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(Circulation. 1997;96:1257-1265.)
© 1997 American Heart Association, Inc.

Articles

Role of Intracellular Ca²⁺ in Activation of Protein Kinase C During Ischemic Preconditioning

Koichi Node, MD; Masafumi Kitakaze, MD; Hiroshi Sato, MD; Tetsuo Minamino, MD; Kazuo Komamura, MD; Yoshiro Shinozaki, BE; Hidezo Mori, MD; ; Masatsugu Hori, MD

From the First Department of Medicine (K.N., M.K., H.S., T.M., K.K., M.H.), Osaka University School of Medicine, Osaka, and the Department of Physiology (Y.S., H.M.), Tokai University, Isehara, Japan.

Correspondence to Masafumi Kitakaze, MD, PhD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail Kitakaze{at}medone.med.osaka-u.ac.jp

	Abstract

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Background Activation of protein kinase C plays an important role in ischemic preconditioning. Given that protein kinase C is activated by an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and that myocardial ischemia and reperfusion increase [Ca²⁺]_i, the effect of transient exposures to Ca²⁺ on infarct size and the effect of administration of EGTA during ischemic and ₁-adrenoceptor–mediated preconditioning on the limitation of infarct size were investigated in the canine heart.

Methods and Results In open-chest dogs, 5 minutes after the completion of either three 5-minute infusions of CaCl₂ or four 5-minute infusions of the ₁-adrenoceptor agonist methoxamine into the coronary artery, the coronary arteries were occluded for 90 minutes; this occlusion was followed by a 6-hour reperfusion in both the Ca²⁺ preconditioning and methoxamine groups. Infarct sizes in the Ca²⁺ preconditioning (15.8±2.3%) and methoxamine (10.1±2.2%) groups were significantly (P<.01) smaller than in the control group (42.5±2.9%), and administration of either an inhibitor of protein kinase C (GF109203X) or an inhibitor of ecto-5'-nucleotidase (,ß-methyleneadenosine 5'-diphosphate) reduced the infarct size–limiting effect of Ca²⁺ preconditioning. Administration of EGTA during ischemic or ₁-adrenoceptor–mediated preconditioning inhibited both the infarct size–limiting effect and the activation of protein kinase C and ecto-5'-nucleotidase induced by these procedures.

Conclusions [Ca²⁺]_i during ischemic and ₁-adrenoceptor–mediated preconditioning plays an important role in the infarct size–limiting effect of these procedures by activating protein kinase C and ecto-5'-nucleotidase in the canine heart.

Key Words: adenosine • calcium • myocardial infarction • pathology • receptors, adrenergic, alpha

	Introduction

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Ischemic preconditioning, achieved by exposure of the heart to brief periods of ischemia, results in marked tolerance of the myocardium to normally lethal ischemic insults.¹ The IS-limiting effect of IP is reduced by exposure to 8-SPT, an antagonist of adenosine receptors.² Furthermore, we have shown³ that activation of ecto-5'-N, which catalyzes 5'-AMP and the synthesis of adenosine, contributes to IP-induced cardioprotection and that activation of PKC is responsible for the activation of ecto-5'-N. Because myocardial ischemia and reperfusion increases [Ca²⁺]_i in the myocardium,^{4 5} it is likely that the brief periods of ischemia during IP also increase [Ca²⁺]_i. Furthermore, we have observed³ that activation of PKC in response to ₁-adrenoceptor stimulation during IP requires intracellular Ca²⁺ and limits IS through activation of ecto-5'-N, suggesting that an increase in [Ca²⁺]_i during IP contributes to the activation of PKC and subsequently, ecto-5'-N, and mediates the IS-limiting effect of this procedure.

To clarify the role of [Ca²⁺]_i in the IS limitation, we examined whether transient exposures to CaCl₂ limit IS through activation of PKC, and subsequently, ecto-5'-N. We also investigated whether administration of the Ca²⁺ chelator EGTA during IP or ₁-adrenoceptor–mediated preconditioning inhibits the IS-limiting effect of these procedures.

	Methods

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Instrumentation
Mongrel dogs (body weight, 16 to 24 kg) were anesthetized with sodium pentobarbital (30 mg/kg body wt IV), intubated, and ventilated with room air mixed with oxygen (100% O₂ at a flow rate of 1.0 L/min). The chest was opened through the left fifth intercostal space, and the heart was suspended in a pericardial cradle. After administration of heparin (500 U/kg IV), we cannulated the LAD and perfused it with blood from the left carotid artery through an extracorporeal bypass tube. CBF in the perfused area was measured with an electromagnetic flow probe attached to the bypass tube, and CPP was monitored at the tip of the coronary artery cannula. A pair of ultrasonic crystals (5 MHz, 2 mm in diameter; Schuessler) was implanted in the left ventricular anterior wall in the endomyocardial segment in the center of the perfused area to measure segment length. We calculated FS from the equation FS=[(EDL-ESL)/EDL]x100%, where EDL and ESL are end-diastolic and end-systolic segment lengths, respectively.

A small-caliber (1 mm), short (70 mm) collecting tube was introduced into a small coronary vein near the center of the perfused area to sample coronary venous blood. Drained venous blood was collected in a reservoir placed at the level of the left atrium and was returned to the jugular vein. The left atrium was catheterized for microsphere injection. The femoral artery was also cannulated for the sampling of reference (control) blood. Hydration was maintained by a slow infusion of saline. Aortic blood pressure was monitored at the tip of the cannula to the femoral artery.

Heart rate averaged 139±2 bpm during control conditions and did not change during the study. The pH and partial pressures of O₂ and CO₂ in the systemic arterial blood before performing the experimental protocols were 7.42±0.02, 104±4 mm Hg, and 37.5±1.9 mm Hg, respectively.

Experimental Protocols
Protocol 1: Effects of IP and ₁-Adrenoceptor–Mediated Preconditioning
After the dogs became hemodynamically stable, four cycles of coronary occlusion for 5 minutes followed by 5 minutes of reperfusion were performed to precondition the myocardium before the onset of 90 minutes of ischemia and a subsequent 6 hours of reperfusion (IP group, n=8). In a second group of dogs (MTX group, n=6), we administered the ₁-adrenoceptor agonist MTX (Sigma Chemical Co) dissolved in saline (40 µg·kg^-1·min^-1; infusion rate, 16.7 µL·kg^-1·min^-1 IC) for four 5-minute periods separated by 5-minute intervals before the onset of 90 minutes of coronary occlusion and 6 hours of reperfusion. In the control group (n=8), 45 minutes after the dogs were hemodynamically stabilized, they were subjected to 90 minutes of coronary artery occlusion and 6 hours of reperfusion. Coronary arterial and venous blood were sampled at 20-minute intervals during 45 minutes of hemodynamic stabilization for the measurement of plasma adenosine, lactate, and norepinephrine concentrations. In addition to the dogs used for the measurement of IS, other animals were used to measure PKC activity in cytosolic and membrane fractions of the endocardium as well as cytosolic and ecto-5'-N activity in the endocardium and epicardium of the LAD area. Animals were killed immediately after the IP (n=5) or ₁-adrenoceptor–stimulated preconditioning (n=5) procedures; control dogs (n=5) were killed 40 minutes after they reached hemodynamic stabilization.

Protocol 2: Effects of EGTA During IP and ₁-Adrenoceptor–Mediated Preconditioning
Because activation of PKC requires Ca²⁺, [Ca²⁺]_i in IP and ₁-adrenoceptor–mediated preconditioning may contribute to the activation of PKC and ecto-5'-N, and subsequently, to the IS-limiting effect. To test this hypothesis, we examined whether administration of EGTA (1.7 µmol·kg^-1·min^-1 IC) beginning 10 minutes before and continuing throughout IP (IP+EGTA group, n=7) and MTX preconditioning (MTX+EGTA group, n=7) reduced the IS-limiting effect. The LAD was then occluded for 90 minutes, after which reperfusion was performed for 6 hours. Animals in the EGTA group (n=7) received EGTA for 45 minutes before coronary occlusion and reperfusion. In another three groups (n=5 for each group), corresponding to the IP+EGTA, MTX+EGTA, and EGTA groups in protocol 1, we measured PKC and ecto-5'-N activities as described for the earlier protocol.

Protocol 3: Effects of Transient Exposures to CaCl₂ on IS and Activities of PKC and Ecto-5'-N
To test whether transient exposures to CaCl₂ (Ca²⁺ preconditioning) mimic the IS-limiting effect of IP, we administered CaCl₂ into the LAD (8 µmol·min^-1·mL^-1 of CBF; infusion rate, 16.7 µL·kg^-1·min^-1) for three periods of 5 minutes each with 5-minute intervals (Ca²⁺ group, n=8). The dogs were then subjected to 90 minutes of coronary occlusion followed by 6 hours of reperfusion. In five other dogs, we measured PKC and ecto-5'-N activities after the Ca²⁺ preconditioning procedure as described for protocol 1. We also examined the effects on IS and the activities of PKC and ecto-5'-N of three additional doses of CaCl₂ (4, 6, and 10 µmol·min^-1·mL^-1 of CBF; infusion rate, 16.7 µL·kg^-1·min^-1) administered into the LAD for three periods of 5 minutes each separated by 5-minute intervals (n=5 for each group). Finally, we also administered CaCl₂ into the LAD (8 µmol·min^-1·mL^-1 of CBF) for one cycle of 5 minutes with a 5-minute interval (n=5). CBF is expressed in milliliters per minute.

Protocol 4: Effects of GF109203X Administration During Ca²⁺ Preconditioning on IS-Limiting Effect and Ecto-5'-N Activities
To examine the role of PKC in Ca²⁺ preconditioning, we concomitantly infused GF109203X, a selective inhibitor of PKC. Infusion of GF109203X (40 ng·kg^-1·min^-1; infusion rate, 16.7 µL·kg^-1·min^-1; Calbiochem) into the LAD was started 5 minutes before and continued during Ca²⁺ preconditioning (Ca²⁺+GF109203X group, n=8). We also determined the effect of GF109203X alone on IS (GF109203X group, n=7); GF109203X was administered for 45 minutes before coronary occlusion. We measured ecto-5'-N activity in the endocardium and epicardium of the LAD area in five animals each corresponding to the Ca²⁺+GF109203X and GF109203X groups.

Protocol 5: Effects of AMP-CP and 8-SPT on the IS-Limiting Effect of Ca²⁺ Preconditioning
To examine the roles of the activation of ecto-5'-N and endogenous adenosine in Ca²⁺ preconditioning, we infused AMP-CP (8 µg·kg^-1·min^-1; infusion rate, 16.7 µL · kg^-1 · min^-1 IC; Sigma), a specific and competitive inhibitor of ecto-5'-N, or 8-SPT (25 µg·kg^-1·min^-1; infusion rate, 16.7 µL·kg^-1 · min^-1 IC; Research Biochemicals) beginning 5 minutes before and continuing throughout the Ca²⁺ preconditioning procedure and resuming for the first 60 minutes of reperfusion (Ca²⁺+AMP-CP group [n=8] and Ca²⁺+8-SPT group [n=7], respectively). We also determined the effects of AMP-CP and 8-SPT alone on IS (AMP-CP group [n=6] and 8-SPT group [n=8]); AMP-CP or 8-SPT was administered for 45 minutes before coronary occlusion and for the first 60 minutes of reperfusion. In other dogs, we measured the ecto-5'-N activity of the endocardium and epicardium in the LAD area (Ca²⁺+8-SPT group [n=5]; 8-SPT group [n=5]).

Protocol 6: Effects of 8-SPT Only During Ca²⁺ Exposure on IS Limitation by Ca²⁺ Preconditioning
To examine whether increased adenosine production in response to Ca²⁺ exposure triggers the IS-limiting effect of Ca²⁺ preconditioning, we began 8-SPT infusion 5 minutes before the administration of Ca²⁺ and continued it until the onset of coronary occlusion (Ca²⁺+8-SPT [pretreatment] group, n=6). In five other dogs, we measured the activity of PKC in cytosolic and membrane fractions of the endocardium as well as the ecto-5'-N activity of the endocardium and epicardium in the LAD area.

Protocol 7: Effects of Transient Exposures to CaCl₂ on IS and PKC and Ecto-5'-N Activities in Chemically Denervated Hearts
To clarify whether norepinephrine release contributes to the IS-limiting effect of Ca²⁺ preconditioning, we subjected dogs that had undergone chemical denervation of the heart to the control (protocol 1) and Ca²⁺-preconditioning (protocol 3) procedures (denervation group [n=7] and Ca²⁺+denervation group [n=7], respectively). Systemic chemical sympathectomy was performed by an intravenous injection of 6-hydroxydopamine (50 mg/kg) 5 days before the experiment. Deleterious side effects of 6-hydroxydopamine were prevented by previous injections of propranolol and phentolamine (1 mg/kg each); three fractional doses of 6-hydroxydopamine (10, 20, and 20 mg/kg) were administered over a 24-hour period. Myocardial tissue from the perfused area of dogs killed immediately after the experiment was sampled for the measurement of norepinephrine. Norepinephrine contents of the myocardium of systemically denervated and innervated dogs were 11±3 and 366±28 pg/mg tissue (mean±SEM, n=5, P<.01), respectively. We measured PKC and ecto-5'-N activities as described for protocol 1 in five animals each, corresponding to the denervation and Ca²⁺+denervation groups.

Protocol 8: Effects of IP and Transient Exposures to CaCl₂ on the Microtubular Structure
Although we needed to confirm that infusion of CaCl₂ would increase [Ca²⁺]_i or that infusion of EGTA would attenuate the increase of [Ca²⁺]_i by IP, we could not measure [Ca²⁺]_i of canine in vivo heart directly. We have reported⁶ that the microtubular structure is sensitive to an increase in [Ca²⁺]_i. Because intracoronary administration of CaCl₂ disrupts microtubules and EDTA prevents the disruption of microtubular structures, we evaluated the change in microtubular structure as a semiquantitative index of [Ca²⁺]_i. In addition to the dogs used for the measurement of IS or the activity of PKC and ecto-5'-N, other animals were used to evaluate the microtubular structure in the endocardium of the LAD area. Animals were killed immediately after IP (n=4), IP+EGTA (1.7 µmol·kg^-1·min^-1 IC, n=3), or CaCl₂ exposure (8µmol · min^-1·mL^-1 of CBF; infusion rate, 16.7 µL kg^-1·min^-1, n=4) or, for the control group (n=4), 40 minutes after hemodynamic stabilization was achieved. Samples were processed, and indirect immunofluorescent staining of microtubules was observed.^{6 7} CBF is expressed in milliliters per minute.

Criteria for Exclusion
To ensure that all the animals included for the analysis of IS data were healthy and exposed to a similar extent of ischemia, we excluded dogs that fulfilled any of the following three criteria: subendocardial collateral flow >15 mL·100 g^-1·min^-1, heart rate >170 bpm, or more than two consecutive attempts required to correct ventricular fibrillation with low-energy DC pulses applied directly to the heart.

Assessment of IS
After 6 hours of reperfusion, the LAD was reoccluded and perfused with autologous blood, and Evans blue dye was injected into a systemic vein to determine the anatomic risk area and the nonischemic area in the heart. The heart was then removed immediately and sliced into serial transverse sections. The nonischemic area was identified by blue stain, and the ischemic region was incubated at 37°C for 20 to 30 minutes in sodium phosphate buffer (pH 7.4) containing 1% tetraphenyl tetrazolium chloride (Sigma). IS was expressed as a percentage of the area at risk.

Measurement of regional myocardial blood flow was determined by using the microsphere technique.^{3 8}

Chemical Analysis
MO₂ was calculated by multiplying CBF by the coronary arteriovenous blood oxygen difference. Lactate was measured by using an enzymatic assay, and the LER was obtained by dividing the coronary arteriovenous difference in lactate concentration by arterial lactate concentration and multiplying by 100%. The calcium concentration in coronary arterial and venous blood was measured by using automated spectrophotometry (Automatic Analyzer 705, Hitachi) with o-cresolphthalein complexone in the presence of 8-hydroxyquinoline.⁹

The methods used for measuring adenosine and norepinephrine concentrations have been described.^{3 10}

Measurements of PKC and Ecto-5'-N Activities
A biopsy specimen (1 to 2 g) of the LAD-perfused myocardium was obtained before sustained coronary occlusion in the various treatment groups. Tissue samples were processed, and the activity of ecto-5'-N was measured by using an enzymatic assay.³ The activity of PKC was measured by using an enzyme assay kit (Amersham) that provides a simple and reliable method of estimating PKC activity without extensive purification of the enzyme.¹¹ Ecto-5'-N and PKC activities are expressed as nanomoles per milligram of protein per minute. The dependence of PKC activity on Ca²⁺ and phospholipids was examined by adding 0.5 mmol/L excess EGTA or eliminating phosphatidylserine from the assay system.¹¹

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance was assessed by using an ANOVA and Bonferroni's test. The effect of endomyocardial collateral blood flow on IS was analyzed by using an ANCOVA, with regional collateral flow in the inner half of the left ventricle wall as the covariant. A probability value of <.05 was considered statistically significant.

	Results

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Mortality and Exclusions
We excluded eight dogs from data analysis because the subendocardial collateral flow was >15 mL·100 g^-1·min^-1. No dogs were excluded because of heart rate >170 bpm. At least one episode of ventricular fibrillation occurred in 36 dogs: ventricular fibrillation that matched the exclusion criterion occurred in 12 of these animals during the 90 minutes of ischemia and in 17 during reperfusion (Table 1).

View this table: [in this window] [in a new window]   Table 1. Number of Dogs Assigned to and Excluded From Each Group for Measurement of Infarct Size

Hemodynamic and Metabolic Parameters
No significant differences in systolic and diastolic blood pressures or heart rate were detected immediately before, during, or after 90 minutes of myocardial ischemia among the various groups of innervated dogs. Heart rate in the denervated dogs was lower than that in the innervated dogs. Although EGTA alone had no effect on baseline systemic and coronary hemodynamic and metabolic parameters except for FS (from 24.3±0.7% to 16.6±0.5%, P<.05 versus the control group), an intracoronary administration of EGTA during IP reduced the coronary arterial and venous difference in adenosine concentration and the extent of coronary hyperemic flow measured after the fourth 5-minute exposure to myocardial ischemia (Table 2). CBF decreased immediately after administration of the fourth dose of MTX (Table 2) but returned to the control level after a further 5 minutes (before ischemia); systolic and diastolic blood pressures and CPP increased and CBF decreased during MTX administration due to ₁-adrenoceptor–mediated vasoconstriction, which did not cause myocardial ischemia as revealed by LER (Table 2). Adenosine concentration in the coronary venous blood increased during an intracoronary MTX infusion; this increase was reduced by EGTA, but the adenosine concentration returned to the control level 10 minutes after the fourth exposure to MTX (Table 2). CBF, FS, MO_2, and the coronary arterial and venous difference in adenosine concentration increased during administration of CaCl₂ but returned to control values 10 minutes after the third exposure to Ca²⁺ (Table 3). CPP, CBF, MO₂, pH in coronary arterial and venous blood, adenosine and norepinephrine concentrations in coronary arterial and venous blood, and LER did not differ significantly among the various groups of innervated dogs immediately before the onset of 90 minutes of ischemia. MO₂ was lower and norepinephrine concentrations in coronary arterial and venous blood were higher in the denervated dogs than in the innervated dogs. Intracoronary infusions of EGTA reduced the Ca²⁺ concentration in the perfused blood from 14.8±1.2 to 2.5±0.8 mg/dL; intracoronary infusion of CaCl₂ increased the Ca²⁺ concentration in the perfused blood from 8.1±0.2 to 19.2±1.2 mg/dL (P<.01).

View this table: [in this window] [in a new window]   Table 2. Coronary Hemodynamic and Myocardial Metabolic Parameters Immediately After the Fourth Cycle of IP and 1-Adrenoceptor–Stimulated Preconditioning

View this table: [in this window] [in a new window]   Table 3. Coronary Hemodynamic and Myocardial Metabolic Parameters Immediately After the Third Cycle of Ca2+ Preconditioning

Changes in Microtubular Structures
Microtubular structures in intact nonischemic hearts appeared throughout the cytoplasm as tortuous, loosely organized filaments composed mainly of longitudinal and transverse filaments. Microtubules encircling the nuclei showed dense staining (Fig 1A). In the IP (Fig 1B) and Ca²⁺ groups (Fig 1C), the microtubular structure was disrupted and observed as dotted spots; staining around the nuclei had disappeared. Intracoronary administration of EGTA during the IP procedure prevented the disruption of microtubules (Fig 1D).

View larger version (126K): [in this window] [in a new window]   Figure 1. Photomicrographs show microtubular staining in endocardium. Microtubules form a filamentous structure throughout the cytoplasm and encircle the nucleus in the control (normal) heart (A). Loss of microtubular staining in the cytoplasm and around the nucleus as well as microtubular fragmentation (dotted spots and short microtubular filaments) are observed immediately after the IP (B) and the CaCl2 infusion (C). Microtubular structures are preserved immediately after the IP procedure with EGTA (D).

PKC and Ecto-5'-N Activities
PKC activity in the membrane fraction of the myocardium was increased in the IP and Ca²⁺ groups relative to the control group (Fig 2). In contrast, PKC activity in the cytosolic fraction did not differ among these three groups (Fig 2). Total PKC activity was not significantly changed by IP or Ca²⁺ exposure. Removal of Ca²⁺ or phospholipid from the PKC assay reduced PKC activity in the cytosolic fraction but not in the membrane fraction of the control group. The increase in PKC activity in the membrane fraction induced by CaCl₂ or IP was also apparent only when the assay was performed in the presence of both Ca²⁺ and phospholipid. Administration of EGTA during IP or ₁-adrenoceptor–mediated preconditioning reduced the increase in PKC activity in the membrane fraction (MTX and MTX+EGTA groups, 26±2 and 9±1 nmol·mg protein^-1·min^-1, respectively; P<.01). Administration of 8-SPT only during Ca²⁺ preconditioning did not reduce PKC activity in the membrane fraction (26±3 nmol·mg protein^-1·min^-1). Preconditioning with Ca²⁺ increased PKC activity in the membrane fraction of chemically denervated hearts (24±2 nmol·mg protein^-1·min^-1; P<.01 versus the control group).

View larger version (33K): [in this window] [in a new window]   Figure 2. Bar graphs show PKC activity in the membrane (A) and cytosolic (B) fractions of the endocardium. PKC activity was measured in the absence (-) or presence (+) of Ca2+ or phospholipid (PL). Data are mean±SEM. *P <.05.

Activation of ecto-5'-N by IP and ₁-adrenoceptor–mediated preconditioning was inhibited by concomitant administration of EGTA (Fig 3). Exposure of both innervated and denervated hearts to CaCl₂ increased ecto-5'-N activity in the epicardium (Fig 3A) and endocardium (Fig 3B) to the values similar to those obtained with IP in innervated hearts. The CaCl₂-induced increase in ecto-5'-N activity was reduced by treatment with GF109203X. The CaCl₂-induced increase in ecto-5'-N activity was not decreased by treatment with 8-SPT alone during the preconditioning procedure (Fig 3).

View larger version (53K): [in this window] [in a new window]   Figure 3. Bar graphs show ecto-5'-N activity in the epicardium (A) and endocardium (B). Data are mean±SEM. *P<.05, **P<.01 vs control.

IS-Limiting Effect of Ca²⁺ Preconditioning and Effect of EGTA on IS-Limiting Effect of IP
The area at risk and collateral flow were similar among all groups. Administration of EGTA reduced the IS-limiting effect of IP and ₁-adrenoceptor–mediated preconditioning (Fig 4). Transient Ca²⁺ exposures mimicked the IS-limiting effect of IP in both innervated and denervated hearts. The IS-limiting effect of Ca²⁺ preconditioning was prevented by intracoronary administration of AMP-CP or 8-SPT both before and after ischemia or of GF109203X during exposure to Ca²⁺; administration of 8-SPT alone during CaCl₂ exposures did not inhibit the IS-limiting effect of Ca²⁺ preconditioning. These results indicate that neither endogenous adenosine nor norepinephrine release in response to CaCl₂ administration is the trigger for Ca²⁺ preconditioning and that activation of PKC contributes to the IS-limiting effects of IP and ₁-adrenoceptor–mediated preconditioning. Similar results were obtained by plotting IS normalized by risk area against the collateral blood flow to the inner half of the LAD-dependent endocardium during the sustained ischemic period (Fig 5).

View larger version (20K): [in this window] [in a new window]   Figure 4. Plots of IS for the various experimental groups. Both individual data points () and mean±SEM ( with bars) are shown. *P<.01, **P<.005 vs control by Bonferroni's test.

View larger version (27K): [in this window] [in a new window]   Figure 5. A and B, Plots of IS expressed as a percentage of risk area versus regional collateral flow during ischemia. IS in hearts subjected to IP or 1-adrenoceptor–stimulated preconditioning was significantly smaller than in the control group at any given value of collateral blood flow (P<.01 by ANCOVA). IS in both innervated and denervated hearts subjected to Ca2+ preconditioning as well as in innervated hearts also exposed to 8-SPT pretreatment was significantly smaller than in the control group at any given value of collateral blood flow (P<.01 by ANCOVA).

Effects of Doses of CaCl₂ on IS and Activities of PKC and Ecto-5'-N
Administration of CaCl₂ at a dose of 4 µmol ·min^-1·mL^-1 of CBF did not mimic the IS-limiting effect of IP (IS, 39.4±4.1%) and did not activate PKC in the membrane fraction of the endocardium (11.6±2.1 nmol·mg protein^-1·min^-1) or ecto-5'-N in the endocardium (43.8±2.5 nmol·mg protein^-1·min^-1). CaCl₂ at a dose of 6 µmol·min^-1·mL^-1 of CBF did mimic the IS-limiting effect of IP (18.3±2.1%, P<.01 versus control group) and activated PKC in the membrane fraction of the endocardium (23.6±1.7 nmol·mg protein^-1 · min^-1, P<.01 versus control group) and ecto-5'-N in the endocardium (58.2±3.6 nmol·mg protein^-1·min^-1, P<.01 versus control group). CaCl₂ at a dose of 10 µmol·min^-1·mL^-1 of CBF consistently induced more than two episodes of ventricular fibrillation. One cycle of CaCl₂ exposure at 8 µmol·min^-1·mL^-1 of CBF also reduced IS (18.4±3.5%, P<.01 versus control group) and activated PKC in the membrane of the endocardium (25.8±1.3 nmol·mg protein^-1·min^-1, P<.05 versus control group) and ecto-5'-N in the endocardium (63.6±3.9 nmol·mg protein^-1·min^-1, P<.05 versus control group). Therefore, doses of 6 to 8 µmol·min^-1·mL^-1 of CBF of CaCl₂ appear adequate for cardioprotection.

	Discussion

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We have shown that exposures of the heart to CaCl₂ induce the translocation of PKC from the cytosolic to the membrane fraction of the endocardium and result in the limitation of IS. We have further demonstrated that activation of ecto-5'-N is important in the PKC-mediated cardioprotection induced by exposure to CaCl₂.

Mechanism of Activation of PKC Induced by Exposure to CaCl₂
An underlying assumption of our study is that exposure of the myocardium to CaCl₂ increases [Ca²⁺]_i. The results of several studies support this hypothesis.^{12 13} De Tombe et al¹⁴ have shown that increases in extracellular Ca²⁺ increase myocardial contractility as a result of increased [Ca²⁺]_i, and Marban et al¹² report that increases in extracellular Ca²⁺ increase the average [Ca²⁺]_i. We also observed that microtubular structures are sensitive to an increase in [Ca²⁺]_i and that intracoronary administration of CaCl₂ disrupts microtubular structures, as shown by immunohistochemical staining.⁶ It is thus likely that an increase in the extracellular Ca²⁺ concentration results in Ca²⁺ influx via Ca²⁺ channels and Na⁺/Ca²⁺ exchanges and a consequent increase in [Ca²⁺]_i in myocardial cells.¹³

The increases in PKC activity in response to exposure to CaCl₂ could be achieved by at least four means. Exposure to CaCl₂ increases MO₂,¹⁵ which may increase adenosine release in the heart.¹¹ Adenosine induces translocation of PKC from the cytosolic to the membrane fraction in a G_i protein–dependent manner,¹⁶ and adenosine mimics the IS-limiting effect of IP in the rabbit heart.¹⁷ Indeed, we have also shown that transient exposures to high doses of adenosine can activate ecto-5'-N and limit IS in the canine heart.¹⁸ However, in the present study, although MO₂ and adenosine release were slightly increased during exposure to CaCl₂, the concomitant administration of 8-SPT during Ca²⁺ preconditioning did not inhibit the IS-limiting effect or activation of PKC and ecto-5'-N. Given that we confirmed that the dose of 8-SPT used in the present study is sufficient to inhibit the cardiovascular effects of endogenous adenosine during myocardial ischemia, the present results suggest that the amount of adenosine released during exposure to CaCl₂ is not sufficient to induce cardioprotection via activation of PKC.

Second, transient exposures to Ca²⁺ may induce myocardial subendocardial ischemia due to an imbalance between myocardial oxygen supply and demand. We did not detect either lactate release or a decrease in the pH of coronary venous blood during Ca²⁺ exposure, suggesting that myocardial ischemia did not occur during Ca²⁺ preconditioning.

Third, exposure to Ca²⁺ may increase the release of norepinephrine,¹⁹ which would then stimulate ₁-adrenoceptors and activate PKC as a result of diacylglycerol formation.¹⁰ However, the concentration of norepinephrine did not change during and after the administration of CaCl₂ in innervated hearts. Furthermore, transient Ca²⁺ exposures limited IS even in denervated hearts. Thus, endogenous norepinephrine does not appear to mediate the effects of Ca²⁺ preconditioning.

The most likely possibility is that exposure to Ca²⁺ increases PKC activity directly by increasing [Ca²⁺]_i. Increases in [Ca²⁺]_i activate phospholipase C,²⁰ the enzyme that catalyzes polyphosphoinositide hydrolysis and thereby generates diacylglycerol.²¹

In this experiment, we administered drugs, including EGTA and CaCl₂, into a coronary bypass tube. Since we perfused the LAD with blood from the left carotid artery through a bypass tube and the left circumflex artery is dominant in the canine heart, systemic hemodynamic and metabolic parameters including systemic blood pressure, dP/dt, and heart rate did not change by infusion of either CaCl₂ or EGTA via the LAD. On the other hand, the FS of the LAD-perfused area increased during infusion of CaCl₂ but returned to baseline 5 minutes after the third transient infusion of CaCl₂ (Table 3).

Cytosolic PKC activity tended to decrease but did not reach statistical significance. Accordingly, total PKC activity tended to increase but showed no significant changes, while PKC in the membrane fraction was markedly activated. There may be two possible reasons for this. First, when a small part of cytosolic PKC is translocated to the plasma membrane, the assay system used in the present study may not detect decreases in cytosolic PKC. In this case, however, we could detect significant changes in PKC activity in the membrane fraction. Another possibility is the contamination of inhibitors in the cytosolic fraction of the total PKC activity. Although it is difficult to address which possibility is more likely, we can conclude that PKC activity of the membrane fraction is increased due to exposures to CaCl₂.

Role of Increases in [Ca²⁺]_i and ₁-Adrenoceptor Activation in IP-Induced Cardioprotection
Myocardial ischemia and reperfusion are characterized by an increase in [Ca²⁺]_i⁵ and the release of norepinephrine, both of which appear to mediate the IS-limiting effect of IP by activating PKC, because activation of PKC plays an important role in IP.^{22 23 24 25} We³ and others^{26 27} have shown that ₁-adrenoceptor stimulation plays a key role in mediating the IS-limiting effect of IP. Furthermore, we have shown that the PKC activation induced by ₁-adrenoceptor stimulation is important for cardioprotection because it activates ecto-5'-N,³ and we have suggested that the activated PKC is Ca²⁺ dependent in the canine heart.²⁸ Therefore, an increase in [Ca²⁺]_i may be required for activation of PKC induced by ₁-adrenoceptor stimulation and may decrease the activation threshold of the enzyme.²⁹ Both pathways, an increase in [Ca²⁺]_i and ₁-adrenoceptor activation, may mediate IP-induced cardioprotection in the canine heart independently or they may be interdependent. Indeed, exposures of cardiac tissue to ₁-adrenoceptor agonists stimulate phosphoinositide hydrolysis, resulting in an increase in [Ca²⁺]_i triggered by inositol 1,4,5-trisphosphate,³⁰ and ₁-adrenoceptor activation enhances intracellular alkalization through Na⁺/H⁺ exchange and increases Ca²⁺ influx as a result of the subsequent activation of Na⁺/Ca²⁺ exchange in adult rat cardiomyocytes.³¹ In turn, PKC activity typically depends on [Ca²⁺]_i.^{20 21} Thus, both increases in [Ca²⁺]_i and ₁-adrenoceptor activation are linked to each other and to activation of PKC and ecto-5'-N. The relative importance of these two pathways may depend on the severity, duration, and number of episodes of transient ischemia during IP.

Pathophysiological Role of Transient Ca²⁺ Overload in Ischemia and Reperfusion Injury
Ca²⁺ overload during ischemia and reperfusion results in reversible or irreversible cellular injury,³² and the administration of EGTA during sustained ischemia and reperfusion protects against such injury.³³ Indeed, an intracoronary infusion of EDTA during the initial 10 minutes of reperfusion attenuates the severity of myocardial stunning in canine hearts.⁶

On the other hand, transient exposures to CaCl₂ before sustained ischemia limited IS, and the administration of EGTA during IP reduced the IS-limiting effect of this procedure. These observations suggest that Ca²⁺ overload during sustained ischemia and reperfusion is deleterious but that transient Ca²⁺ overload before sustained ischemia may induce cardioprotection.

Transient Ca²⁺ overload induced by brief periods of exposure to 10 mmol/L (Ca²⁺)_o causes contractile dysfunction and ATP depletion in perfused ferret hearts.³⁴ In the present study intracoronary infusions of CaCl₂ increased the Ca²⁺ concentration in the perfused blood from 0.07 to 0.17 mmol/L, but transient CaCl₂ infusion at a low dose did not cause contractile dysfunction.

	Selected Abbreviations and Acronyms

 

AMP-CP = ,ß-methyleneadenosine 5'-diphosphate [Ca2+]i = intracellular Ca2+ concentration CBF = coronary blood flow CPP = coronary perfusion pressure ecto-5'-N = ecto-5'-nucleotidase FS = fractional shortening IP = ischemic preconditioning IS = infarct size LAD = left anterior descending coronary artery LER = lactate extraction ratio MTX = methoxamine MO2 = myocardial oxygen consumption PKC = protein kinase C 8-SPT = 8-sulfophenyltheophylline

	Acknowledgments

 
We thank Noriko Tamai, Kayoko Yoshida, Yukiyo Nomura, Kiyomi Okuto, Shinya Suzuki, and Makoto Hasegawa for technical assistance.

	Footnotes

 
Presented in part at the 67th Annual Scientific Sessions of the American Heart Association, Dallas, Tex, November 11-14, 1994, and the 17th Congress of the European Society of Cardiology, Amsterdam, Netherlands, August 20-24, 1995. Published in abstract form (Circulation. 1994;90[suppl I]:I-209).

Received October 31, 1996; revision received February 24, 1997; accepted February 28, 1997.

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