administered through the aortic cannula at a rate of 0.2 ml/min by use of a Harvard infusion pump. Heart Preparation The current study conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH, Publication No , revised 1985). Male Sprague- Dawley rats that weighed 250 to 300 g were anesthetized by an intraperitoneal injection of 30 mg/kg pentobarbital. After intraperitoneal injection of 500 U/kg heparin sodium, hearts were removed and retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with Krebs-Henseleit (KH) buffer, which consisted of the following (mmol/l): NaCl 118, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, CaCl 2 1.8, NaHCO 3 25, and glucose 11. The buffer was saturated with 95% O 2 /5% CO 2 (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 heart performance analyzer (Digi-Med model 210, version 1.01, Micro-Med) via a pressure transducer. Hearts were paced at 350 bpm except during periods of Ca 2 depletion in all groups. The index of myocardial function was determined with left ventricular developed pressure, which was calculated from the difference between left ventricular peak systolic pressure and end-diastolic pressure. Left ventricular systolic pressure, end-diastolic pressure, and the derivative of left ventricular pressure ( dp/dt) were recorded continuously as a function of time by personal computer (International Computer Technology, Inc). After perfusion with the oxygenated KH buffer for an equilibration period of 25 minutes, a 3-way stopcock above the aortic root was used to perfuse the Ca 2 -free KH buffer for 10 minutes, followed by a KH buffer that contained Ca 2 for 10 minutes, to induce a typical Ca 2 PD. 2 The coronary effluent was collected in a beaker, and the coronary flow was determined volumetrically at the pretreatment; just before being subjected to Ca 2 depletion; during the Ca 2 depletion period; and at 1, 3, 5, and 10 minutes during Ca 2 repletion. Experimental Protocol After equilibration, hearts were randomly divided into experimental groups. Group 1: Normal Control and Vehicle Control Hearts (n 6) were perfused for 45 minutes with KH buffer as a normal control for different experimental groups. Rat hearts were pretreated for 6 minutes with vehicle (0.01% DMSO, n 6) followed by a 5-minute washout, after which the hearts were subjected to the Ca 2 PD. The amounts of DMSO used in vehicle control experiments did not affect any hemodynamic parameters, lactate dehydrogenase (LDH) release, ATP content, or cell morphology (data not shown). Group 2: Ca 2 PD After a 25-minute perfusion with normal KH buffer, hearts (n 8) were subjected to Ca 2 -free KH buffer for 10 minutes followed by KH buffer that contained Ca 2 for 10 minutes. Group 3A: Ca 2 Preconditioning and Calcium Paradox This group was designed to determine whether CPC elicits protection against Ca 2 PD. Hearts (n 8) were perfused for 3 cycles of 1 minute each with Ca 2 -free KH buffer followed by 5 minutes with Ca 2 -containing KH buffer; then, the hearts were subjected to Ca 2 PD as in group 2. Group 3B: 5-HD, CPC, and Ca 2 PD The rationale for this experiment was that CPC activates MitoK ATP channels, which results in attenuation of cellular injury. To test this, 5-HD, a specific blocker of MitoK ATP channels, was used during CPC. Hearts (n 8) were perfused in a manner similar to that used for group 3A, except that 5-HD (100 mol/l) was infused for 4 minutes before CPC. The dose of 5-HD was chosen on the basis of a previous report. 8 Group 3C: Chelerythrine Chloride, CPC, and Ca 2 PD This group tested whether the inhibition of PKC during CPC also abrogates the protection. Chelerythrine chloride was added instead of 5-HD. Hearts (n 8) were perfused in a manner similar to that used in group 3A, except that chelerythrine chloride (6.4 mol/l), a specific PKC inhibitor, was infused through the aorta at a rate of 0.2 ml/min for 4 minutes before CPC. The dose of chelerythrine chloride was chosen on the basis of a previous report. 7 Group 4: Role of MitoK ATP Channels in Cardiac Protection Diazoxide, a potent opener of the MitoK ATP channel, was used during Ca 2 PD. Diazoxide opens the sarcolemmal K ATP channel at a higher concentration (855 mol/l K 1 2), whereas it opens the MitoK ATP channel at a low concentration (0.4 mol/l K 1 2). 10 This group will determine whether this specific opener of MitoK ATP channels mimics the effect of CPC. An inhibitor of mitochondrial K ATP channels 5-HD was used to determine whether MitoK ATP channels play an important role in the prevention of cell injury. It has been reported that 5-HD abolishes the protective effects of K ATP channel openers, although it has no effect on the ADP-shortening activity, which suggests its high specificity for MitoK ATP channels. 11 Group 4A: Diazoxide and Ca 2 PD Hearts were perfused with KH buffer that contained diazoxide for 6 minutes and then washed out for 5 minutes. After the hearts were washed, they were subjected to the Ca 2 PD. A dose response of diazoxide with 1, 30, 40, 80, and 100 mol/l was studied (n 6 to 10 hearts for each subgroup). We found 80 mol/l diazoxide to have the optimal effect on the Ca 2 PD injury. Group 4B: 5-HD, Diazoxide, and Ca 2 PD Hearts (n 8) were perfused similarly to group 4A except 5-HD (100 mol/l) was infused before the use of diazoxide. Group 5: Influence of PKC on MitoK ATP Channel To determine whether the direct activation of PKC protects the myocardium independent of MitoK ATP channels, we used the PKC activator PMA. Group 5A: PMA and Ca 2 PD Hearts (n 8) were perfused with KH buffer that contained PMA (100 nmol/l) for 6 minutes followed by a 5-minute washout before the Ca 2 PD. The dose of PMA was chosen on the basis of a previous report. 11 Group 5B: Chelerythrine Chloride, Diazoxide, and Ca 2 PD To determine whether diazoxide activates MitoK ATP channels via a PKC signaling pathway, hearts (n 8) were perfused by a method similar to group 4A except chelerythrine chloride (6.4 mol/l) was

3 1158 Diazoxide-Mediated Preconditioning Against Ca 2 Overload Injury infused through the aorta at the rate of 0.2 ml/min for 4 minutes before diazoxide was added. Group 6: Verapamil or Nifedipine, Diazoxide, and Ca 2 PD These experiments tested the hypothesis that diazoxide is linked to transient Ca 2 increase, which was responsible for the cardioprotection available through the PKC signaling pathway. The Ca 2 entry was blocked by verapamil or nifedipine, which are both L-type Ca 2 channel blockers. This group was similar to group 4A except that verapamil (2 mol/l) or nifedipine (0.001 mol/l) was infused at a rate of 0.2 ml/min for 4 minutes before diazoxide administration (n 6 to8). After a 5-minute washout, Ca 2 PD was instituted. The doses of verapamil and nifedipine were chosen on the basis of a previous report. 12,13 Measurement of LDH LDH, an indicator of myocardial tissue injury, was determined in coronary effluent 2 by a coupled-enzyme spectrometric technique by use of a Sigma assay kit. Measurement of enzyme activity was based on the oxidation of lactate and the rate of increase in absorbance at 340 nm. Measurement of Tissue ATP After termination of the experiment, hearts were cut into transverse slices and immediately frozen in liquid nitrogen until they were freeze-dried. Fifty to 100 mg of freeze-dried tissue 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 the spectrophotometric method. 2 Immunofluorescence Microscopy Subcellular localization of PKC isoforms after various interventions were performed by immunofluorescence staining and were compared with control hearts as previously described. 12,14 Control and specimens with previous treatments were harvested just before Ca 2 depletion. Left ventricular tissue was blotted and embedded in OCT compound, rapidly frozen in liquid nitrogen, and then stored at 70 C until use. 15 Transverse 5- m cryosections were prepared with a cryostat (Jung Frigocut 2800E, Leica) and collected on poly-l-lysine coated slides. All sections were fixed for 10 minutes in a 70% acetone/30% methanol mixture at 20 C, rinsed in PBS, and incubated in 10% normal goat serum in PBS for 30 minutes to block nonspecific binding. Primary antibodies (rabbit polyclonal antibodies against PKC isoforms PKC-, PKC- I, PKC- II, PKC-, PKC-, PKC-, PKC-, and PKC- ) were diluted with PBS that contained 0.1% BSA. Sections were incubated for 1 hour at room temperature with diluted primary antibodies and then washed with PBS 3 times (3 minutes each). Sections were then incubated for 45 minutes with indocarbocyanine-conjugated goat anti-rabbit IgG, then washed once with 0.1% Triton X-100 in PBS for 3 minutes, and washed twice with PBS (2 minutes each). Nuclear staining was achieved with bis-benzamide (10 mg/ml in PBS) for 30 seconds and washed with PBS 3 times (2 minutes each). Sections were mounted with a medium (Antifade, Oncor) and 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 fluorescence microscope with a TCS 4D confocal scanning attachment (Leica, Inc). Fluorescence was excited by the 568-nm line of krypton laser, and the emission was recorded at 568 to 580 nm. Morphological Examination Heart tissue was taken from the left ventricular free wall at the end of experimental protocols and was cut into 1.0-mm pieces that were fixed with 2.5% buffered glutaraldehyde. A semiquantitative estimate of cell damage was performed on 1- m-thick sections. Three randomly chosen blocks from each heart were examined for quantification of cell damage without prior knowledge of the treatment. Approximately 500 cells were analyzed in each heart, and 1 of 3 degrees of cell damage was assigned to each cell. Cell morphology was assessed according to the following classification 2,15 : (1) normal: compact myofibers with uniform staining of nucleoplasm, well-defined rows of mitochondria between the myofibrils, and nonseparation of opposing intercalated disks; (2) mild damage: same as above, except some vacuoles were present adjacent to the mitochondria; and (3) severe damage: reduced staining of cytoplasmic organelles, clumped chromatin material, wavy myofibers, and granularity of cytoplasm; the cells with contraction band necrosis were added to this category. Statistical Analysis All values are expressed as mean SEM. Group comparisons were done by ANOVA (Statview 4.0.). All groups were analyzed simultaneously with the Bonferroni-Dunn test. A difference of P 0.05 was considered significant. Results Ca 2 Paradox During Ca 2 PD, hearts were bleached of normal reddish color, which indicated the loss of intracellular contents. Then the hearts underwent severe contracture and lost their contractile function (Table 1). At the end of the Ca 2 -free period, LDH release was not different than the normal control values. Immediately on Ca 2 repletion, LDH release increased multifold and peaked after 3 minutes of Ca 2 repletion (Figure 1). Similarly, the maximum LDH release was also observed in Ca 2 PD hearts versus other groups (Figure 1). Coronary flow was significantly reduced at the end of Ca 2 PD (Table 1). Tissue ATP content in Ca 2 PD hearts was obviously depleted compared with the control hearts (Figure 2). In Ca 2 PD hearts, most of the cells were hypercontracted, and the cell membranes were ruptured, which resulted in the loss of mitochondria, myoglobin, and other cellular proteins (Figure 3B, Table 2). After a 10-minute period of Ca 2 -free perfusion, tissue showed increased interfibrillar space and a marked separation in region of the intercalated disks in hearts treated without diazoxide (Figure 3L). Effects of MitoK ATP Channel and PKC and Ca 2 Preconditioning on Ca 2 PD Intracellular ATP was preserved in diazoxide-treated hearts (Figure 2). However, no significant differences existed in LDH release and tissue ATP levels between diazoxide- and CPC-treated hearts after Ca 2 repletion. After Ca 2 repletion, most of the hearts pretreated with PMA, CPC, or diazoxide exhibited a weak beat. In diazoxide- or CPC-treated hearts, maximum LDH release was reduced significantly (P 0.05) compared with Ca 2 PD hearts (Figure 1). At the end of Ca 2 repletion, a significant improvement in coronary flow and LVEDP was observed in diazoxidetreated, PMA-treated, and CPC hearts versus hearts subjected to Ca 2 PD alone. The cellular structure was also markedly preserved in the diazoxide-treated and CPC hearts. In diazoxide-pretreated hearts, 49.3% of cells were normal and 17.4% and 33.3% were mildly and severely damaged, respectively (Table 2). After a 10-minute period of Ca 2 -free perfusion, tissue showed considerable separation of cells at the intercalated disks. This separation of the intercalated disks

4 Wang and Ashraf May 28, TABLE 1. Effect of Various Interventions on Hemodynamic Parameters in Hearts Subjected to Ca 2 PD Group Equilibration Treatment Pre Ca 2 PD Post Ca 2 PD Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm CPC Ca 2 PD (n 8) LVDP, mm Hg * LVEDP, mm Hg * CF, ml/min * HR, bpm * CPC 5-HD Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm CPC CHE Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm DE Ca 2 PD (n 10) LVDP, mm Hg * LVEDP, mm Hg * CF, ml/min * HR, bpm * 5-HD DE Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm CHE DE Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm PMA Ca 2 PD (n 8) LVDP, mm Hg * LVEDP, mm Hg * CF, ml/min * HR, bpm * VL DE Ca 2 PD (n 8) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm NE DE Ca 2 PD (n 6) LVDP, mm Hg LVEDP, mm Hg CF, ml/min HR, bpm NE indicates nifedipine; LVDP, left ventricular developed pressure; and CF, coronary flow. Values are mean SEM. *P 0.05 vs post Ca 2 PD group; P 0.05 vs equilibration.

5 1160 Diazoxide-Mediated Preconditioning Against Ca 2 Overload Injury pretreated with chelerythrine. The contractile function in hearts treated with chelerythrine and diazoxide was lost. LVEDP, maximum LDH release, coronary flow, and tissue ATP levels were similar to the Ca 2 PD group (Table 1, Figures 1 and 2). Morphological damage was also significantly higher in hearts pretreated with chelerythrine (Table 2, Figure 3). Figure 1. Effect of various interventions on temporal LDH release. in diazoxide-pretreated hearts was at a lower scale (Figure 3K) than that in control Ca 2 PD hearts. In a separate series of experiments, we determined the effect of the 5-HD on the cardiac protection elicited by CPC. The hearts treated with 5-HD during CPC stopped beating. In chelerythrine-treated hearts during CPC, LVEDP and LDH release were significantly increased and CF and tissue ATP levels were similar to those of the Ca 2 PD group (Table 1, Figures 1 and 2). Similarly, during CPC, 5-HD treated hearts underwent drastic morphological changes (Table 2, Figure 3). Similarly, the blockade of MitoK ATP channels with 5-HD during diazoxide treatment caused a loss of contractile function during Ca 2 repletion. Effect of Diazoxide on PKC Activation To determine whether MitoK ATP channel opening by diazoxide is mediated via PKC signaling pathways, hearts were Figure 2. Effect of various interventions on ATP contents in hearts subjected to Ca 2 PD. Diazoxide and Blockade of L-Type Ca 2 Channel To determine whether a transient increase in [Ca 2 ] i during diazoxide infusion triggers cardioprotection, we attempted to reduce Ca 2 entry during diazoxide treatment. The protective effect of diazoxide was abolished in hearts pretreated with verapamil or nifedipine (Table 1). Maximum LDH release was significantly higher, and loss of tissue ATP content was similar to nontreated hearts (Figures 1 and 2). In separate experiments, the hearts treated with only verapamil or nifedipine did not exhibit any beneficial effects (data not shown). Immunocytochemical Distribution of PKC Isoforms After Various Interventions Representative results from the immunohistochemical study are shown in Figure 4. PKC-, which was diffusely distributed in the cytoplasm of cells in control, was distinctly localized in the cardiac cell sarcolemma. PKC- was positively localized in the mitochondrial sites, intercalated disks, and nuclei of myocytes in diazoxide-treated hearts. PKC- was localized in the intercalated disks of 90% of cells in diazoxide-pretreated hearts. PKC- I staining was observed in the nuclear region. PKC-, PKC-, PKC-, and PKC- I showed diffuse nonspecific immunofluorescence in heart treated with 5-HD, chelerythrine, or verapamil during diazoxide-pretreatment, respectively. (Only PKC- isoforms were shown in hearts treated with 5-HD, chelerythrine, or verapamil, respectively, during diazoxide-pretreatment.) Staining with PKC- II, PKC-, PKC-, and PKC- was less intense and weaker than with PKC-, -, - and - I isoforms after diazoxide treatment. Discussion The main findings of the present study are as follows: (1) MitoK ATP channel is the effector of Ca 2 preconditioning. (2) The protective effect caused by MitoK ATP channel activation is mediated by PKC signaling pathways. (3) Diazoxide effect is blocked by verapamil or nifedipine, inhibitors of L-type Ca 2 channels. (4) Translocation of PKC- to the cell membrane and PKC- to the mitochondrial sites was dominant, although PKC- I and PKC- were distributed in the nuclear region and intercalated disks, respectively, in diazoxide-pretreated hearts. Role of the Diazoxide on Cardioprotection The activation of the sarcolemmal K ATP channel shortens the action potential duration subsequent to increased K efflux and decreased Ca 2 influx via voltage-gated channels, which leads to attenuation of cellular damage during ischemia. 16 However, several studies from other laboratories showed a poor correlation between sarcolemmal K currents and cardioprotection by ATP-sensitive K channel openers (KCOs). 8

6 Wang and Ashraf May 28, Figure 3. Effect of different interventions on the cell morphology. A, Normal heart. Nuclei showed uniform distribution of chromatin material (arrowhead). Compact and well-preserved myofibers were observed ( 400). B, Ca 2 PD heart. All cells were so hypercontracted that the membranes were ruptured. As a result, mitochondria were extruded from the cells (arrowhead; 400). C, Heart treated with CPC. Most of myocytes were well preserved with the exception of some damaged cells (arrowhead; 400). D, Heart treated with 5-HD and CPC. All myocytes were severely damaged ( 400). E, Heart treated with chelerythrine (CHE) and CPC. All myocytes were severely damaged ( 400). F, Diazoxide (DE)- pretreated heart. Myocytes were significantly preserved compared with nontreated calcium paradox heart (B). A few damaged cells were also observed (arrowhead; 400). G, Heart treated with DE and 5-HD. All cells were damaged similar to Ca 2 PD ( 400). H, Heart treated with DE and CHE. All cells were damaged as with the Ca 2 PD ( 400). I, PMA-pretreated heart. Most of myocytes were well preserved, with the exception of some damaged cells (arrowhead; 400). J, Heart treated with DE and verapamil (VL). All cells were damaged similarly to the Ca 2 PD ( 400). K, Heart treated with DE. After a 10-minute period of Ca 2 -free perfusion, the separation of the intercalated disk is rarely seen (arrowhead; 400). L, After a 10-minute period of Ca 2 -free perfusion without DE, a marked separation in the region of the intercalated disk is frequently observed (arrowhead; 400). Recognition of the MitoK ATP channel as an intracellular receptor for KCOs adds a new dimension to the KCO pharmacology. 10 Recently, the MitoK ATP channel has been proposed as the site of cardioprotective action. 17 Diazoxide is reported to be a specific opener of MitoK ATP channels. 18,19 Although the physiological functions of MitoK ATP channel are unclear, Szewczyk 20 pointed out that the opening of the MitoK ATP channel partially compensates the membrane po-

7 1162 Diazoxide-Mediated Preconditioning Against Ca 2 Overload Injury TABLE 2. Semiquatitative Estimate of Morphological Damage in Hearts Subjected to Ca 2 PD After Various Interventions Degree of Cell Damage, % of cells Group (n 4) Zero (Normal) Mild Severe Normal control Ca 2 PD CPC Ca 2 PD * * * 5-HD CPC Ca 2 PD CHE CPC Ca 2 PD DE Ca 2 PD * * * 5-HD DE Ca 2 PD CHE DE Ca 2 PD PMA Ca 2 PD * * * VL DE Ca 2 PD Values are mean SEM. *P 0.05 vs Ca 2 PD. tential, which enables additional protons to be pumped out to form a H electrochemical gradient for both ATP synthesis and Ca 2 transport. Mitochondria are known to play at least 2 roles that are essential for cell survival: ATP synthesis and maintenance of Ca 2 homeostasis. During aerobic physiological conditions, a micromolar increase in Ca 2 concentration stimulates the Krebs cycle and NADH redox potential, which is essential for ATP synthesis. High-energy production is essential to support the action of ATP-dependent ion pumps, such as Na /K and Ca 2 pumps, 21 to maintain membrane intactness and calcium ion gradients across various cell membranes, including the sarcolemma, sarcoplasmic reticulum, and mitochondria. 22 Xu and colleagues. 23 proposed that glycolytically derived ATP is functionally coupled to Ca 2 transport by cardiac sarcoplasmic reticulum. Combined, it is possible that the enhancement of ATP production in the preconditioned heart by diazoxide maintains the Ca 2 homeostasis. The action of the electrophoretic K uniport and the electroneutral K /H exchange by opening the MitoK ATP channel is believed to be primarily responsible for the maintenance of potassium homeostasis within the mitochondrion and the control of intramitochondrial osmotic pressure and mitochondrial volume, which are important in the modulation of metabolic processes. 24 Janczcwski and colleagues 25 and Wendt-Gallitelli and Isenberg 26 reported that Ca 2 transient in the mitochondrial matrix is important for ATP synthesis. A Ca 2 increase within the physiological range stimulates the activity of 3 enzymes: pyruvate dehydrogenase, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase. Ca 2 -induced increases in NADH redox potential might increase the rate of ATP synthesis. 27,28 Ca 2 transient increase also activates Ca 2 -dependent PKC isoforms, which leads to cardioprotection. In addition, mitochondria may participate directly in Ca 2 homeostasis by means of separate influx and efflux pathways located within the inner mitochondrial membrane, 27,29 which may also regulate mitochondrial volume by opening MitoK ATP channels. The total mitochondrial capacity for Ca 2 accumulation is at least several times larger than that of the sarcoplasmic reticulum, 30 which suggests a key mitochondrial role for the maintenance of Ca 2 homeostasis during pathological conditions when cytosolic Ca 2 concentration increases substantially. An interesting finding of our study was that verapamil and nifedipine abolished the protective effect of diazoxide. This finding was consistent with our previous report that the protective effect of calcium preconditioning was abolished with verapamil. 12 It is possible that diazoxide-induced cardiac protection is also shared by [Ca 2 ] i -triggered pathways. Diazoxide may also open sarcolemmal channels in smooth muscle. 31 However, it may seem to be selective in the heart and liver mitochondria. Also, diazoxide is a potent vasodilator 31 and has little effect on the duration of cardiac action potential. 9,32 The opening of MitoK ATP channels by diazoxide may be a necessary component for higher ATP production to support an increased workload in the heart. 10 Thus, a major goal in this study was to preserve as much membrane function and energy production as possible to delay calcium overload and cell damage. Therefore, in light of these functions, it is reasonable to propose that the opening of MitoK ATP channels by diazoxide elicits a unique cardiac protection by delaying Ca 2 overload injury. Role of PKC on the Activation of the MitoK ATP Channel by Diazoxide and Its Cardioprotection PKC is a key enzyme in the regulation of signal transduction and cell growth and differentiation in many cell types. 33,34 The role of PKC in preconditioning has been well established by several investigations. 35,36 The current study demonstrates that Ca 2 plays an important role in the activation of various second messenger pathways including PKC. This is supported by the current data that the blockade of Ca 2 increase by verapamil inhibits PKC activation and its effect on MitoK ATP channels. Thus, it is clear that Ca 2 influx from the exterior of cells is required for PKC and MitoK ATP channel activation. The latter observation can only be substantiated by direct measurement of [Ca 2 ] i transient. However, the participation of Ca 2 in the diazoxide-induced protection may have some relevance because diazoxide is also a known inhibitor of phosphodiesterase, 37,38 and this enzyme degrades camp. 39 Therefore, an increase in camp influences cellular functions. It has been reported that inhibition of phosphodiesterase causes an increase in camp in myocytes, which, in turn, activates protein kinases that modulate L-type Ca 2 channels such that additional channels open during each depolarization, which leads to an elevated Ca 2 transient. 40 The role of Ca 2 in mediating protection against lethal ischemia and Ca 2 overload via PKC has been established by our previous studies 2,12,15,41 and by others as well. 42,43 Administration of PKC activator PMA produces cardioprotection against Ca 2 PD similar to that by diazoxide. This finding was also supported by Sato and colleagues 11 who pointed out that the activity of the MitoK ATP channel could be regulated by PKC in intact heart cells. Exposure to PMA potentiated and accelerated the effect of diazoxide. The effects of PMA were blocked by the MitoK ATP channel blocker 5-HD, which was verified with simultaneous recordings of membrane current and flavoprotein fluorescence. In the latter studies, DeWeille and colleagues 44 also demonstrated that stimulation of PKC by the

8 Wang and Ashraf May 28, Figure 4. Immunofluorescent staining of PKC isoforms. A, Negative control (no PKC antibody). B, PKC- in control hearts. Diffuse cytoplasmic distribution of the isoform is observed. C, PKC- in a DE-treated heart. Immunofluorescence is observed in the sarcolemma (arrow). D, PKC- in DE-treated heart. PKC- is positively localized in the mitochondrial sites between myofibers (arrow), intercalated disks (arrowhead), and nuclei (not shown). PKC- in control hearts is similar to panel B (data not shown). E, PKC- in heart treated with DE and 5-HD. A diffuse and nonspecific immunofluorescence is observed. F, PKC- in hearts treated with DE and CHE; no specific localization is observed. G, PKC- in hearts treated with DE and VL; no specific localization is observed. H, PKC- in DE-treated heart; PKC- is prominently distributed in the intercalated disks (arrow). PKC- in control hearts is similar to panel B (data not shown). I, PKC- I staining was observed in the nuclear region (arrow). All magnifications 400 except confocal imaging, which is Micrographs in panels C, D, H, and I were taken with a confocal microscope, and others were taken with an immunofluorescent microscope. phorbol ester PMA activated the K ATP channel. Therefore, the PKC-mediated pathway appears to be important in preconditioning of the isolated rat heart. Because the K ATP channel from cardiac mitochondria is 2000 times more sensitive to diazoxide than the channel from cardiac sarcolemma, 10 it is reasonable to speculate that PKC activation might phosphorylate both the sarcolemmal K ATP and MitoK ATP channels. Diazoxide perhaps stimulates both PKC and MitoK ATP channels, thus conferring a greater protection as suggested by increased ATP preservation and better cardiac function. The inhibition of MitoK ATP channel-mediated effects by chelerythrine further reinforced the notion that PKC regulates intracellular processes not only by direct phosphorylation of numerous rate-limiting enzyme targets but also by initiating other protein kinase cascades. 45,46 Although diazoxide activation of PKC appears to be Ca 2 dependent, there may be cross talk between different second messenger systems, both Ca 2 -dependent and -independent, which may act synergistically to induce protection. Given the strategic importance of mitochondria in the cardiac cell, the mitochondrial site of action deserves further molecular characterization. Translocation of PKC activity from cytosolic to particulate compartments is commonly used as an index of PKC activation. 47 We used immunohistochemistry to determine subcellular localization of PKC isoforms. Although this technique primarily provides only qualitative data, the use of isoformspecific anti-pkc antibodies allows the assessment of both isoform-selective activation and compartmentation. 48 We confirmed the movement of PKC-, PKC-, PKC-, and PKC- I in diazoxide-pretreated hearts. These results are consistent with our previous report that a transient rise in Ca 2 as a result of Ca 2 preconditioning mediated the translocation of PKC- to the cell membrane. 12 An interesting finding of this study is that PKC- was predominantly translocated to the mitochondrial sites after diazoxide treatment. Because PKC-, PKC-, and PKC- I are Ca 2 -insensitive isoforms, it appears that diazoxide affects both Ca 2 -dependent and -independent pathways. The question whether diazoxide in-

9 1164 Diazoxide-Mediated Preconditioning Against Ca 2 Overload Injury creases the [Ca 2 ] i transient remains unanswered in this study because we did not directly measure intracellular [Ca 2 ] i during the pretreatment period. However, the diazoxide effect was blocked by verapamil and nifedipine, which suggests Ca 2 influx through these channels by diazoxide treatment. Because no reduction in cellular injury was observed in the hearts subjected to the Ca 2 PD, it is unlikely that myocardium injury is reduced with the concentration of the Ca 2 channel blockers used in this study. The activation of Ca 2 - dependent PKC- strongly suggests a rise in [Ca 2 ] i, which was attributed to Ca 2 influx from the extracellular space. Ca 2 could also be released from preloaded mitochondria by opening of MitoK ATP channels. 49 The translocation of PKC- isoform is also consistent with our previous studies with CPC in which a modest increase in [Ca 2 ] i played an important role in PKC-mediated pathways. 12 PKC- does not require Ca 2 for activation but requires diacylglycerol (DAG) and phosphatidylserine, a component of the plasma membrane. 32,48 Transient increase in [Ca 2 ] i as stated above activates Ca 2 - dependent isoforms of PKC and phospholipase C releasing 1,4,5-inositol triphosphate and DAG, which can stimulate Ca 2 -independent isoforms of PKC, such as PKC- and PKC-. 50 The role of DAG in the activation of PKC is supported by our previous studies 14 in which hearts were treated with SAG, a precursor of DAG, and by our current study with diazoxide. However, translocation of PKC to sarcolemma or mitochondrial sites was inhibited in hearts after treatment with 5-HD or chelerythrine. Thus it is reasonable to suggest that diazoxide, in addition to its effect on the MitoK ATP channel, activates PKC through the Ca 2 -mediated pathway. The significance of PKC is further indicated by the translocation of its isoform to the intercalated disks in diazoxide-treated hearts. It is known that weakening of intracellular junctions occurs at intercalated disks during Ca 2 depletion. 51 The translocation of PKC- to the intercalated disks in diazoxide-treated hearts may suggest that PKC- might modulate myocardial function through cell-to-cell interaction. In light of these findings, the presence of PKC- in the cell junctions may contribute to effective communication and ion exchange that reduces Ca 2 PD injury. 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