Role of Intracellular Ca2+ in Activation of Protein Kinase C During Ischemic Preconditioning
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 α1-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 α1-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 (α,β-methyleneadenosine 5′-diphosphate) reduced the infarct size–limiting effect of Ca2+ preconditioning. Administration of EGTA during ischemic or α1-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 α1-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.
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 α1-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 α1-adrenoceptor–mediated preconditioning inhibits the IS-limiting effect of these procedures.
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]×100%, 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.
Protocol 1: Effects of IP and α1-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 α1-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 α1-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 α1-Adrenoceptor–Mediated Preconditioning
Because activation of PKC requires Ca2+, [Ca2+]i in IP and α1-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.
MV̇o2 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
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
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.
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⇓).
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 α1-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, MV̇o2, 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, MV̇o2, 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. MV̇o2 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).
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⇓).
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 α1-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).
Activation of ecto-5′-N by IP and α1-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⇓).
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 α1-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 α1-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⇓).
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.
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 MV̇o2,15 which may increase adenosine release in the heart.11 Adenosine induces translocation of PKC from the cytosolic to the membrane fraction in a Gi 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 MV̇o2 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 α1-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 α1-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 others26 27 have shown that α1-adrenoceptor stimulation plays a key role in mediating the IS-limiting effect of IP. Furthermore, we have shown that the PKC activation induced by α1-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 α1-adrenoceptor stimulation and may decrease the activation threshold of the enzyme.29 Both pathways, an increase in [Ca2+]i and α1-adrenoceptor activation, may mediate IP-induced cardioprotection in the canine heart independently or they may be interdependent. Indeed, exposures of cardiac tissue to α1-adrenoceptor agonists stimulate phosphoinositide hydrolysis, resulting in an increase in [Ca2+]i triggered by inositol 1,4,5-trisphosphate,30 and α1-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 α1-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
|[Ca2+]i||=||intracellular Ca2+ concentration|
|CBF||=||coronary blood flow|
|CPP||=||coronary perfusion pressure|
|LAD||=||left anterior descending coronary artery|
|LER||=||lactate extraction ratio|
|MV̇o2||=||myocardial oxygen consumption|
|PKC||=||protein kinase C|
We thank Noriko Tamai, Kayoko Yoshida, Yukiyo Nomura, Kiyomi Okuto, Shinya Suzuki, and Makoto Hasegawa for technical assistance.
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.
- Copyright © 1997 by American Heart Association
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