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(Circulation. 1998;97:786-794.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Tachycardia Preconditions Infarct Size in Dogs

Role of Adenosine and Protein Kinase C

Raúl J. Domenech, MD; Pilar Macho, MD; Diego Vélez, MD; Gina Sánchez, BQ; Xueliang Liu, MD; ; Naranjan Dhalla, MD

From Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile (R.J.D., P.M., D.V., G.S.), and the Institute of Cardiovascular Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, Canada (X.L., N.D.).

Correspondence to Dr Raúl J. Domenech, Facultad de Medicina, Campus Oriente, Universidad de Chile, Casilla 16038, Santiago 9, Chile.

	Abstract

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Background—Myocardial ischemic preconditioning is a well-known phenomenon, however there is scant information in regard to nonischemic preconditioning.

Methods and Results—We studied in anesthetized dogs the preconditioning effect of tachycardia and the mediation of adenosine and protein kinase C in this process. In a control group the anterior descending coronary artery was occluded for 60 minutes and reperfused for 270 minutes. Heart rate was kept constant at 120±5 cycles/min and aortic pressure changes were damped. The infarct size (necrotic volume/risk region volumex100) was 15.8±1.5%. In another group of dogs a similar protocol was followed, but five periods of tachycardia (213±12 cycles/min), 5 minutes in duration each, with 5 minutes of intervening periods at control heart rate, were induced previous to the coronary occlusion. The infarct size was reduced by 46% (P<.001) with respect to the nonpreconditioned group. This effect was not due to changes in collateral flow nor risk region size. During tachycardia, myocardial interstitial adenosine increased about twofold (P<.05); no metabolic, hemodynamic, or ECG evidences of ischemia were observed and the transmural vasodilatory reserve was preserved. The blockade of adenosine receptors with 8 phenyltheophylline, before or after the preconditioning tachycardia, reverted its protecting effect but it did not modify infarct size in nonpreconditioned dogs. No changes in cytosolic or particulate protein kinase C activity or translocation of -, ß-, -, and - protein kinase C isozyme by effect of tachycardia or ischemia were observed between preconditioned and nonpreconditioned dogs.

Conclusions—Tachycardia, in the absence of ischemia, mimics the preconditioning effect of ischemia in the dog. This effect is mediated by adenosine but not by changes in protein kinase C activity or its translocation.

Key Words: adenosine • myocardial infarction • preconditioning • protein kinase C

	Introduction

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Myocardial ischemic preconditioning described by Murry et al¹ in 1986 is a well-known phenomenon that occurs in several species. The mechanism of preconditioning is not known yet, however the current hypothesis is that one or more substances released by the ischemic preconditioning episode modulate the myocardial response to further ischemia.² Accordingly, the triggering effect of the brief episode of ischemia must be related to a metabolic alteration secondary to an imbalance between the oxygen supply and demand and, specifically for ischemic preconditioning, to a decrease in blood supply. However, it is reasonable to think that preconditioning could also be obtained by increasing the demand instead of decreasing the blood supply without the production of ischemia. Thus Koning et al³ described a preconditioning effect of tachycardia without ischemia in the pig heart mediated by ATP-dependent potassium channels. Because ischemic preconditioning in several species, including the dog, appears to be mediated through adenosine,^{2 4} and because myocardial interstitial adenosine appears also to increase in the dog heart during enhanced myocardial oxygen consumption due to several maneuvers including pacing, and exercise,^{5 6 7 8} we studied in the dog the presumptive preconditioning effect of tachycardia without myocardial ischemia and the participation of adenosine and protein kinase C (PKC) in this effect.

	Methods

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The present study was conducted in accordance with the "Position of the American Heart Association on Research and Animal Use" and the guidelines of the Animal Care Committee of the Faculty of Medicine, University of Chile.

Surgical Preparation
One hundred fifty-seven adult mongrel dogs of either sex weighing 15 to 18 kg were anesthetized with sodium pentobarbital (30 mg/kg IV). The trachea was intubated and the lungs ventilated with a volume and frequency regulated pump (Braun respirator) using room air added of a mixture of O₂ 95% and CO₂ 5% as necessary to maintain normal values of PO₂, Pco₂, and pH in the arterial blood as assessed by measurements in the aortic root blood every twenty minutes (Radiometer BMS 3Mk2 Blood Microsystem). Sodium bicarbonate was administered occasionally according to pH and base excess values. Pressure in the root of the aorta was measured with an extracorporeal pressure transducer (Statham P23Db) connected to a polyethylene catheter implanted through the right brachycephalic trunk. Pressure in the left ventricular cavity was measured with a catheter-tip manometer (Millard Instruments) implanted through the apex of the heart, and the first derivative of left ventricular pressure in time was obtained. Flow was measured in the anterior descending coronary artery, immediately below the first diagonal branch, with an electromagnetic flow probe connected to a Nihon Kohden flowmeter. Flow was expressed as per 100 g of tissue of the risk region that was demarked and weighed as detailed below. A plastic snare was implanted around the artery to occlude it, below the flow probe. We selected this site for the occlusion because pilot experiments demonstrated that the smaller infarct size so obtained prevented excess episodes of ventricular fibrillation and hemodynamic instability. A thin polyethylene catheter was implanted into the great coronary vein to obtain blood samples for the analysis of oxygen and lactate content. To damp changes in aortic pressure during the experiments and between animals, the systemic arterial circulation was connected through the left brachycephalic trunk to a large reservoir filled with homologous, heparinized, thermoregulated, constantly stirred blood. Mean aortic pressure in the root of the aorta was maintained between 80 and 90 mm Hg by adjusting the level of the reservoir. This procedure, in pilot experiments, prevented ventricular dilatation and severe left ventricular failure after inducing the infarct. The heart rate was controlled by producing a complete atrioventricular blockade⁹ followed by electrical stimulation of the outflow tract of the right ventricular wall at 120 cycles/min. The oxygen consumption of the heart was calculated by the product of coronary blood flow, as measured with the flowmeter, times the difference in oxygen content between the root of the aorta and the great coronary vein and was expressed per 100 g of tissue of the left ventricular wall. Piezoelectric crystals were implanted in the left ventricular wall irrigated by the anterior descending coronary artery to measure segment length as described below. Limb leads of the ECG, pressures, left ventricular dP/dt, segment length, and coronary flow were continuously recorded on a Nihon Kohden physiograph. After the surgical procedure, 500 U/kg of heparin was administered intravenously.

Experimental Design
Dogs were assigned to 10 groups as described below.

Group 1.
Nonpreconditioned dogs (n=35). Seventy minutes (20 minutes for stabilization followed by 50 minutes equivalent to the lapse of time required for preconditioning in other groups) after finishing the surgical preparation, the anterior descending coronary artery was occluded with the snare for 60 minutes followed by 270 minutes of reperfusion. The heart rate was kept constant during the experiment at 120±5 cycles/min (Fig 1). Coronary arteriovenous differences of oxygen content and lactate were measured from samples drawn from the root of the aorta and the great coronary vein every 5 minutes during the baseline period up to the time of the coronary occlusion.

View larger version (23K): [in this window] [in a new window]   Figure 1. Experimental design to test the preconditioning effect of tachycardia and the mediation of adenosine. T indicates tachycardia period of 5 minutes; 8PT, 8 phenyltheophylline; and REP, reperfusion.

Group 2.
Preconditioned dogs (n=30). The same protocol as in group 1 was performed, but before the occlusion of the anterior descending coronary artery, five periods of tachycardia at a rate of 213±12 cycles/min, 5 minutes in duration each, were induced with electrical stimulation starting 20 minutes after finishing the preparation. Each tachycardic period was followed by 5 minutes of baseline heart rate at 120±7 cycles/min (Fig 1). Coronary arteriovenous differences of oxygen content and lactate were measured from samples drawn from the root of the aorta and the great coronary vein during baseline heart rate and during each period of tachycardia and intervening control heart rate periods.

Group 3.
Preconditioned dogs plus adenosine receptor blockade before preconditioning (n=10). A protocol similar to group 2 was followed, but adenosine receptors were blocked with the intravenous administration of 8 phenyltheophylline (8PT) (Sigma Chemical), 7.5 mg/kg, immediately before preconditioning (Fig 1).

Group 4.
Preconditioned dogs plus adenosine receptor blockade after preconditioning (n=10). A protocol similar to group 3 was followed but 8PT was administered after preconditioning and before ischemia (Fig 1).

Group 5.
Dogs not preconditioned plus adenosine receptor blockade at a time equivalent to the blockade time in group 3, called early blockade (n=10). This protocol was performed as a control for group 3 to discard the blocking effect of the agent (Fig 1).

Group 6.
Dogs not preconditioned plus adenosine receptor blockade at a time equivalent to the blockade time in group 4, called late blockade (n=9). This group was studied as a control for group 4 to discard the blocking effect of the agent (Fig 1).

Group 7.
Measurements of myocardial interstitial adenosine with microdialysis in preconditioned dogs (n=21). Microdialysis probes were implanted into the myocardium as described below and the dialysate concentration of adenosine was used as an index of myocardial interstitial adenosine.¹⁰ The main purpose of these experiments was to test for changes in interstitial adenosine concentration by effect of the increase in myocardial oxygen consumption during tachycardia. Because the measurements of adenosine with microdialysis required 2 hours for stabilization of adenosine concentration in the dialysate,¹⁰ the period of time before ligating the coronary artery was prolonged in these dogs as compared with other groups; therefore they are considered as a separate group.

Group 8.
PKC activity and its isozymes in preconditioned dogs (n=10). In dogs preconditioned as in group 2, myocardial samples from the normal and risk regions were obtained by punch biopsies at the following different times during the procedure: baseline period after 20 minutes of stabilization, immediately after the last tachycardic period, 50 minutes into the ischemic period, and 50 minutes into the reperfusion period. Samples were immediately frozen in liquid nitrogen and processed as detailed below.

Group 9.
PKC activity and its isozymes in nonpreconditioned dogs (n=8). In dogs not preconditioned (as group 1), myocardial samples were obtained in a similar way as in group 8 as a control for this group.

Group 10.
Coronary vascular reserve during tachycardia (n=10). The coronary vascular reserve during tachycardia was assessed as the transmural capacity for flow to increase by effect of adenosine during tachycardia. Coronary flow was measured with the microspheres technique in four layers across the left ventricular wall as previously reported¹¹ and briefly described below. Flow was measured first at the control heart rate (120 cycles/min), then tachycardia at 213 cycles/min was induced with electrical stimulation, as detailed above for preconditioned dogs, and flow was measured again with the microspheres during the last tachycardic period. Finally, adenosine was infused into the anterior descending coronary artery (1 mg/min) through a thin catheter and after stabilization, the five periods of tachycardia were induced again and another measurement of transmural flow was performed with the microspheres during the last period.

Measurement of Collateral Myocardial Blood Flow to the Ischemic Region
Collateral blood flow to the ischemic left ventricular free wall was measured with the radioactive microspheres technique¹¹ 30 minutes into the 60-minute occlusion period. Carbonized plastic microspheres (15±5 µm diameter, New England Nuclear) labeled with ⁴⁶ Sc, ⁸⁵Sr, or ⁵⁷Co were suspended in isotonic saline added of 0.01% Tween 80, ultrasonicated, stirred, and flushed manually into the left atrium while a reference flow was withdrawn from the brachial artery as previously described.¹¹ The amount of microspheres injected was enough to obtain at least 500 microspheres in the region of the ischemic myocardium in which collateral flow was measured in order to avoid measurement errors >10% at the 95% confidence level.¹² After the experiments, transmural pieces of myocardium were obtained from the center of the ischemic region. Each sample was weighed, and its radioactivity (Cm) and the radioactivity of the blood collected from the reference samples (Cr) were measured in a gamma spectrometer equipped with a multichannel analyzer (Packard Auto Gamma 5500). Regional collateral flow (Qm) was calculated as Qm=Qr · Cm · Cr^-1, where Qr is the flow rate of the reference sample (10 mL/min). Flow values are expressed per gram of tissue.

Measurement of Myocardial Interstitial Adenosine
In dogs of group 7, microdialysis probes (CMA-20) with a 10-mm window for diffusion were implanted into the risk region of the heart (region irrigated by the anterior descending coronary artery) and perfused with Krebs-Henseleit buffer at 2 µL/min.¹⁰ A period of 2 hours was allowed for stabilization of adenosine levels in the interstitium after which the following samples were collected: during 20 minutes as baseline value; during preconditioning (integrated sample during the five periods of tachycardia); during three periods, 20 minutes each, during ischemia; and during three periods, 20 minutes each, during reperfusion. The samples were analyzed for adenosine by the high-performance liquid chromatography technique immediately after being obtained as described by Van Wylen et al.¹⁰

Measurement of Protein Kinase C Activity and Its Isozymes Content
The cytosolic and particulate fractions of the tissue homogenate were separated according to the modified method described previously^{13 14 15} ; all procedures were carried out at 4°C. The tissue sample from nonpreconditioned and preconditioned dogs (groups 8 and 9) was minced in 1 mL buffer A (50 mmol/L Tris/HCl, 0.25 mol/L sucrose, 10 mmol/L EGTA, 4 mmol/L EDTA, 20 µg/mL leupeptin, 200 U/mL aprotinin, pH 7.5) and homogenized in a Polytron (Brinkmann PT3000) at setting 8 for 2x30 seconds, then sonicated for 2x15 seconds. The homogenate was centrifuged at 105 000g for 60 minutes in a Beckman ultracentrifuge (Beckman J2-HS). The resulting supernatant (the cytosolic fraction) was kept on ice until use for assay. The pellet was resuspended in 1 mL of buffer A with 1% Triton X-100. After incubation on ice for 60 minutes, the resuspended pellet was centrifuged at 105 000g for 60 minutes. The supernatant was used as the particulate fraction. PKC activities in cytosolic and particulate fractions were determined immediately thereafter.

PKC activity was determined with a PKC assay kit (Upstate Biotechnology Inc). In the present experiments, okadaic acid was used in PKC activity assay of nonpurified cytosolic and particulate fractions from the ventricular tissue.¹⁶ It should be noted that okadaic acid is a highly specific inhibitor of type 1 and type 2A phosphatases and reveals the nature of endogenous PKC-inhibitory activity in heart causing a general stimulation of protein phosphorylation without affecting any of the relevant protein kinases.^{17 18} Each assay tube included substrate (10 µL), inhibitor (10 µL), lipid activator (10 µL), enzyme preparation (10 µL), and okadaic acid (2 µmol/L). The reaction was initiated by the addition of [g-³²P]ATP (10 µL) and allowed to proceed at 30°C for 10 minutes. The reaction mixture (25 µL) was removed onto the P81 phosphocellulose paper for 30 seconds. After the phosphocellulose papers were washed three times (5 minutes each time) with 0.75% phosphoric acid and one time with acetone, respectively, these were put into scintillation vials and the radioactivity was counted in a scintillation counter for measuring the incorporation of ³²P from [g-³²P] into synthesized substrate, which is more specific for PKC than the histone H-1 protein.^{13 19}

The relative contents of PKC-, -ß, -, and - isozymes were measured by 8% mini–SDS-PAGE and Western blot analysis²⁰ of the cytosolic and particulate fractions. The concentration of protein in the fractions was adjusted to 1 mg/mL with the homogenizing buffer, and the SDS-PAGE loading was added into the homogenizing buffer (one part loading buffer to two parts homogenate). The protein loads for nonpreconditioning and preconditioning groups were the same (10 µL in each well). The electrophoresis was carried out at 200 V for 40 to 45 minutes. The proteins in both fractions separated by SDS-PAGE were electroblotted to Immobilon-P transfer membrane (Millipore Co) in a transfer buffer, which contained 25 mmol/L Tris-HCl, 120 mmol/L glycine, and 20% methanol (vol/vol) for the determination of relative protein contents. The transferred membranes were shaken for 2 hours in blocking buffer, which contained TBS (10 mmol/L Tris-HCl, 150 mmol/L NaCl) and 5% fat-free powdered milk, then incubated for 1 hour at room temperature with polyclonal anti–PKC-, -ß, - and - isozyme antibodies (1:100 Life Technologies), respectively. The transferred membranes were subsequently incubated with biotinylated anti-rabbit IgG (1:500; Amersham) for 40 minutes and then finally with streptavidin conjugated horseradish peroxidase (1:500; Amersham) for 30 minutes. The blots were rinsed in the TBS-T (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.2% Tween 20) three times (5 minutes each time) between each of the preceding steps. For chemiluminescent detection, the membrane sheets were dipped into luminal substrate solution (Amersham) and the chemilumigrams were developed on Hyperfilm-ECL (Amersham) to visualize PKC isozymes. The normal exposure time ranged from 2 to 5 minutes. The relative content of PKC isozyme was determined by the model GS-670 Imaging Densitometer (Bio-Rad Co) with the Image Analysis Software Version 1.0.

Measurement of Regional Myocardial Function
In all dogs, pairs of ultrasonic crystals were positioned into the midmyocardial layer of the risk region for the continuous measurement of regional systolic and diastolic segment dimensions by sonomicrometry (Triton Technology Inc). Shortening fraction was calculated as end-diastolic length-end-systolic length/end-diastolic length.¹¹

Measurement of Infarct and Risk Region Sizes
The size of the infarction relative to the risk zone was measured with the triphenyltetrazolium staining technique. After the experiments were finished, the hearts were excised. The right coronary artery and the circumflex coronary artery were perfused from the aorta with a solution of Evans blue dye in saline. The anterior descending coronary artery was perfused, from the place where it had been ligated, with a solution of 1% triphenyltetrazolium chloride. The perfusions were done simultaneously at a pressure of 90 mm Hg. After perfusion, the left ventricle was cut into seven to nine slices of 8-mm thickness, each parallel to the AV groove, and weighed. The slices were incubated in a 1% solution of triphenyltetrazolium for 10 minutes and then kept in 10% formaldehyde solution for 24 hours. In each slice, the volume of the nonrisk region (stained blue), the risk region (stained red), and the necrotic region (not stained) were obtained by measuring with planimetry the corresponding areas on the cross surface of each slice and multiplying them by the thickness of the slice. These volumes were added across the slices to obtain the corresponding total volumes of the three regions. The magnitude of the infarction was expressed by the volume of the necrotic region as percent of the volume of the risk region. The risk region was expressed as percent of the total left ventricular volume. The weight of the risk region was calculated by multiplying the weight of the left ventricular wall times the percent volume of the risk region from the volume of the left ventricular wall. This weight was used to express flow measured with the flowmeter in the anterior descending artery as per 100 g of tissue.

Criteria for Exclusion
To avoid differences in the infarct size consequent to different exposure to ischemia, we used the following criteria for exclusion: (1) collateral flow to the ischemic region >0.20 mL/min/g; (2) >3 consecutive attempts required to convert ventricular fibrillation.

Statistical Analysis
The difference in size of the necrotic and the risk regions between the groups was analyzed by ANOVA followed by Student-Newman-Keuls tests. The changes in oxygen consumption, coronary arteriovenous oxygen difference, and lactate extraction between the periods of baseline heart rate and those of tachycardia were analyzed by Student's paired t test. Hemodynamics variables, regional flows during control, tachycardia and tachycardia plus adenosine, the content of adenosine in the dialysate, and PKC activity and its isozyme content in the biopsies were compared by ANOVA for repeated measurements. The influence of collateral blood flow on the magnitude of the infarction was assessed by ANCOVA. The null hypothesis was discarded at the level of P<.05.

	Results

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Of the 157 dogs, 4 dogs died during the surgical intervention before the experimental protocol was performed. Of the 153 surviving dogs, 37 developed ventricular fibrillation at least once during ischemia or reperfusion and were defibrillated with electrical discharge applied through the thorax. Fifteen of them matched the criteria for exclusion because more than three consecutive attempts to convert ventricular fibrillation were required: 4 dogs in group 7, 3 dogs in each of groups 1 and 8; 2 dogs in group 2; and 1 dog in each of groups 3, 5, and 9. Eight dogs matched the criteria for exclusion due to collateral blood flow to the ischemic region >0.20 mL/g per minute: 2 dogs in each of groups 2 and 7; 1 dog in each of groups 1, 4, 8, and 9. All dogs that matched the criteria for exclusion are not considered in results.

Table 1 shows the evolution in time of the hemodynamic variables in groups 1 through 6 in which the effects of preconditioning and of adenosine receptors blockade were studied. In the preconditioned groups, an average of the five tachycardic periods was computed for the hemodynamic variables. Apart from the periods of pacing at high heart rate for preconditioning (213±12 cycles/min), heart rate was maintained constant by electrical pacing at 120±3 cycles/min during the baseline period, during the intervening periods between tachycardic periods, and during ischemia and reperfusion in all the groups. Considering all the groups together, mean aortic pressure decreased by 5.1±1.2 mm Hg. However, this change was not statistically significant for any group by ANOVA for repeated measurements. In the preconditioned group (group 2) left ventricular systolic pressure during the tachycardic period decreased by 10.4% (P<.05). Left ventricular end-diastolic pressure did not change during tachycardia but increased significantly during ischemia in all the groups although to a less extent in preconditioned dogs without adenosine receptors blockade (group 2, P<.05). Left ventricular maximal dP/dt did not significantly change during the procedures in any group. Diastolic and systolic segment lengths did not significantly change during tachycardia, but both increased in all the groups during ischemia. The shortening fraction decreased during ischemia and reperfusion in all the groups. Coronary blood flow to the risk area, as measured with the flowmeter, increased significantly during tachycardia in the preconditioned groups and during early reperfusion in all the groups but with no significant differences between them.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Data

Table 2 shows the effect of tachycardia on coronary flow and metabolic variables in preconditioned dogs (group 2). Because there were no significant changes in these variables in group 1 (nonpreconditioned dogs), the results are shown only for group 2 and were analyzed as changes from baseline values. An average of the five tachycardic episodes was computed for the metabolic variables because there were no differences between them. Tachycardia increased coronary flow to the risk area by 38.3% (P<.01), the oxygen consumption of the left ventricular wall by 64.5% (P<.002), and the coronary arteriovenous difference in oxygen content by 19.6% (P<.001). The oxygen content of the arterial blood did not change and the oxygen content of the venous coronary blood decreased by 23.4% (P<.001). Lactate extraction did not change and the ECG did not show ischemic signs.

View this table: [in this window] [in a new window]   Table 2. Effect of Tachycardia on Coronary Blood Flow and Metabolic Variables in Preconditioned Dogs (n=26)

Fig 2 shows the distribution of coronary blood flow across four layers of the left ventricular wall as measured with radioactive microspheres during control heart rate, tachycardia, and during tachycardia plus the intracoronary infusion of adenosine (group 10). During control heart rate, flow was similar across the left ventricular wall with an inner/outer flow ratio of 1.20±0.05. During tachycardia, flow increased in all the layers and the inner/outer flow ratio did not change (1.19±0.06). During the infusion of adenosine flow increased in all the layers, although preferentially toward the outer ones, revealing the persistence of vasodilatory reserve across the wall.

View larger version (47K): [in this window] [in a new window]   Figure 2. Coronary vasodilatory reserve across the left ventricular wall during tachycardia (group 10, n=10). Control indicates flow during control heart rate; Tachycardia, flow during tachycardia; Tachycardia+adenosine, flow during tachycardia plus adenosine infusion; I, inner; IM, inner middle; OM, outer middle; and O, outer layer of the left ventricular wall. *P<.05 compared with control; #P<.05 compared with tachycardia.

Fig 3 shows the sizes of the infarct regions in groups 1 through 6. There were no significant differences in the risk region volumes (as percent of the left ventricular volume) between the groups (45.4±2.0%, 45.2±1.8%, 44.8±2.7%, 48.3±3.1%, 49.5±2.5%, and 48.2±5.7% from groups 1 to 6, respectively, not shown). The necrotic region as percent of the risk region was significantly smaller in preconditioned dogs (group 2) as compared with the nonpreconditioned dogs (group 1): 8.6±1.4% versus 15.8±1.5%, respectively, P<.001. This effect was reverted by adenosine receptor blockade induced whether before tachycardia (group 3: 14.3±1.6%) or after tachycardia (group 4: 17.5±2.5%). The early (group 5) and late (group 6) administration of 8PT in nonpreconditioned dogs (at times equivalent to its administration before or after tachycardia in preconditioned dogs of groups 3 and 4, respectively) did not modify the size of the infarction observed in the nonpreconditioned group (15.5±1.9% versus 15.8±1.5%) and are presented as one group in Fig 3.

View larger version (44K): [in this window] [in a new window]   Figure 3. Infarct size expressed in terms of necrotic region volume as percent of the risk region volume [NR/RR (%)]. NoPC indicates nonpreconditioned dogs (group 1); PC, preconditioned dogs (group 2); PC+8PT before PC, preconditioned dogs plus 8 phenyltheophylline (8PT) before tachycardia (group 3); PC+8PT after PC, preconditioned dogs plus 8PT after tachycardia (group 4); and NoPC+8PT, nonpreconditioned dogs plus early or late 8PT administration (groups 5 and 6, respectively, depicted as one group). *P<.001 compared with the NoPC group. Numbers in parenthesis represent numbers of dogs.

Fig 4 shows the regression of infarct size on transmural collateral blood flow during ischemia in nonpreconditioned dogs (group 1), in preconditioned dogs (group 2), in preconditioned dogs with adenosine receptor blockade before and after tachycardia (groups 3 and 4 as one group), and in nonpreconditioned dogs with early and late adenosine receptor blockade (groups 5 and 6 as one group). For any value of collateral flow, the necrotic region, as percent of the risk region, was smaller in preconditioned dogs without adenosine receptor blockade compared to the other groups (P<.02 by ANCOVA).

View larger version (20K): [in this window] [in a new window]   Figure 4. Regression of infarct size, expressed in terms of necrotic region volume as percent of the risk region volume [NR/RR (%)] on collateral coronary flow to the ischemic region. For any value of collateral flow the infarct size is smaller in preconditioned dogs (group 2, ) than in nonpreconditioned dogs, (group 1, ), dogs preconditioned plus blockade of adenosine receptors, (groups 3 and 4, , as one group) and dogs nonpreconditioned plus blockade of adenosine receptors, (groups 5 and 6, , as one group). ANCOVA revealed a significant downward shift in the regression relation for the preconditioned dogs () compared with the other groups, P<.02.

Fig 5 shows the dialysate adenosine concentrations in preconditioned dogs (group 7). Adenosine increased during ischemia about fivefold (P<.05). Tachycardia induced a moderate (twofold) but significant increase (P<.05) of adenosine concentration.

View larger version (38K): [in this window] [in a new window]   Figure 5. Dialysate adenosine concentration during the procedure in preconditioned dogs (group 7, n=15). B indicates baseline; PC, tachycardia; I-1, I-2, and I-3, samples collected during the first, second, and third periods (20 minutes each) of ischemia, respectively; and R-1, R-2, and R-3, samples collected during the first, second, and third periods (20 minutes each) of reperfusion, respectively. *P<.05 compared with baseline.

Table 3 shows the PKC activities in nonpreconditioned and preconditioned dogs (groups 8 and 9). No significant changes of PKC activities were found in the cytosolic or particulate fraction through the procedure nor between the groups. No significant translocation of PKC-, -ß, -, and - were observed between groups (not shown).

View this table: [in this window] [in a new window]   Table 3. Protein Kinase C Activities in Nonpreconditioned and Preconditioned Dog Hearts

	Discussion

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Our results show that an increase in the metabolic demand of the myocardium induced by tachycardia, in the absence of simultaneous ischemia, decreases the infarct size produced by a prolonged coronary occlusion followed by reperfusion. This effect is mediated by adenosine but not by PKC. The absence of demand-induced ischemia during tachycardia is supported by the lack of changes in myocardial lactate production, left ventricular end-diastolic pressure, segment length and ECG repolarization signs, and by the preservation of the inner/outer flow ratio and the transmural coronary vasodilatory reserve, including the subendocardium. Furthermore, previous experiments show that increases in heart rate up to 300 cycles per minute in anesthetized and conscious dogs do not exhaust the transmural coronary vasodilatory reserve.^{21 22 23 24 25} During tachycardia, in our experiments, the coronary arteriovenous oxygen difference increased by 20%, corresponding to an increase of oxygen extraction from 54.5% to 65.2% and a decrease of 24% in the coronary venous oxygen content. These changes are less than those reported in healthy dogs during exercise in spite of a maintenance of coronary vascular reserve.^{26 27 28} Accordingly, the preconditioning effect that we observed was not mediated by ischemia but by a physiological increase in the metabolic rate of the heart.

Our results cannot be explained by differences in hemodynamic conditions between the groups during the procedure. The significant smaller increase in left ventricular end-diastolic pressure during ischemia in preconditioned dogs without adenosine receptor blockade is in agreement with a smaller infarct size. Besides, the segment lengths did not increase during tachycardia, discarding the possibility of preconditioning by effect of ventricular wall distension as reported by Ovize et al.²⁹ Our results cannot be explained by changes in transmural collateral blood flow because for any value of collateral flow measured during the occlusion of the coronary artery, the infarct size was smaller in the preconditioned dogs. Besides, the risk region measured as percent of the total left ventricular volume was similar in all groups and therefore this variable cannot account for the difference in size of the necrotic region between the groups.

There is scant research on the effects of tachycardia on preconditioning. There is evidence that an extreme increase in heart rate preconditions the degree of ischemia, hemodynamics alterations, and the incidence of arrhythmias in rabbits and dogs.^{30 31} However, in the above experiments^{30 31} overpacing was used to induce ischemia and therefore a presumptive preconditioning effect induced by the increase in myocardial oxygen consumption cannot be discriminated from that induced by ischemia. On the other hand, Koning et al³ recently reported that rapid ventricular pacing protects the myocardium against infarction through nonischemic ATP-dependent potassium channel activation in pigs. Our present results in dogs agree with the preconditioning effect of tachycardia reported by Koning et al³ and also with the smaller protective effect of this preconditioning procedure as compared with ischemic preconditioning with brief ischemic episodes. Thus in his original experiments in dogs, Murry et al¹ reported that four brief coronary occlusions, 5 minutes in duration each separated by 5 minutes of reperfusion, and followed by 40 minutes of sustained occlusion, produced a fourfold decrease of the infarct size; in the present experiments, we only obtained a reduction to one half of the infarct size.

Vegh et al³² reported a lower incidence of extrasystoles, ventricular tachycardia, and ventricular fibrillation episodes induced by coronary occlusion in anesthetized dogs with ischemic preconditioning. We did not find a preconditioning effect of tachycardia on arrhythmias as assessed by the number of extrasystoles, episodes of ventricular fibrillation, or sustained ventricular tachycardia between groups including those dogs not computed for infarct size comparison because of irreversible ventricular fibrillation. Our results agree with those of Przyklenk and Kloner,³³ who did not find a preconditioning effect of ischemia on arrhythmias after a sustained coronary occlusion in dogs anesthetized with the same anesthetic agent that we used (pentobarbitone).

The suppression of the preconditioning effect of tachycardia by adenosine receptor blockade induced whether before or after tachycardia shows that in dogs this effect is mediated through adenosine and, besides, that adenosine receptors must be occupied by the agonist during ischemia for the protective effect to persist. This result is analogous to that reported in ischemic preconditioning in rabbits by Thornton et al,³⁴ who demonstrated the necessity of adenosine receptor activation during the prolonged ischemic period. Our results in nonischemic preconditioning are also analogous to those of Auchampach and Gross⁴ in ischemic preconditioning in dogs in the sense that this type of preconditioning is also reverted by adenosine receptors blockade. An increase in adenosine formation during enhanced myocardial metabolism obtained by different maneuvers has been reported by several authors.^{5 6 7 8 35} Thus Saito et al⁵ showed an increase in adenosine content in myocardial biopsies during cardiac pacing in dogs, Watkinson et al⁷ found a positive correlation between adenosine concentration in pericardial fluid and cardiac work during stellate ganglion stimulation in anesthetized dogs and during treadmill exercise in awake dogs, and McKenzie et al⁸ found an increase in myocardial adenosine content during enhanced cardiac performance induced by aortic constriction or isoproterenol administration in the absence of myocardial ischemia in open chest dogs. Hall et al³⁵ reported that an increase in myocardial work, in the absence of demand-induced ischemia, resulted in accumulation of adenosine and AMP in the interstitium. The increase in myocardial interstitial adenosine during tachycardia that we observed was about twofold and agree with that reported by Saito et al⁵ and Hall et al,³⁵ who measured adenosine in myocardial biopsies and in the interstitium (microdialysis), respectively. This increase should be enough to induce the protective infarct size–limiting effect because it has been reported that maximal activation of adenosine A₁ receptors is obtained at nanomolar concentration levels.³⁶ Furthermore, as Niroomand et al reported,³⁷ it is probable that the effect of ischemic preconditioning may be due to an enhanced response of the adenosine A₁ receptor pathway by overactivation of G_i protein, mainly during the reperfusion period before the prolonged ischemia, and this may occur also in nonischemic preconditioning. Finally, the adenosine concentration in the dialysate may not represent its concentration near the plasma membrane; this depends to a large extent on the state of the myocyte metabolism, which is greater during tachycardia than during ischemic preconditioning. It has been reported that ischemic preconditioning causes a slower degradation of adenine nucleotides and less production of purine nucleosides, including adenosine, in the ischemic myocardium,^{1 38 39 40} although an activation of ecto-5'nucleotidase may increase adenosine concentration at the plasma membrane and induce preconditioning when the ischemic preconditioning procedure is used.⁴¹

While it is clear from our results that the administration of an adenosine receptor antagonist abolishes the protective effect of pacing-induced tachycardia completely, other mechanisms of protection may also be involved, such as activation of adrenergic receptors, because a nonischemic preconditioning effect through adrenoceptor stimulation with exogenous norepinephrine or release of endogenous catecholamines has been demonstrated.^{42 43} We think, however, that this mechanism does not have an important participation in our results because the basal release of norepinephrine in the myocardium in open chest dogs does not change significantly over a wide range of pacing frequencies.⁴⁴

Our results do not support the participation of an increase in PKC activity or changes in its isozymes translocation in tachycardia-induced preconditioning. We cannot compare these results with those obtained in ischemic preconditioning in dogs because in this species a controversy exists as to the participation of PKC in ischemic preconditioning.^{41 45} Nevertheless, our results suggest that in tachycardia-induced preconditioning, an increase in PKC activity, through the activation of phospholipase C or D and the production of diacylglycerol, is not a main pathway to produce myocardial protection in dogs as it is in ischemic preconditioning in rabbits and rats.^{46 47}

In summary, tachycardia without ischemia mimics the infarct size-limiting effect of ischemic preconditioning in the dog. This effect is mediated by adenosine but not by PKC translocation.

	Acknowledgments

 
This work was supported in part by FONDECYT grants 1940296 and 1970305. We gratefully acknowledge the skillful technical assistance of Juan Carlos Fuenzalida and Guillermo Arce, Jr.

Received June 26, 1997; revision received October 13, 1997; accepted October 16, 1997.

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