Inhibition of Sarcolemmal Na+,K+-ATPase Activity Reduces the Infarct Size–Limiting Effect of Preconditioning in Rabbit Hearts
Background The inhibition of sarcolemmal Na+,K+-ATPase activity is closely related to ischemic myocardial cell injury. However, the involvement of this enzyme in preconditioning has not been determined.
Methods and Results We assessed the effect of ischemia on sarcolemmal Na+,K+-ATPase activity. Control and preconditioned rabbits were subjected to 0, 10, 20, 30, and 60 minutes of coronary occlusion. Ten to 60 minutes of ischemia reduced Na+,K+-ATPase activity, whereas preconditioning preserved the activity of this enzyme only during the first 20 minutes of ischemia. To determine whether the preservation of Na+,K+-ATPase activity in the early phase of ischemia contributed to limiting the infarct size, additional rabbits underwent 30 minutes of occlusion followed by 3 hours of reperfusion with or without pretreatment with digoxin, an inhibitor of Na+,K+-ATPase. Infarct size in animals pretreated with digoxin in the absence of preconditioning did not differ from that in controls. It was markedly reduced by preconditioning, whereas digoxin reduced the infarct size–limiting effect. Moreover, preconditioning increased sarcolemmal Na+-Ca2+ exchange activity in rabbits subjected to 20 minutes of ischemia, whereas digoxin diminished this increase.
Conclusions Preconditioning preserves the ischemia-induced reduction in sarcolemmal Na+,K+-ATPase activity in the early phase of ischemia in rabbit hearts. Inhibition of Na+,K+-ATPase activity reduces the infarct size–limiting effect of preconditioning with a loss of increased Na+-Ca2+ exchange activity, implying that this preservation is responsible for the cardioprotective effect of preconditioning.
Ischemic preconditioning protects the myocardium against subsequent sustained ischemic insults. The protective effects of preconditioning include a limitation of infarct size,1 a suppression of life-threatening ventricular arrhythmias induced by ischemia or reperfusion,2 3 and an improvement in regional wall-motion abnormalities after reperfusion.4 Considerable attention has focused on the involvement of altered sarcolemmal function in preconditioning. In this context, increased 5′-nucleotidase activity,5 the opening of ATP-sensitive potassium (KATP) channels,6 7 and the activation of protein kinase C8 9 have all been studied; however, the biochemical mechanism responsible for preconditioning is still unknown.
During ischemia, myocardial cell injury is accompanied by an increase in [Na+]i and a decrease in [K+]i.10 These ionic alterations are caused by the activation of a Na+-H+ exchange due to intracellular acidosis and the inhibition of Na+,K+-ATPase caused by reduced intracellular ATP stores. This increase in [Na+]i leads to an increase in [Ca2+]i (Ca2+ overload) via a reverse Na+-Ca2+ exchange; this initiates a destructive chain of ischemic events. Yet interestingly, changes in Na+,K+-ATPase activity associated with Na+-Ca2+ exchange activity during myocardial ischemia have not been determined in preconditioning.
Our objective was to compare the activity of Na+,K+-ATPase between control and preconditioned rabbit hearts made ischemic for varied times. We also examined the effect of digoxin, an inhibitor of Na+,K+-ATPase, on sarcolemmal Na+,K+-ATPase as well as Na+-Ca2+ exchange activities and infarct size in control and preconditioned animals.
Female Japanese White rabbits (weight, 2.5 to 3.2 kg) were anesthetized with 30 mg/kg sodium pentobarbital, intubated, then mechanically ventilated with 100% oxygen by use of a volume-cycled respirator (model SN-485-5; Shinano Apparatus). A fluid-filled catheter was placed in the right femoral artery and was connected to a transducer (Statham p-23Db; Gould) for measurement of arterial pressure. Ventilation was maintained at 15 to 20 breaths per minute, and tidal volume was ≈40 mL. The respiratory rate was adjusted to keep the blood pH in the physiological range. A left thoracotomy was performed in the fourth intercostal space, and the pericardium was opened. A 2-0 silk thread was then passed around the circumflex branch of the left coronary artery, with its ends being threaded through a small polyethylene tube. Precordial electrocardiography was monitored by use of bipolar chest leads. Rabbits were allowed ≥20 minutes to reach a steady state after surgical preparation. Coronary occlusion was produced by pulling the snare and clamping it with a mosquito hemostat. Reperfusion was produced by releasing the clamp. Myocardial ischemia was confirmed by ST-segment elevation of the ECG as well as observation of regional cyanosis over the myocardial surface. Reperfusion was confirmed by reactive hyperemia over the surface after the snare was released.
First, the time courses of ischemic change in Na+,K+-ATPase activity were evaluated in 32 control and 31 preconditioned rabbits. Ischemic preconditioning was elicited by a 5-minute occlusion of the circumflex coronary artery followed by a 10-minute reperfusion. Sustained ischemia was then induced for 0, 10, 20, 30, and 60 minutes (n=6, 6, 7, 6, and 6, respectively). Control animals did not undergo coronary occlusion and reperfusion before 0, 10, 20, 30, and 60 minutes of sustained ischemia (n=6, 6, 7, 6, and 7, respectively).
Second, the effects of digoxin and of dobutamine, another inotropic agent that does not act through Na+,K+-ATPase inhibition, on sarcolemmal Na+,K+-ATPase activity were compared in 13 control and 13 preconditioned animals subjected to 20 minutes of sustained ischemia: 7 animals treated with 0.3 mg/kg digoxin without preconditioning, 7 treated with digoxin before preconditioning, 6 treated with 10 μg·kg−1·min−1 dobutamine without preconditioning, and 6 treated with dobutamine before preconditioning. Digoxin was administered intravenously as a bolus infusion 30 minutes before sustained ischemia. A dose of 0.3 mg/kg was chosen because in our preliminary study it was found to provide stable and maximal left ventricular contractility for ≥1 hour without digitalis-induced ventricular arrhythmias. In this preliminary study, a 2F micromanometer-tipped catheter (Miller Instruments) was inserted into the left ventricle through the right carotid artery to measure left ventricular peak positive dP/dt. This parameter of left ventricular contractility increased by 29±2% (mean±SE) compared with the baseline value 30 minutes after the injection of 0.3 mg/kg digoxin. Dobutamine was administered intravenously as a continuous infusion from 30 minutes before sustained ischemia until the end of ischemia. The dose of dobutamine (10 μg·kg−1·min−1) was found to have an inotropic effect equivalent to that produced by 0.3 mg/kg digoxin (left ventricular peak positive dP/dt increased by 28±1% compared with the baseline value 30 minutes after the start of the infusion).
Third, the effect of digoxin on sarcolemmal Na+-Ca2+ exchange activity was also evaluated in four additional groups of animals: 5 animals treated with saline, 6 treated with saline before preconditioning, 5 treated with 0.3 mg/kg digoxin without preconditioning, and 6 treated with digoxin before preconditioning. These animals were then subjected to 20 minutes of sustained ischemia.
After each procedure, the heart was quickly excised, with the coronary artery remaining occluded, and the aortic root was clamped with a mosquito hemostat. The heart was placed on ice and then perfused with iced saline by intraventricular injection to demarcate the ischemic (nonperfused) and nonischemic (perfused) regions. The free wall of the right ventricle was removed, and the left ventricle was then divided transmurally into ischemic and nonischemic regions. The tissue samples were frozen in liquid nitrogen and stored at −80°C until membrane preparation.
We also evaluated the effects of digoxin and dobutamine on the limitation of infarct size by preconditioning. Six groups of animals underwent the same surgical preparations as used in the sarcolemmal study and were then subjected to 30 minutes of coronary occlusion followed by 3 hours of reperfusion. These groups included 9 animals treated with saline, 7 treated with saline before preconditioning, 8 treated with 0.3 mg/kg digoxin without preconditioning, 8 treated with digoxin before preconditioning, 8 treated with 10 μg·kg−1·min−1 dobutamine without preconditioning, and 7 treated with dobutamine before preconditioning.
With minor modifications, cardiac sarcolemmal membranes from ischemic and nonischemic regions were prepared separately according to the method of Jones et al11 and Maisel et al.12 Briefly, the heart tissue was minced and homogenized in 10 vol/wt of membrane buffer (25 mmol/L Tris-HCl, 10 mmol/L MgCl2, and 1 mmol/L EDTA at pH 7.5) by use of a Polytron PT10 for 10 seconds at half-maximal speed. This crude homogenate was treated with 750 mmol/L NaCl and centrifuged at 14 000g for 20 minutes. The pellet was then resuspended and washed once in buffer containing 10 mmol/L NaHCO3 and 10 mmol/L histidine. This material was vigorously homogenized in buffer containing 0.25 mol/L sucrose, 10 mmol/L histidine, and 1 mmol/L EDTA (pH 7.5) by use of the Polytron for three 20-second bursts at half-maximal speed and was then centrifuged at 45 000g for 30 minutes. The pellet was then resuspended, washed, and centrifuged at 17 000g for 20 minutes. The resulting supernatant was centrifuged at 210 000g for 60 minutes. This final pellet was resuspended in membrane buffer as purified sarcolemmal membranes and stored at −80°C. For measurement of Na+-Ca2+ exchange activity, sarcolemmal membrane was prepared by using the buffer without EDTA at each step to avoid its Ca2+ chelating action.
Protein concentrations were measured according to the method of Lowry et al13 with bovine serum albumin used as a standard.
Measurement of Na+,K+-ATPase Activity
Na+,K+-ATPase activity was assayed according to the method of Jones et al.11 Approximately 10 μg of purified membrane was preincubated in 50 mmol/L histidine, 3 mmol/L MgCl2, 100 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L NaN3, and 1 mmol/L EGTA (pH 7.4), with or without 1 mmol/L ouabain, at 37°C for 15 minutes. The reaction was started by adding ATP to a final concentration of 3 mmol/L and was continued at 37°C for 30 minutes. Inorganic phosphate liberated from ATP was quantified colorimetrically by measuring the absorbance at 700 nm. Na+,K+-ATPase activity measured with 1 mmol/L ouabain was subtracted from that obtained without ouabain.
Measurement of Na+-Ca2+ Exchange Activity
Na+-Ca2+ exchange activity was measured according to the method of Reeves and Sutko.14 Approximately 15 μg of membrane vesicle was equilibrated with 160 mmol/L NaCl and 20 mmol/L MOPS/Tris (pH 7.4) at 37°C for 30 minutes. A 5-μL aliquot of vesicle was placed as a bead on the side of a polystyrene test tube containing 30 μmol/L 45CaCl2 (New England Nuclear) in 100 μL of 160 mmol/L KCl and 20 mmol/L MOPS/Tris (pH 7.4) at 37°C. The vesicle was mixed with the dilution medium by spinning the tube. 45Ca2+ uptake was terminated 2, 5, 10, and 60 seconds after mixing by diluting the contents of the tube with 5 mL of ice-cold 200 mmol/L KCl, 20 mmol/L MOPS/Tris, and 0.1 mmol/L EGTA (pH 7.4). The vesicle was harvested on a Whatman GF/C glass fiber filter and washed with two additional 5-mL aliquots of the quenching medium. Radioactivity on the filter was determined by use of a liquid scintillation counter. Control incubation was also performed simultaneously in which 160 mmol/L NaCl was substituted for KCl in the dilution medium so that there was no Na+ gradient from inside to outside the vesicle. 45Ca2+ uptake measured without Na+ gradient was subtracted from that obtained with Na+ gradient to estimate the uptake of 45Ca2+ due to Na+-Ca2+ exchange.
Determination of Infarct Size
For each infarct-size study, at the end of the 3 hours of reperfusion, the heart was rapidly excised and mounted on a Langendorff apparatus by the aortic root. The snare was retightened and 0.5% phthalocyanine blue pigment was infused into the perfusate to demarcate the area at risk as the tissue without blue dye. The heart was then removed, frozen, and cut into slices 2 mm thick. The slices were weighed and the area at risk (nonblue area) was cut, weighed, and incubated at 37°C for 15 minutes in 1% triphenyltetrazolium chloride (TTC) in pH 7.4 buffer. TTC stained the noninfarcted myocardium a brick-red color. The area at risk and the area of infarction were then determined by planimetry, corrected for the weight of each tissue slice, and summed for each heart.
Data are expressed as mean±SE. The hemodynamic changes and Na+,K+-ATPase activity (ischemic versus nonischemic region) within each group were compared by use of a paired t test. The differences in hemodynamics and enzyme activities between groups were analyzed by one-way ANOVA using Fisher’s least significant difference as the post hoc test. The differences in time-activity curves of the Na+-Ca2+ exchange between the ischemic and nonischemic regions were analyzed by two-way ANOVA with repeated measures. In the infarct-size study, the differences between groups were compared by one-way ANOVA with Scheffé’s post hoc test. A level of P<.05 was accepted as statistically significant.
The hemodynamic data from our enzymatic study are summarized in Table 1⇓. There were no significant differences between control and preconditioned animals in mean arterial pressure or heart rate at baseline or at the end of 10, 20, 30, and 60 minutes of sustained ischemia. At the end of each period of sustained ischemia, the mean arterial pressure tended to decrease compared with that at baseline. In animals pretreated with digoxin or dobutamine, there were also no significant differences in mean arterial pressure or heart rate at baseline or at the end of 20 minutes of ischemia between control and preconditioned groups. Table 2⇓ shows the hemodynamic changes during the infarct-size study. There were no significant differences in mean arterial pressure or heart rate at baseline or at the end of sustained ischemia between the six groups. In digoxin-pretreated and dobutamine-pretreated animals without preconditioning ischemia, the mean arterial pressure before sustained ischemia was significantly higher than that in control animals.
The time dependency of the ischemic changes in Na+,K+-ATPase activity and the effects of digoxin and dobutamine on this enzyme activity are summarized in Table 3⇓. Na+,K+-ATPase activity was progressively reduced after 10 to 60 minutes of ischemia. Preconditioning significantly preserved the reduction of the activity of this enzyme during the first 20 minutes of sustained ischemia (the reduction ratio of enzyme activity in the ischemic region relative to that in the nonischemic region: 10 minutes, 23±7% versus 4±3%, P<.05; 20 minutes, 36±3% versus 4±9%, P<.05).
The intravenous administration of digoxin did not change the ischemia-induced reduction in Na+,K+-ATPase activity in control animals, whereas it abolished the preservation of the activity of this enzyme in preconditioned animals; there was no difference in percent reduction in activity between these two groups (38±4% versus 32±6%). On the other hand, the administration of dobutamine did not affect the activity of this enzyme in either control or preconditioned animals (33±5% versus 5±5%; P<.05).
Na+-Ca2+ Exchange Activity
Fig 1⇓ shows time-activity curves of the sarcolemmal Na+-Ca2+ exchange in control and preconditioned animals with and without digoxin pretreatment. There was no significant difference in Na+-Ca2+ exchange activity between ischemic and nonischemic regions in control animals. Interestingly, however, Na+-Ca2+ exchange activity in the ischemic region was significantly increased compared with that in nonischemic regions in preconditioned animals. Digoxin pretreatment did not affect Na+-Ca2+ exchange activity in control animals, whereas it diminished the preconditioning-induced increase in this activity.
Data from the study of infarct size are summarized in Table 4⇓. Body weights, left ventricular weights, and the area at risk, expressed as a percent of the left ventricle, did not differ significantly among the experimental groups. The infarct-size results, expressed as a percent of the area at risk, are shown in Fig 2⇓. Preconditioning significantly reduced the size of the infarct compared with the control group (12.5±2.4% versus 44.3±3.9%; P<.05). The administration of 0.3 mg/kg digoxin had no effect on infarct size, but it significantly reduced the beneficial effect of preconditioning on limitation of infarct size (33.5±3.3% versus 12.5±2.4%; P<.05). On the other hand, dobutamine pretreatment had no effect on infarct size either in control or preconditioned animals.
Fig 3⇓ shows the relationship between infarct size and risk-zone size in protected groups (7 preconditioned animals and 7 treated with dobutamine and preconditioning) and the respective control groups (9 control animals and 8 treated with dobutamine). The regression lines had different slopes (0.28 with r=.47 in protected groups and 0.65 with r=.71 in control groups) with positive x intercepts.15 16 It is apparent that there is minimal overlap in data points between protected and control groups. These observations indicate that the smaller infarcts in protected groups are not merely the result of smaller risk zones.
The present study demonstrated that the activity of sarcolemmal Na+,K+-ATPase progressively diminished after 10 to 60 minutes of sustained ischemia. Importantly, these reductions were abolished by preconditioning (≤20 minutes of sustained ischemia) in rabbit hearts. These alterations in sarcolemmal Na+,K+-ATPase activity may simply reflect functional or structural damage to the sarcolemma depending on the severity of the myocardial ischemia. However, it is possible that the preserved enzyme activity in preconditioned hearts has a beneficial effect on preventing further progression of ischemic injury, especially since the inhibition of Na+,K+-ATPase activity is known to trigger cardiac injury during myocardial ischemia.
During myocardial ischemia, a reduction in intracellular ATP stores inhibits Na+,K+-ATPase activity, which elevates intracellular [Na+], thereby raising intracellular [Ca2+] via a reverse Na+-Ca2+ exchange. The resultant increase in [Ca2+]i activates sarcolemmal phospholipases and proteases that release membranous phospholipid degradation products, whose detergent properties impair the integrity of the cell membrane.17 18 19 We hypothesized that the preservation of Na+,K+-ATPase activity in the early phase of sustained ischemia in preconditioned hearts helps to protect the heart against further ischemic injury. To test this hypothesis, we evaluated the effect of a functional inhibitor of Na+,K+-ATPase, digitalis glycoside, on the preconditioning-mediated limitation of infarct size.
Effects of Na+,K+-ATPase Blockade on Cardioprotection of Preconditioning
We found that digoxin pretreatment did not affect infarct size in the absence of preconditioning. By contrast, such pretreatment reduced the beneficial effect of preconditioning on limiting infarct size. Importantly, these changes in infarct size as a result of digoxin pretreatment were associated with those in sarcolemmal Na+,K+-ATPase activity. Although digoxin had no effect on the ischemia-induced reduction in Na+,K+-ATPase activity in control animals, it diminished the preservation of the preconditioning-induced activity of this enzyme. These findings strongly suggest that the cardioprotective effect of preconditioning is achieved by the preservation of sarcolemmal Na+,K+-ATPase.
However, because digoxin exerts an inotropic effect on the myocardium, we considered it possible that digoxin was blocking the infarct size–limiting effect of preconditioning by increasing myocardial oxygen demand rather than by inhibiting Na+,K+-ATPase activity. To exclude this possibility, we compared the effects of another inotropic agent, dobutamine, on Na+,K+-ATPase activity as well as infarct size in control and preconditioned animals. The dose of dobutamine used in the present study provided the same inotropic effect as did digoxin before and during myocardial ischemia. Nevertheless, dobutamine pretreatment did not alter Na+,K+-ATPase activity in the early phase of sustained ischemia or infarct size either in the presence or absence of preconditioning. These observations indicate that the inhibition of Na+,K+-ATPase is a specific mechanism that is important in preventing the cardioprotective effect of preconditioning and that an increase in oxygen demand is not just a nonspecific mechanism to block preconditioning.
Effect of Preconditioning on Na+-Ca2+ Exchange Activity
Previous studies20 21 demonstrated that adenosine is a key endogenous mediator involved in the cardioprotective mechanism of ischemic preconditioning. Other studies also revealed that the KATP channel is a target of adenosine A1 stimulation and that the opening of this channel is involved in the cardioprotective effects of preconditioning in dogs,6 7 22 pigs,23 and rabbits.24 25 Activation of KATP channels shortens the action-potential duration and antagonizes membrane depolarization.26 These effects would be expected to interfere with the entry of Ca2+ into cells via voltage-regulated calcium channels and in consequence prevent a variety of ischemic insults by a reduction in Ca2+ overload. However, because [Ca2+]i is regulated by various ionic channels and transporters in the sarcolemma and the sarcoplasmic reticulum, it is unlikely that KATP channel activation alone contributes to lowering [Ca2+]i and thus reducing infarct size in preconditioned hearts.
As mentioned above, during myocardial ischemia an increase in [Na+]i caused by the inhibition of Na+,K+-ATPase leads to an increase in [Ca2+]i via a reverse Na+-Ca2+ exchange. It is possible that the cardioprotective effect of preconditioning relates to an alteration in Na+-Ca2+ exchange activity. Therefore, in the present study, we also compared sarcolemmal Na+-Ca2+ exchange activity in the early phase of sustained ischemia in control and preconditioned animals. In control animals, the Na+-Ca2+ exchange activity in the ischemic myocardium was comparable to that in the nonischemic myocardium. Interestingly, however, Na+-Ca2+ exchange activity increased in ischemic compared with nonischemic myocardium in preconditioned animals. Furthermore, a blockade of Na+,K+-ATPase by pretreatment with digoxin was accompanied by prevention of this preconditioning-induced increase in Na+-Ca2+ exchange activity. Although the actual changes in [Ca2+]i in these hearts were not measured in the present study, our observations suggest that these alterations in sarcolemmal function might contribute profoundly to the infarct size–limiting effect of preconditioning by reducing Ca2+ overload.
In summary, preconditioning preserves the ischemia-induced reduction in sarcolemmal Na+,K+-ATPase activity in the early phase of sustained ischemia in rabbit hearts. This preservation is accompanied by an increase in Na+-Ca2+ exchange activity. Inhibition of Na+,K+-ATPase activity with digoxin prevents the infarct size–limiting effect of preconditioning in combination with a loss of increased Na+-Ca2+ exchange activity. These findings suggest that preservation of Na+,K+-ATPase activity in the early phase of sustained ischemia is responsible for protection of the heart against further ischemic injury.
Dr Iwase’s current address is Cardiovascular Section, Takeda Hospital, Kyoto, Japan.
- Received October 28, 1996.
- Revision received December 18, 1996.
- Accepted January 20, 1997.
- Copyright © 1997 by American Heart Association
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
Shiki K, Hearse DJ. Preconditioning of ischemic myocardium: reperfusion-induced arrhythmias. Am J Physiol. 1987;253:H1470-H1476.
Hager JM, Hale SL, Kloner RA. Effects of preconditioning ischemia on reperfusion arrhythmias after coronary artery occlusion and reperfusion in the rat. Circ Res. 1991;68:61-68.
Cohen MV, Liu GS, Downey JM. Preconditioning causes improved wall motion as well as smaller infarcts after transient coronary occlusion in rabbits. Circulation. 1991;84:341-349.
Kitakaze M, Hori M, Takashima S, Sato H, Inoue M, Kamada T. Ischemic preconditioning increases adenosine release and 5′-nucleotidase activity during myocardial ischemia and reperfusion in dogs: implications for myocardial salvage. Circulation. 1993;87:208-215.
Gross GJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res. 1992;70:223-233.
Grover GJ, Sleph PG, Dzwonczyk S. Role of myocardial ATP-sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A1-receptors. Circulation. 1992;86:1310-1316.
Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol. 1994;266:H1145-H1152.
Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning: ionic alterations. Circ Res. 1993;72:112-125.
Jones LR, Maddock SW, Besch HR Jr. Unmasking effect of alamethicin on the (Na+,K+)-ATPase, β-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J Biol Chem. 1980;255:9971-9980.
Maisel AS, Motulsky HJ, Insel PA. Externalization of β-adrenergic receptors promoted by myocardial ischemia. Science. 1985;230:183-186.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Reeves JP, Sutko JL. Sodium-calcium ion exchange in cardiac membrane vesicles. Proc Natl Acad Sci U S A. 1979;76:590-594.
Ytrehus K, Liu Y, Tsuchida A, Miura T, Liu GS, Yang X-M, Herbert D, Cohen MV, Downey JM. Rat and rabbit heart infarction: effects of anesthesia, perfusate, risk zone, and method of infarct sizing. Am J Physiol. 1994;267:H2383-H2390.
Tsuchida A, Liu Y, Liu GS, Cohen MV, Downey JM. α1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res. 1994;75:576-585.
Sedlis SP, Corr PB, Sobel BE, Ahumada GG. Lysophosphatidylcholine potentiates Ca2+ accumulation in rat cardiac myocytes. Am J Physiol. 1983;13:H32-H38.
Atsma DE, Lars Bastiaanse EM, Jerzewski A, Van der Valk LJM, Van der Laarse A. Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition. Circ Res. 1995;76:1071-1078.
Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991;84:350-356.
Thornton JD, Liu GS, Olsson RA, Downey JM. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation. 1992;85:659-665.
Auchampach JA, Gross GJ. Adenosine A1 receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol. 1993;264:H1327-H1336.
Van Winkle DM, Chien GL, Wolff RA, Soifer BE, Kuzume K, Davis RF. Cardioprotection provided by adenosine receptor activation is abolished by blockade of the KATP channel. Am J Physiol. 1994;266:H829-H839.
Toombs CF, Moore TL, Shebuski RJ. Limitation of infarct size in the rabbit by ischaemic preconditioning is reversible with glibenclamide. Cardiovasc Res. 1993;27:617-622.
Tan HL, Mazon P, Verberne HJ, Sleeswijk ME, Coronel R, Opthof T, Janse MJ. Ischaemic preconditioning delays ischaemia induced cellular electrical uncoupling in rabbit myocardium by activation of ATP sensitive potassium channels. Cardiovasc Res. 1993;27:644-651.