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(Circulation Research. 1997;80:790-799.)
© 1997 American Heart Association, Inc.

Articles

Ca²⁺ as a Mediator of Ischemic Preconditioning

Hiroshi Miyawaki, , Muhammad Ashraf

From the Department of Pathology and Laboratory Medicine, University of Cincinnati (Ohio) Medical Center.

Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Ave, Cincinnati, OH 45267-0529.

	Abstract

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Abstract We tested the hypothesis that elevation of [Ca²⁺]_i during ischemic preconditioning (IPC) stimulates protein kinase C (PKC), which confers the protection against the ischemic injury. Langendorff-perfused rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion (I/R). In preconditioned groups, hearts were subjected to either IPC, consisting of 5-minute global ischemia and 10-minute reperfusion, or high-Ca²⁺ preconditioning (HCPC), ie, the 5-minute perfusion of higher Ca²⁺ perfusate (2.3 mmol/L Ca²⁺) followed by 10-minute perfusion of normal perfusate (1.8 mmol/L Ca²⁺), and then were subjected to I/R. A significant functional recovery and decreased lactate dehydrogenase release were observed in HCPC and IPC hearts compared with ischemic control hearts. ATP contents of preconditioned hearts were significantly higher than those of the ischemic control hearts. The cell structure in preconditioned hearts was preserved better than that in the ischemic control hearts. Furthermore, the activation and translocation of PKC from cytoplasm to sarcolemma were observed in the preconditioned hearts. Verapamil administered during IPC significantly attenuated the salutary effects of IPC. Administration of chelerythrine, a specific PKC inhibitor, completely abolished the HCPC- and IPC-induced cardioprotection. The translocation of PKC by IPC was blocked by verapamil or chelerythrine. Immunohistochemical study using rabbit polyclonal antibody against PKC isoforms indicated that stress induced by IPC or HCPC evoked the translocation of PKC and PKC to the cell membrane. Translocation of PKC isoforms was attenuated by the treatment with verapamil or chelerythrine. These results demonstrate that (1) a transient increase in [Ca²⁺]_i during IPC is an important trigger for the activation of PKC, which is responsible for cardioprotection; (2) the elevation of [Ca²⁺]_i during IPC, at least partly, resulted from Ca²⁺ entry via voltage-dependent Ca²⁺ channel; and (3) activation and translocation of PKC and PKC occur during IPC and HCPC and may be important in preconditioning.

Key Words: preconditioning • ischemia • intracellular Ca²⁺ • protein kinase C

	Introduction

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Brief periods of myocardial ischemia and reperfusion render the myocardium tolerant to a subsequent sustained ischemia. This phenomenon has been known as IPC.¹ Since IPC is a strong endogenous protective mechanism, triggering factors and effectors of IPC have been under intense investigation. At present, several endogenous agonists such as adenosine,² norepinephrine,^{3 4} and bradykinin^{5 6} have been demonstrated to be triggers for IPC. Recently, activation of PKC, which is known to be a key player in numerous intracellular signal transduction pathways,^{7 8} is believed to be a central mechanism in the protection of IPC across various species.^{9 10 11 12}

We have demonstrated that Ca²⁺ preconditioning¹³ (repetitive Ca²⁺ depletion and repletion for short duration) conferred cardioprotection against ischemia/reperfusion injury¹⁴ via the activation of PKC, including Ca²⁺-dependent isoforms elicited by a transient increase in [Ca²⁺]_i. Since a brief period of ischemia/reperfusion causes an increase in [Ca²⁺]_i,^{15 16 17 18 19 20} we postulate that an increase in [Ca²⁺]_i during IPC also triggers activation of PKC responsible for the cardioprotection. The present study, for the first time, focused on and demonstrated the crucial role of Ca²⁺ as the trigger for IPC.

The purpose of the present study was to test the hypothesis that an increase in [Ca²⁺]_i induced by IPC triggers PKC signaling pathways, leading to protection against ischemic injury. We further tested whether (1) elevation of [Ca²⁺]_i by brief augmentation of [Ca²⁺]_o mimics IPC, (2) administration of a Ca²⁺ antagonist during IPC attenuates the protective effects of IPC, and (3) specific isoforms of PKC are involved with "Ca²⁺-mediated" preconditioning.

	Materials and Methods

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Materials
Verapamil hydrochloride was obtained from Research Biochemical Intl. Chelerythrine chloride was from Calbiochem-Novabiochem Co. 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; ß1, 656 to 671; ß2, 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. Verapamil and chelerythrine were directly administered through the aortic cannula at a rate of 0.2 mL/min by a Harvard infusion pump.

Heart Preparation
Sprague-Dawley male rats weighing 220 to 250 g were anesthetized with 40 mg/kg sodium pentobarbital. After an intravenous injection of 300 U heparin, the heart was rapidly excised via a midsternal thoracotomy and arrested in the ice-cold Krebs-Henseleit buffer containing (mmol/L) NaCl 118, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.8, NaHCO₃ 25, and glucose 11. The heart was attached to a Langendorff apparatus via an aorta for retrograde perfusion with Krebs-Henseleit buffer maintained at 37°C and pH 7.4 and saturated with a gas mixture of 95% O₂/5% CO₂. The coronary perfusion pressure was maintained at 80 mm Hg. A water-filled latex balloon connected to a pressure transducer (Gould P23Db) was introduced into the left ventricle via the mitral valve to record isovolumic left ventricular pressure. The balloon volume was adjusted to achieve a stable left ventricular end-diastolic pressure of 5 to 10 mm Hg during initial equilibration. The first derivative of left ventricular pressure (±dP/dt) was obtained from a differentiator (model 7P20, Grass Instrument Co). Heart rate, left ventricular pressure, and dP/dt were monitored on a Grass 7D polygraph. Right atrial pacing at 320 bpm was performed throughout the experiment, except during ischemia. The pulmonary artery was cannulated for collection of coronary effluent. Hearts were maintained at 37°C during the ischemic period in a water-jacketed beaker. The coronary effluent was collected in a beaker, and the flow was determined volumetrically at the end of equilibration and during the pretreatment and reperfusion periods.

Experimental Protocol
After equilibration, hearts were randomly divided into nine groups.

Group 1: Normal Control
Hearts (n=5) were perfused for 90 minutes with KH buffer as a normal control for different experimental groups.

Group 2: Ischemic Control
Hearts (n=6) were perfused with KH buffer for 20 minutes and were subjected to 40-minute global ischemia followed by 30-minute reperfusion.

Group 3: IPC
In this group, hearts (n=7) were preconditioned with 5-minute global ischemia followed by 10-minute reflow and then subjected to I/R.

Group 4: IPC and Verapamil
Hearts were infused with verapamil, an L-type Ca²⁺ channel blocker, at 8x10^-3 µmol/min for 5 minutes each before and after preconditioning ischemia (n=6). The final concentration of verapamil was 0.63 µmol/L. After a 5-minute washout period, hearts were subjected to I/R. Verapamil is reported to reduce the early increase in [Ca²⁺]_i-dependent fluorescence transients during ischemia,¹⁶ and the same dose of verapamil was found to block the temporary rise in contractility caused by Ca²⁺ preconditioning.¹⁴

Group 5: IPC and Chelerythrine
The protocol was similar to group 3, except chelerythrine chloride, a specific PKC inhibitor,²¹ was infused at 4x10^-2 µmol/min for 10 minutes before and after preconditioning ischemia (n=6). The final concentration of chelerythrine was 3.2 µmol/L. The dose of chelerythrine was chosen on the basis of a previous report by Speechly-Dick et al.⁹

Group 6: HCPC
Hearts (n=7) were perfused for 5 minutes with KH buffer containing 2.3 mmol/L Ca²⁺ followed by 10 minutes of normal Ca²⁺-containing KH buffer (1.8 mmol/L), and then the hearts were subjected to I/R. Intracellular Ca²⁺ is known to correlate with [Ca²⁺] in the perfusate,²² and this difference in [Ca²⁺] in the perfusate was determined to produce an 30% to 50% increase in +dP/dt, as observed in Ca²⁺ preconditioning.¹⁴

Group 7: HCPC and Chelerythrine
The protocol was similar to that for group 7, except chelerythrine chloride was infused through the aorta at 4x10^-2 µmol/min (3.2 µmol/L) during HCPC (n=6).

Group 8: Verapamil Alone
The protocol was similar to group 2, except verapamil was infused at 8x10^-3 µmol/min (0.63 µmol/L) for 15 minutes, followed by 5 minutes of a washout period. After that, hearts were subjected to I/R (n=5).

Group 9: Chelerythrine Alone
The protocol was similar to group 2, except chelerythrine was infused at 4x10^-2 µmol/min (3.2 µmol/L) for 15 minutes before global ischemia and reperfusion (n=5).

Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in coronary effluent. This was assayed by a coupled-enzyme spectrophotometric technique using a Sigma assay kit. Measurement of enzyme activity was based on oxidation of lactate and the rate of increase in absorbance at 340 nm. The accumulated amount of LDH released during 30 minutes of reperfusion was obtained by integrating the area underneath the individual time course curve for 30 minutes of reperfusion.^{14 23}

Measurement of Tissue ATP
The heart was immediately frozen in liquid nitrogen and was freeze dried for 24 hours. 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.^{14 23}

Measurement of PKC Activity
PKC activity was measured with a modified method as previously described.^{24 25} At the end of the pretreatment period, hearts were excised, immediately frozen in liquid nitrogen, and stored at -70°C until use. Twenty milligrams of the freeze-dried tissues of the hearts was used for analysis. The left ventricles were dissected and minced. The minced tissue was homogenized with 1.5 mL of homogenizing buffer containing (mmol/L) Tris 50, EDTA 4, and mercaptoethanol 15, pH 7.5. The homogenate was subjected to differential centrifugation at 1500g for 10 minutes. The supernatant was collected and centrifuged at 100 000g for 1 hour, and it served as the cytosolic fraction. The membrane pellet was resuspended in 1.5 mL homogenizing buffer containing 0.3% Triton X-100 and mixed gently for 1 hour on the ice. The homogenate was centrifuged at 100 000g for 1 hour, and the supernatant contained the membrane fraction. PKC activity was measured after incubation for 10 minutes at 30°C in a reaction mixture containing 5 mmol/L Tris, 10 mmol/L MgCl₂, 0.15 mol/L KCl, 0.2 mmol/L dithiothreitol, 0.2 mg/mL BSA, 2.0 mmol/L ATP, and 100 µmol/L substrate peptide [Ser²⁵]PKC(19-31).²⁶ Phosphorylation of the Ser residue in the PKC substrate peptide catalyzed by the catalytic subunit of PKC at pH 7.5 causes a spectral change at 430 nm. PKC activity was analyzed by absorbance at 430 nm in a Gilford spectrophotometer. The activity of PKC was expressed as nmol/mg dry wt.

Immunofluorescence Microscopy
Subcellular localization of PKC isoforms after various interventions was visualized by immunocytochemical staining as previously described.^{5 11 14} In these experiments, hearts in different experimental groups were perfused as described above and harvested just before prolonged ischemia. Three to four hearts from each group (control, IPC, IPC with verapamil, HCPC, and HCPC with chelerythrine) were selected for immunohistochemistry. Left ventricular tissue was blotted and embedded in OCT compound, rapidly frozen in liquid nitrogen, and stored at -70°C until use. Six cryosections, 5 µm in thickness, were prepared with a cryostat (Jung Frigocut 2800E, Leica) and collected on poly-L-lysine–coated slides. All sections were fixed in a 70% acetone/30% methanol mixture at -20°C for 10 minutes, 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, PKCß1, PKCß2, PKC, PKC, PKC, PKC, and PKC) were diluted with PBS containing 0.1% BSA. Sections were incubated for 1 hour at room temperature with diluted primary antibodies and then washed with PBS three times. Sections were then incubated for 45 minutes with indocarbocyanine-conjugated goat anti-rabbit IgG. After that, sections were washed once with 0.1% Triton X-100 in PBS and twice with PBS. Nuclear staining was achieved with bis-benzamide (10 mg/mL in PBS) for 30 seconds and washed with PBS three times. Sections were mounted with a medium (Antifade, Oncor) and were either examined and photographed with a microscope equipped with fluorescence optics (BH-2 with a PM-CBSP camera, Olympus) or stored at 4°C.

Morphological Examination
Tissue from the midventricular wall was cut into 1.0-mm pieces, fixed in 2.5% buffered glutaraldehyde, postfixed in 1% buffered osmium tetroxide, and processed for transmission electron microscopy. A semiquantitative estimate of cell damage was carried out on 1-µm-thick sections. Five randomly chosen blocks from each heart were examined for quantification of cell damage without prior knowledge of the treatment. Approximately 1500 cells were analyzed in each heart, and one of three degrees of cell damage was assigned to each cell. Cellular changes were assessed according to the following classification: (1) normal (compact myofibers with uniform staining of nucleoplasm, well-defined rows of mitochondria between the myofibrils, and no separation 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 bands were added in this category).^{14 23}

Statistical Analysis
Data were expressed as mean±SE. Group comparisons were performed by ANOVA with multiple comparisons or t test when appropriate. A difference of P<.05 was considered significant.

	Results

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Preconditioning Effect on Heart Function
In Table 1, a significant decrease in recovery of left ventricular developed pressure, left ventricular dP/dt, and coronary flow upon reperfusion was observed in ischemic control hearts. Functional recovery was accelerated in IPC hearts after ischemia and reperfusion in contrast to ischemic control hearts. The protective effect of HCPC was similar to IPC. Pretreatment of IPC hearts with verapamil significantly attenuated the preconditioning effect on heart function. During verapamil treatment, left ventricular developed pressure was significantly decreased (75.5±4.2% of equilibration value). To determine whether the beneficial effects of IPC and HCPC were mediated by PKC, administration of chelerythrine, a specific PKC inhibitor, during IPC and HCPC abolished the beneficial effects of both IPC and HCPC.

View this table: [in this window] [in a new window]   Table 1. Effects of Preconditioning on Heart Function in Hearts Subjected to I/R

Preconditioning Effect on LDH Release and ATP Contents
In ischemic control hearts, a multifold increase in LDH release (21.9±1.4 U/g) was observed upon reperfusion (Fig 1), and ATP levels were reduced to 6.1±0.7 µmol/g dry wt compared with the normal control value of 20.3±0.8 µmol/g dry wt (Fig 2). In IPC hearts, LDH release was significantly decreased (7.0±0.7 U/g), and ATP contents were markedly preserved (13.8±0.8 µmol/g dry wt) compared with those in ischemic control hearts. Verapamil given during IPC neither reduced LDH release (18.9±2.3 U/g) nor preserved ATP content (7.9±0.6 µmol/g dry wt). Administration of chelerythrine during IPC and HCPC completely reversed the beneficial effects of preconditioning. Pretreatment with verapamil alone did not exert a significant effect on the ischemic injury compared with no pretreatment in ischemic control hearts.

View larger version (59K): [in this window] [in a new window]   Figure 1. Effect of preconditioning on LDH release. LDH loss is significantly reduced in hearts subjected to I/R after IPC or HCPC compared with ischemic control hearts. The release of LDH was not reduced in hearts treated with verapamil (VR) compared with nontreated IPC hearts. Treatment with chelerythrine (CH), a PKC inhibitor during IPC and HCPC, completely blocked the protection. Values are mean±SEM. *P<.05 vs ischemic control.

View larger version (52K): [in this window] [in a new window]   Figure 2. Effects of preconditioning on tissue high-energy phosphates (ATP). ATP depletion is increased in IPC hearts treated with verapamil (VR) compared with nontreated IPC hearts. ATP contents in preconditioned hearts treated with chelerythrine (CH) are nearly similar to those in ischemic control hearts. Values are mean±SEM. *P<.05 vs ischemic control.

Preconditioning Effect on Cell Damage
The cellular structure of the normal control hearts was well maintained (Fig 3A). Cell damage was severe in the ischemic control hearts (Fig 3B). The semiquantitative estimate of cell injury showed that only 12.9±2.7% of the cells were normal and that 13.4±2.6% and 73.3±4.3% of the cells were mildly and severely damaged, respectively (Table 2). The cellular structure was extremely well preserved in the IPC hearts: 66.2±5.7% of the cells were normal; 13.0±2.1% and 20.8±3.9% were mildly and severely damaged, respectively. The degree of cell damage was much less than that in the ischemic control hearts. The cellular structure in the HCPC hearts was also well preserved, similar to that in IPC hearts (Fig 3F), and 67.9±4.4% of the cells were normal; 14.6±2.6% and 17.5±2.1% were mildly and severely damaged, respectively. In IPC hearts treated with verapamil, the cellular damage indicated by intracellular vacuolization and hypercontraction was clearly observed (Fig 3D); 25.0±3.6% of cells were normal, and 23.2±2.9% and 51.9±3.6% were mildly and severely damaged, respectively. Treatment with chelerythrine during the IPC or HCPC period also abolished their beneficial effects on the cellular structure. The cell damage (Fig 3E and 3G) was almost similar to that in the ischemic control group.

View larger version (172K): [in this window] [in a new window]   Figure 3. A, Light micrograph of normal (nonischemic) heart showing compact and well-preserved myofibers. Nuclei show uniform distribution of chromatin material (magnification x320). B, Light micro-graph of ischemic control heart subjected to I/R showing extensive damage to the myofibers. Many myofibers are hypercontracted releasing intracellular contents (arrowhead) and form contraction bands (arrow) (x320). C, Light micrograph of IPC heart subjected to I/R. The majority of cells are normal with distinct nuclei. A few cells have undergone contraction band formation (arrow) (magnification x320). D, Light micrograph of heart pretreated with verapamil during IPC and then subjected to prolonged ischemia and reperfusion. The cellular damage is clearly observed. Intracellular vacuolization and hypercontraction (arrow) are observed. A few normal cells are also seen (magnification x320). E, Light micrograph of heart treated with chelerythrine during IPC and then subjected to prolonged ischemia and reperfusion. Many cells (arrow) are severely damaged. However, a few normal-appearing cells are also seen (asterisk) (magnification x320). F, Light micrograph of heart preconditioned with high Ca2+ and then subjected to longer ischemia and reperfusion. Overall, myofibers are well preserved, similar to IPC (upper section of panel); however, a few cells are severely damaged (arrow, lower section of panel) (magnification x320). G, Light micrograph of heart preconditioned with high Ca2+ with simultaneous treatment of chelerythrine before prolonged ischemia and reperfusion. Many cells are severely damaged (arrow). However, a few normal cells are also observed (upper left) (magnification x320). H, Light micrograph of nonpreconditioned heart treated with verapamil before ischemia and reperfusion. Most of the cells are severely damaged (arrow). Some normal cells are also observed at the bottom (magnification x320).

View this table: [in this window] [in a new window]   Table 2. Semiquantitative Estimate of Morphological Damage in Hearts Subjected to I/R and Various Interventions

Effect of Preconditioning on PKC Activity
Translocation of PKC from cytoplasm to the plasma membrane has been recognized as an index of PKC activation. We determined the distribution of PKC activity after IPC and HCPC with or without various treatments (Fig 4). A prominent increase in PKC activity of the membrane fraction was observed in IPC and HCPC hearts (5.60±0.27 and 5.91±0.46 nmol/mg dry wt, respectively) compared with control hearts (1.33±0.09 nmol/mg dry wt) (Fig 4, left). On the other hand, chelerythrine completely blocked the preconditioning-induced increase in PKC activity of the membrane fraction (IPC with chelerythrine, 1.47±0.09 nmol/mg dry wt; HCPC with chelerythrine, 1.31±0.15 nmol/mg dry wt). Treatment with verapamil also significantly reduced PKC activity of membrane fraction in IPC hearts (Fig 4, left). The significant reduction in PKC activity of the cytosolic fraction was observed in preconditioned hearts with ischemia or high Ca²⁺ (Fig 4, right).

View larger version (29K): [in this window] [in a new window]   Figure 4. Effect of IPC and HCPC on PKC activity after various treatments. Left, IPC and HCPC caused a prominent increase in PKC activity of membrane fraction. Treatment with chelerythrine (CH), a PKC inhibitor during preconditioning, completely abolished the effect. Hearts treated with Ca2+ blockers during IPC also showed marked reduction in PKC activity of membrane fraction compared with IPC hearts. Right, There were no significant differences in PKC activity of the cytosolic fraction. VR indicates verapamil. Values are mean±SEM of five experiments. *P<.05 vs control.

Subcellular Localization of PKC Isoforms After Preconditioning
Representative results from the immunohistochemical study are shown in Fig 5. PKC, which was diffusely distributed in the cytoplasm in control hearts (Fig 5B), was distinctly localized in the plasma membrane of preconditioned hearts (Fig 5C and 5E). Furthermore, HCPC stimulated its translocation to the nuclear region. Translocation of PKC observed in preconditioned hearts did not take place after the treatment with verapamil (Fig 5D) or chelerythrine (Fig 5F). The distribution pattern of PKC is shown in Fig 5G through 5I. HCPC caused its translocation from the cytoplasm (Fig 5G) to the sarcolemma but not to the nuclear region (Fig 5H). Translocation of PKC to either the sarcolemma or nuclear region in the myocardium after HCPC was prevented by chelerythrine (Fig 5I). Among other PKC isoforms, PKCß1 staining was primarily perinuclear or nuclear under all conditions, ie, in all experimental groups. PKC staining in the intercalated disk was observed in >90% of cells, and its pattern of staining was unchanged by IPC or HCPC. However, preconditioning stimuli increased its translocation to the nucleus.

View larger version (120K): [in this window] [in a new window]   Figure 5. Immunofluorescent staining of PKC isoforms. A, Negative control (no PKC antibody). B, PKC in control heart. Diffuse cytoplasmic distribution of the isoform is observed. C, PKC in an IPC heart. Immunofluorescence is observed predominantly at the plasma membrane (arrow). D,PKC in an IPC heart treated with verapamil. Immunofluorescence is diffuse throughout the cytoplasm. E, PKC in an HCPC heart. Sarcolemmal (arrow) and intracellular (arrowhead) distributions are pronounced. F, PKC in an HCPC heart treated with chelerythrine. In contrast to the HCPC heart (panel E), immunofluorescence is diffuse throughout the cytoplasm. G, PKC in control heart. Diffuse cytosolic distribution is observed. H, PKC in an HCPC heart. PKC is positively localized in the sarcolemma (arrow). I, PKC in an HCPC heart treated with chelerythrine. A diffuse cytoplasmic distribution of PKC is observed, and its translocation to the plasma membrane is rarely observed. Original magnifications x320.

	Discussion

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The major findings of the present study are as follows: (1) a transient elevation of [Ca²⁺]_o (HCPC) mimics IPC; (2) blocking of Ca²⁺ during IPC attenuates the protection by affecting the translocation of PKC; (3) cardioprotection is mediated by the PKC signaling pathway and is blocked with a specific inhibitor of PKC, chelerythrine; and (4) and isoforms of PKC are likely involved in preconditioning.

Increase in Intracellular Ca²⁺ and IPC
The main hypothesis of the present study was that Ca²⁺ is an important intracellular trigger or mediator of IPC. This hypothesis was formulated from our previous study,¹⁴ in which Ca²⁺ preconditioning,¹³ characterized by transient stress induced by repetitive Ca²⁺ depletion and repletion of short duration, provided a strong protection against ischemic injury. In our previous study,¹⁴ we showed that blockade of Ca²⁺ entry with verapamil, amiloride, or low-Na⁺ perfusion during Ca²⁺ preconditioning attenuated the activation of PKC, and eventually the protection was lost.

A number of studies have shown that [Ca²⁺]_i increases after the induction of myocardial ischemia. It is generally believed that a high Ca²⁺ influx during ischemia/reperfusion causes pathological changes in the ischemia/reperfusion injury,^{17 27 28 29 30 31} although it is noteworthy that brief ischemia also evokes a transient and mild elevation of [Ca²⁺]_i.^{15 16 17 18 19 20 32 33} Smith et al³² have recently demonstrated that [Ca²⁺]_i measured by surface fluorometry using indo 1 showed a 2- to 4-fold increase during 5-minute preconditioning ischemia in the isolated rat heart. Thus, it is reasonable to propose that the [Ca²⁺]_i increase in preconditioning ischemia/reperfusion is a strong trigger for IPC.

The results from the present study that administration of the L-type Ca²⁺ channel antagonist verapamil during IPC attenuates the IPC-induced protection support our hypothesis. In addition, the experimental maneuvers that result in transient elevation of [Ca²⁺]_i mimic IPC. In the latter experiments, a brief infusion of high-Ca²⁺ medium yielded results similar to IPC. Therefore, it appears that Ca²⁺ plays an important role in activating intracellular pathways leading to protection. Although we did not measure [Ca²⁺]_i during the pretreatment period, the increase in [Ca²⁺]_i during IPC was blocked by verapamil by reducing the Ca²⁺ influx through the Ca²⁺ channel.^{16 33 34} It is also possible that cellular acidosis is reduced by verapamil during IPC, which may lead to secondary inhibition in the Na⁺-Ca²⁺ exchanger linked to the Na⁺-H⁺ exchanger.^{29 34} Consequently, the lack of adequate stress by Ca²⁺ resulted in attenuation of protection by IPC.

Mechanisms of Ca²⁺-Induced PKC Activation
Recently, enough evidence has been accumulated that PKC activation is essential for the protection induced by IPC.^{9 10 11 12 25} In the present study, we demonstrated that the pretreatment with a specific PKC inhibitor abolished the protective effects of a brief Ca²⁺ loading during HCPC or IPC. The measurement of PKC activity (Fig 3) further strengthened our hypothesis that a transient increase in [Ca²⁺]_i led to translocation of PKC from the cytoplasm to the cell membrane. The redistribution of PKC was completely blocked by chelerythrine, and it resulted in loss of protection. The blockade of Ca²⁺ entry by verapamil also indirectly affected the translocation of PKC. Therefore, it is likely that appropriate Ca²⁺ stress renders the hearts tolerant to ischemia through the activation of PKC.

The link between Ca²⁺ and PKC activity appears to be stronger. [Ca²⁺]_i may directly activate the Ca²⁺-dependent isoforms of PKC (conventional PKC)^{35 36} or may stimulate the activation of PKC through the G_q protein^{37 38} linked to Ca²⁺-dependent PLC.^{37 39} As a result of PLC activation, inositol 1,4,5-triphosphate is produced from PIP₂, thus increasing cytosolic free Ca²⁺ through Ca²⁺ release from the sarcoplasmic reticulum. DAG is also produced from PIP₂ and maintains activation of PKC.⁷

Recent studies have indicated that norepinephrine^{3 4 10} and bradykinin^{5 6} are strongly implicated in IPC of the isolated rat heart. These endogenous substances also stimulate the phosphatidylinositol cycle⁴⁰ and produce DAG and inositol 1,4,5-triphosphate, causing elevation of [Ca²⁺]_i. Adenosine produced by Ca²⁺ stress may be involved with Ca²⁺-PKC–mediated protection. In rat hearts, the role of adenosine in IPC has been questioned since Liu and Downey⁴¹ reported that an adenosine A₁ receptor agonist could induce the protection but that pretreatment with an adenosine receptor blocker failed to prevent the protection. However, recently, Headrick² has elegantly demonstrated that interstitial adenosine levels during IPC are so high in the rat that higher concentration of adenosine A₁ antagonist is required to block the protection. Thus, adenosine is obviously involved with IPC in the rat. In our previous studies,^{13 14} Ca²⁺ preconditioning by repeated Ca²⁺ depletion and repletion simultaneously caused a multifold increase in adenosine release¹³ and the activation of PKC.¹⁴ Therefore, it is convincing that Ca²⁺ stress by HCPC also releases endogenous adenosine, which induces beneficial effects on subsequent prolonged ischemia/reperfusion⁴² or Ca²⁺ paradox¹³ via receptor-mediated mechanisms. Adenosine further increases the production of inositol phosphate compounds,⁴³ suggesting that it is also coupled to PKC. Adenosine is also reported to activate PKC through the A₁ receptor–G_i protein signaling pathway.⁴⁴ Thus, it is likely that Ca²⁺, adenosine, and PKC are interlinked and exert a strong influence in the preconditioning phenomenon.

Translocation and Activation of PKC Isoforms With IPC and HCPC
We used immunohistochemistry to determine subcellular localization of PKC isoforms. We confirmed the existence of PKC, PKCß1, PKC, and PKC in adult rat hearts, consistent with the previous studies.^{11 45 46 47} Among PKC isoforms, PKC and the Ca²⁺-independent isoform PKC were prominently translocated from the cytoplasm to the plasma membrane after IPC and HCPC. These results were consistent with our previous study,¹⁴ in which both PKC and PKC were activated and translocated to the cell membrane after Ca²⁺ stress by Ca²⁺ preconditioning. This is partly in contrast to the study by Mitchell et al,¹¹ in which only PKC was translocated to the sarcolemma with the preconditioning stimulus of ischemia or ₁-receptor activation in the isolated rat. This variation could be due to differences in timing of sample collection in these studies. We harvested hearts just after 5-minute ischemia and 10-minute reperfusion in IPC hearts, in contrast to the study by Mitchell et al, in which hearts were harvested after only 2-minute ischemia but without 10-minute reperfusion. Because higher [Ca²⁺]_i transients are still observed in the early phase of reperfusion³² after brief preconditioning ischemia, it is possible that Ca²⁺-related activation of PKC was lacking in the latter study without reperfusion. This might have influenced the translocation of conventional PKC.

PKC does not require Ca²⁺ for activation but requires DAG and a component of the plasma membrane, phosphatidylserine.^{7 48} The reason for the activation of the Ca²⁺-independent isoform PKC by high-Ca²⁺ stress is unknown. However, as discussed previously, Ca²⁺ stress may be interlinked with the phosphatidylinositol pathway through Ca²⁺-dependent PLC, which results in the production of DAG from PIP₂. Alternatively, Ca²⁺ stress might also stimulate the production of DAG from other precursors, such as phosphatidylcholine and glycerol,⁴⁹ and then novel PKC (PKC) can be activated by DAG.

Another interesting finding was that the translocation of PKC to the nucleus was highly conspicuous in HCPC hearts. This phenomenon might be associated with the "second window of preconditioning"^{50 51} by Ca²⁺ stress through regulation of gene transcription,⁸ because the acute preconditioning effects of HCPC were similar to IPC.

PKC and PKCß1 were distributed in the intercalated disk and perinuclear region of the control heart, respectively, but did not migrate to other intracellular organelles after preconditioning stimuli. In agreement with our results, PKC might modulate myocardial function through cell-to-cell interaction, and PKCß1 might be involved with protein transportation to the nucleus.⁵² However, these isoforms do not appear to contribute to protection by classical preconditioning. Further studies are required to determine the isoform-specific function of PKC in myocardium with or without preconditioning stimuli.

Meanwhile, Przyklenk et al⁵³ have failed to detect the translocation of PKC from the cytosol to the cell membrane in in vivo dog hearts after preconditioning ischemia by immunohistochemical methods. This finding is in sharp contrast to our findings and those of Mitchell et al.¹¹ The reason for the discrepancy among different studies is obviously due to species differences and experimental protocols. So far, numerous studies in animal and human myocardium^{12 54} suggest that PKC activation plays a pivotal role in the mechanism of preconditioning against the lethal ischemic injury.

Possible Mechanisms of Cardioprotection by PKC
Although PKC activation plays an important role in the preconditioning phenomenon, it is not presently clear how PKC affords cardioprotection to the hearts. However, the most plausible explanation for PKC-mediated protection is that PKC attenuates the deleterious [Ca²⁺]_i increase that occurs during prolonged ischemia and reperfusion. PKC can activate K_ATP,^{55 56 57} which leads to shortening of the action potential duration and, thus, reduction of Ca²⁺ influx. It is especially enforced in the study by Light et al,⁵⁷ in which PKC altered the stoichiometry of ATP binding to K_ATP, resulting in PKC-mediated activation of K_ATP at near physiological ATP concentrations. This finding has an important implication for IPC, because significant reduction of ATP concentration, which usually triggers the opening of K_ATP, has not been observed during brief preconditioning ischemia.⁵⁸ Involvement of K_ATP with IPC in the rat is controversial⁵⁹ ; however, Schultz et al⁵⁹ have recently confirmed that glibenclamide, a blocker of K_ATP, is able to abolish the protection by IPC in the isolated rat heart, thus suggesting a role of K_ATP in preconditioning.

Other possible mechanisms for the reduction of [Ca²⁺]_i increase by PKC are accelerated Ca²⁺ uptake from cytosol by sarcoplasmic reticulum through activation of sarcoplasmic reticular (Ca²⁺-Mg²⁺) ATPase,⁶⁰ inhibition of the L-type Ca²⁺ channel,^{61 62 63} and stimulation of the sarcolemmal Ca²⁺ pump.⁶⁴ The increase of myocardial Ca²⁺ sensitivity⁵⁸ and phosphorylation of contractile proteins^{65 66 67 68} by activated PKC may also contribute to modulation of myocardial function in hearts subjected to prolonged ischemia and reperfusion.

Conclusions
We have demonstrated that the transient increase in [Ca²⁺]_i during IPC is a trigger for the induction of IPC and that [Ca²⁺]_i effectively activates PKC, which modulates different ion channels, leading to the protection. These findings appear to be significant and clinically relevant and require further investigation for the development and manipulation of pharmacological agents eliciting preconditioning effects under clinical conditions.

	Selected Abbreviations and Acronyms

 

DAG = diacylglycerol HCPC = high-Ca2+ preconditioning I/R = 40-minute global ischemia followed by 30-minute reperfusion IPC = ischemic preconditioning KATP = ATP-sensitive K+ channel KH = Krebs-Henseleit LDH = lactate dehydrogenase PIP2 = phosphatidylinositol 4,5-bisphosphate PKC = protein kinase C PLC = phospholipase C

	Acknowledgments

 
This study was supported by grants HL-23597 and HL-55678 from the National Institutes of Health, National Heart, Lung, and Blood Institute. The authors would like to express their thanks to Dr Robert Fontaine for his expert technical assistance and suggestions regarding the immunohistochemical study.

Received December 20, 1996; accepted March 27, 1997.

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