Cardioplegia containing specific adenosine-triphosphate

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1 The Adenosine-Triphosphate Sensitive Potassium- Channel Opener Pinacidil Is Effective in Blood Cardioplegia Jennifer S. Lawton, MD, Peng-Wie Hsia, PhD, and Ralph J. Damiano, Jr, MD Department of Surgery, Medical College of Virginia, Richmond, Virginia Background. This study was designed to evaluate the adenosine-triphosphate sensitive potassium channel opener pinacidil as a blood cardioplegic agent. Methods. Using a blood-perfused, parabiotic, Langendorff rabbit model, hearts underwent 30 minutes of normothermic ischemia protected with blood cardioplegia (St. Thomas solution [n 8] or Krebs-Henseleit solution with pinacidil [50 mol/l, n 8]) and 30 minutes of reperfusion. Percent recovery of developed pressure, mechanical arrest, electrical arrest, reperfusion ventricular fibrillation, percent tissue water, and myocardial oxygen consumption were compared. Results. The percent recovery of developed pressure was not different between the groups ( and for hyperkalemic and pinacidil cardioplegia, respectively). Pinacidil cardioplegia was associated with prolonged electrical and mechanical activity ( and minutes), compared with hyperkalemic cardioplegia ( and minutes, respectively; p < 0.05). Pinacidil cardioplegia was associated with a higher reperfusion myocardial oxygen consumption ( versus ml/100 g myocardium/ beat; p < 0.05) and a higher percent of tissue water (79.6% 0.7% versus 78.6% 1.2%; p < 0.05). Conclusions. Systolic recovery was not different between groups, demonstrating comparable effectiveness of pinacidil and hyperkalemic warm blood cardioplegia. (Ann Thorac Surg 1998;66:768 73) 1998 by The Society of Thoracic Surgeons Cardioplegia containing specific adenosine-triphosphate (ATP) potassium channel openers has been documented to provide an effective form of myocardial protection in various animal models [1 4]. The pharmacologic opening of the ATP potassium channel results in potassium efflux from the myocyte and a shortening of the action potential duration. Reduction in the duration of the action potential decreases the plateau phase, and thus decreases calcium influx. This leads to contractile failure and therefore energy conservation during ischemia [4, 5]. Adenosine-triphosphate potassium channels are inhibited by intracellular ATP. They open during myocardial ischemia and are part of the cell s natural response to a limited supply of energy substrate [4, 6]. Crystalloid cardioplegia containing ATP potassium channel openers (PCOs) has been demonstrated to provide superior or equivalent myocardial protection when compared with hyperkalemic arrest [2, 3]. The PCO pinacidil has been demonstrated to be an effective cardioplegic agent in an acutely injured isolated rabbit heart model and hypothermic arrest [7]. However, there is little information on the efficacy of PCOs in warm blood cardioplegia. The benefits of blood cardioplegia have been clearly demonstrated in the literature and include rapid cardiac Accepted for publication March 31, Address reprint requests to Dr Damiano, The Milton S. Hershey Medical Center, PO Box 850, Hershey, PA ( damiano@surg.hmc. psu.edu). arrest in an oxygenated environment, oncotic constituents to prevent or minimize myocardial edema, improved oxygen-carrying and buffering capacity, improved capillary flow distribution, limitation of systemic hemodilution, and endogenous free radical scavenging [8 10]. Because of these advantages, most cardiac surgeons incorporate blood cardioplegia into their myocardial protection regimen. This study was performed to evaluate the effectiveness of the PCO pinacidil in blood cardioplegia during normothermic arrest in an isolated rabbit heart model. Material and Methods Experimental Preparation Adult New Zealand white rabbits of either sex weighing 3.0 to 5.0 kg were anesthetized intramuscularly with acepromazine (1 mg/kg) and xylazine (17.5 mg/kg), followed by ketamine (62.5 mg/kg). Each study involved a support animal and a study animal. The support animal was prepared, continuously monitored, and ventilated while on an extracorporeal circuit for parabiotic support of the isolated heart, as previously described [2]. After support animal preparation, the donor animal was heparinized and ventilated through a tracheostomy. The animal then underwent rapid sternotomy. A 2-0 silk suture was placed into the transverse sinus and tied to secure closure of both cavas and the pulmonary veins. A rapid cardiectomy was performed. The heart was im by The Society of Thoracic Surgeons /98/$19.00 Published by Elsevier Science Inc PII S (98)

2 Ann Thorac Surg LAWTON ET AL 1998;66: PINACIDIL IS EFFECTIVE IN BLOOD CARDIOPLEGIA 769 mersed in iced Plasmalyte (Baxter Healthcare Corp, Deerfield, IL) while the pulmonary artery was opened and a vent (polyethylene tubing, internal diameter 0.86 mm; Clay Adams, Parsippany, NJ) was placed into the left ventricle through a left atriotomy. The aorta was cannulated and coronary perfusion instituted through the Langendorff column. A fluid-filled latex balloon was placed into the left ventricle and secured with a pursestring suture in the mitral annulus for continuous pressure monitoring, as previously described [2]. An oximetric catheter (Fiberoptic Oximetric catheter; Abbott Laboratories, Chicago, IL) was inserted into the coronary sinus by a right atriotomy and secured. This catheter was connected to an oximeter (model 1270; Shaw Oximetrix, Mountain View, CA) and calibrated daily using a co-oximeter (model IL-482; Instrument Laboratories, Lexington, MA). Two right atrial electrodes were positioned for continuous pacing, and two left ventricular epicardial bipolar electrodes were positioned for continuous electrogram monitoring, as described previously [2]. The pressure and electrogram waveforms were digitized online using an AT-CODAS system (DATAQ Instruments, Akron, OH) at a sampling rate of 1,000 Hz. The heart was enclosed in an air bath surrounded by a glass-jacketed water beaker. Myocardial temperature was monitored throughout the experiment using a temperature probe (model 0112; Shiley Inc, Irvine, CA) placed in the right ventricle. Myocardial temperature was maintained at 37 C by adjusting the water bath (Model 71, Polyscience, Niles, IL) temperature. Coronary flow was monitored using an inline flow probe located directly above the aortic inflow (model 2N; Transonic Systems, Ithaca, NY) and an ultrasonic blood flow meter (model 101; Transonic Systems). Experimental Protocol Hearts were excluded from the study if they did not obtain a developed pressure of 80 mm Hg at an enddiastolic pressure of 10 mm Hg, or if the baseline developed pressure did not remain stable during the 30 minutes after instrumentation. After the 30-minute equilibration period, baseline electrolytes, hematocrit, hemoglobin, arterial blood oxygen saturation, and arterial blood gases were recorded for the support animal and any abnormalities were corrected. Left ventricular pressure-volume relationships were measured. A wide range of volumes were infused into the intraventricular latex balloon to generate enddiastolic pressures (EDP) of 0, 2.5, 5, 10, 15, and 20 mm Hg. After baseline data acquisition, fluid was adjusted in the latex balloon to obtain an EDP of 5 mm Hg before the ischemic period. Sixteen hearts were randomly assigned to receive either hyperpolarizing blood cardioplegia with pinacidil or depolarizing blood hyperkalemic cardioplegia for myocardial protection during a 30-minute period of global normothermic ischemia. Hyperpolarizing blood cardioplegia consisted of Krebs-Henseleit solution (in mmol/l distilled water: NaCl, 118.5; NaHCO 3, 25.0; KCl, 3.2; Table 1. Cardioplegic Solutions a Variable Pinacidil Hyperkalemic ph po pco b Hct Osmolarity a Values are represented as mean standard error of the mean. b p versus pinacidil. Hct hematocrit; PCO 2 carbon dioxide tension; PO 2 oxygen tension. MgSO 4, 1.2; KH 2 PO 4, 1.2; glucose, 5.5; and CaCl 2, 2.5) with pinacidil (50 mol/l, n 8) in a 1:2 mixture with rabbit blood. Depolarizing blood cardioplegia consisted of St. Thomas solution (n 8) in a 1:2 concentration with rabbit blood, with a final concentration of 20 mmol/l KCl. Rabbit blood was obtained at the time of cardiectomy from the donor rabbit. The ph, oxygen tension, carbon dioxide tension, and hematocrit of the cardioplegia solutions were similar and ranged between 7.45 and 7.66, 98 and 230, 18 and 34, and 18 and 25, respectively (Table 1). Pinacidil was provided by Leo Pharmaceuticals, Ballerup, Denmark. St. Thomas solution (in mmol/l: NaCl, 110.0; CaCl 2, 1.6; MgCl 2, 16.0; and KCl, 16.0) was provided by Abbott Laboratories (North Chicago, IL). Sodium bicarbonate (8.4%) was added to the St. Thomas solution (0.5 ml/50 ml) to correct the ph to 7.8. Heparin (12.5 U/mL) was added to each of the cardioplegia solutions. At the start of the ischemic period, the Langendorff perfusion column was clamped and 50 ml of normothermic (37 C) blood cardioplegia was infused from a height of 80 cm H 2 O through a separate column. The cardioplegic effluent was collected and discarded. Both the time until cessation of mechanical activity and the time until electrical quiescence were recorded. Cessation of mechanical activity was defined as the absence of a developed pressure. After 30 minutes of normothermic global ischemia, the Langendorff column was unclamped and the heart reperfused for 30 minutes. If ventricular fibrillation persisted after reperfusion, the heart was defibrillated (Model K84, Electrodyne Co., Inc., Westwood, MA). Electrolytes, hematocrit, hemoglobin, arterial blood oxygen saturation, and arterial blood gas measurements of the support animal were repeated to ensure stability. After 30 minutes of reperfusion, data were collected at the identical balloon volumes used during baseline preischemic data acquisition. At the conclusion of the experiment, the left ventricle, together with the septum and the right ventricular free wall, were separated, blotted, and weighed to obtain wet weight. The samples were dried until a constant dry weight was achieved. Percent tissue water (%TW) was determined using the following equation: %TW (wet weight dry weight)/wet weight. Myocardial Oxygen Consumption The coronary sinus venous saturation was recorded and myocardial oxygen consumption was calculated at each

3 770 LAWTON ET AL Ann Thorac Surg PINACIDIL IS EFFECTIVE IN BLOOD CARDIOPLEGIA 1998;66: EDP during baseline data acquisition, reperfusion, and postischemic data acquisition. Total myocardial oxygen consumption (ml/100 g myocardium/beat) was calculated as the product of coronary blood flow and the arteriovenous oxygen content difference and normalized for heart weight and heart rate [11]. Left ventricular thebesian flow was excluded because it was negligible in this model [12]. Because the right ventricle remained vented and unloaded throughout the experiment, the oxygen consumption of the right ventricle was considered to be minimal and constant [12]. All animals received humane care in Association for Assessment and Accreditation of Laboratory Animal Care-accredited (#00036), United States Department of Agriculture-registered (#52-R-007) facilities in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 85-23, revised 1985). Data Analysis Left ventricular systolic and diastolic pressures were determined from the digitized data files using software developed in our laboratory. END-SYSTOLIC PRESSURE. The end-systolic pressure (ESP) of a beat was defined as the maximum point of the digitized pressure waveform. The average ESP was calculated by averaging the ESPs of 10 beats. Average ESPs were obtained for each balloon volume at baseline and postreperfusion. The ESP versus balloon volume data for baseline and postreperfusion data were fitted to the linear ESP-volume relationship below using a leastsquares linear regression algorithm: ESP E max (BV) k, where E max is the slope, k is the y-intercept, and BV is the balloon volume. END-DIASTOLIC PRESSURE. The EDP of a beat was defined as the point at which the increase in slope of the pressure waveform exceeded a threshold of 0.5 mm Hg/ms. This was confirmed visually for each beat. The average EDP was calculated by averaging the EDPs of 10 beats, and was obtained for each balloon volume at baseline and postreperfusion. The EDP versus balloon volume data were fitted to the linear EDP-volume relationship below using a leastsquares linear regression algorithm: EDP m (BV BV 0, where m is the slope and BV 0 is the balloon volume corresponding to an EDP of zero, or the x-intercept [13]. The mean linear regression coefficient for the diastolic pressurevolume curves was and for the hyperkalemic and the pinacidil groups, respectively. Thus, a linear representation of the diastolic pressure-volume relationships was appropriate over the limited range of end-diastolic volumes examined in this model [14]. DEVELOPED PRESSURE. The left ventricular developed pressure was obtained by subtracting the EDP from the ESP for each data point. An average of 10 data points was used for each balloon volume. The developed pressure versus balloon volume data for baseline and postreperfusion were fitted to a linear pressure-volume relationship using the following linear regression algorithm: DP ESP EDP (E max BV k) m (BV BV 0 ), where DP is the developed pressure. PERCENT RECOVERY OF DEVELOPED PRESSURE. The percent recovery of developed pressure was calculated as the percentage of the post-reperfusion average developed pressure to the baseline average developed pressure at the same balloon volume. This was calculated for each of the postreperfusion matched balloon volumes. The average percent recovery of developed pressure was the integral of change in developed pressure over the change in balloon volume and was obtained using the trapezoidal rule [15] and the following equation: Bvmin Average % Rec DP DP 100 BV max DP postreperfusion d BV/(Bv DP baseline max BV min, where % Rec DP is the percent recovery of developed pressure, BV max is the maximum postreperfusion matched balloon volume, and BV min is the minimum postreperfusion matched balloon volume [3]. Statistical Analysis Values are represented as the mean standard error of the mean. Analysis of variance (or Kruskall-Wallis when appropriate) was used for multiple comparisons of means with a Dunnett s test for individual comparisons. A Fisher s exact test was used to compare mutually exclusive data where appropriate. A Student s t test (or Mann-Whitney test when appropriate) was used to compare two means. A paired Student s t test was used to compare means before and after an intervention. Differences were considered statistically significant when p was less than Statistical analysis was performed using Sigma Stat (version 1.01; Jandel Corp, San Rafael, CA). Results Cardioplegia Delivery and the Development of Electromechanical Arrest The mean time to infuse the blood cardioplegia solution was minutes for the hyperkalemic group and minutes for the pinacidil group (not significant). Electrical and mechanical arrest occurred most rapidly in the hyperkalemic group ( and minutes). This was significantly shorter than the time until electrical and mechanical arrest in the pinacidil group ( and minutes; p 0.05). Reperfusion Arrhythmias Upon release of the Langendorff column clamp, no hearts protected with hyperkalemic blood cardioplegia underwent ventricular fibrillation. However, 50% of the hearts in the pinacidil group fibrillated upon reperfusion. This

4 Ann Thorac Surg LAWTON ET AL 1998;66: PINACIDIL IS EFFECTIVE IN BLOOD CARDIOPLEGIA 771 Table 2. Postischemic Recovery of Systolic and Diastolic Function a Cardioplegia % Recovery of Developed Pressure Baseline Slope b (mm Hg/mL) Reperfusion Slope (mm Hg/mL) Hyperkalemic c 50 mol/l pinacidil a Data are represented as mean standard error the mean. b Slope (m) is the slope of y m (BV BV 0 ), where BV balloon volume and BV 0 the balloon volume corresponding to an end-diastolic pressure of zero. c p versus baseline value. difference was not statistically significant (p 0.077). After initial cardioversion, all hearts in the pinacidil group remained in normal sinus rhythm for the remainder of the reperfusion period. Postischemic Recovery of Systolic and Diastolic Function After 30 minutes of reperfusion, the hearts protected with hyperkalemic blood cardioplegia recovered a mean 52.3% 5.9% of their preischemic developed pressure. The hearts protected with pinacidil blood cardioplegia developed a mean 52.8% 6.9% of their preischemic developed pressure (not significant) (Table 2). The mean baseline slopes of the linear diastolic pressure-volume relationships were not different between groups (Table 2). After ischemia and reperfusion, the hearts in the hyperkalemic group had a significant increase in the mean slope of the linear diastolic pressurevolume relationships (p 0.001). In contrast, the mean slope of the linear diastolic pressure-volume relationships for the pinacidil group did not significantly increase above its baseline value (p 0.068). Reperfusion Coronary Blood Flow The mean baseline preischemic coronary blood flow before ischemia at an EDP of 5 mm Hg was not significantly different between groups (Fig 1). Upon reperfusion, the coronary blood flow in the hyperkalemic group increased significantly above baseline level for 1 minute and then the coronary blood flow declined until it reached a level significantly below baseline level after 6 minutes of reperfusion. The coronary blood flow remained significantly below baseline level until 25 minutes of reperfusion, when it returned to baseline levels. The coronary blood flow in the pinacidil group also increased above baseline level upon reperfusion. However, this increase continued until 11 minutes of reperfusion, when the coronary blood flow returned to near baseline levels. Between 1 and 25 minutes of reperfusion, the coronary blood flow in the pinacidil group remained significantly higher than that in the hyperkalemic group. Fig 1. Reperfusion coronary blood flow is represented versus time. Time 5 is baseline, time 0 is reperfusion, time 5 is 5 minutes after reperfusion, and so on. Values are represented as mean standard error of the mean (*p 0.05 versus St. Thomas by analysis of variance.) Reperfusion Myocardial Oxygen Consumption The baseline preischemic myocardial oxygen consumption was not different between groups (Fig 2). The reperfusion myocardial oxygen consumption in the hyperkalemic group remained near baseline level for the entire reperfusion period. The reperfusion myocardial oxygen consumption in the pinacidil group increased significantly above that in the hyperkalemic group after 2 minutes of reperfusion, and this persisted until 20 minutes of reperfusion. Myocardial Tissue Water The mean percent tissue water after reperfusion in the hyperkalemic group was significantly lower ( ) than that in the pinacidil group ( ; p 0.05). Comment Traditional hyperkalemic cardioplegia arrests the myocyte at depolarized membrane potentials. At these potentials, energy-consuming ion transport pumps are activated and these harmful transmembrane ion fluxes can exacerbate ischemic injury. To overcome the shortcomings of hyperkalemic cardioplegia, numerous adjuncts Fig 2. Myocardial oxygen consumption (MVO2) is represented versus time. Time 0 is baseline, time 1 is 1 minute after reperfusion, and so on. Values are represented as mean standard error of the mean (*p 0.05 versus St. Thomas by analysis of variance.)

5 772 LAWTON ET AL Ann Thorac Surg PINACIDIL IS EFFECTIVE IN BLOOD CARDIOPLEGIA 1998;66: have been developed to improve myocardial protection, including hypothermia, blood-enriched solutions, and substrate enhancement. The pharmacologic opening of ATP potassium channels arrests the cell close to its resting membrane potential (hyperpolarized arrest), which approximates the natural resting state of the myocyte. At this membrane potential, transmembrane ion gradients are largely balanced and net ion flux is minimal. Theoretically, this provides a more physiologic environment in which to provide ischemic protection. This may make potassium channel openers more ideal cardioplegic agents than traditional hyperkalemic solutions. Potassium channel openers in crystalloid solutions have provided superior myocardial protection, compared with hyperkalemic cardioplegia [3]. However, several investigators have documented the benefits of blood cardioplegia in both experimental models and clinical trials [10, 16, 17]. This study investigated the effectiveness and the characteristics of the PCO pinacidil as a cardioplegic agent in warm blood cardioplegia in an isolated blood-perfused rabbit heart model. Development of Electromechanical Arrest Hyperkalemic blood cardioplegia provided a more rapid electrical and mechanical arrest than pinacidil blood cardioplegia. This is similar to our previous results with crystalloid PCO cardioplegia [2, 3]. The equivalent recovery of systolic function between the two groups indicates that pinacidil was able to provide comparable protection despite prolonged mechanical and electrical activity. Reperfusion Arrhythmias There was an increased, although not statistically significant, incidence of ventricular fibrillation with pinacidil in this study. Our previous experiments using crystalloid cardioplegia demonstrated a statistically significant increased incidence of ventricular fibrillation with PCOs [2]. Further studies are needed to characterize this proarrhythmic trend with PCOs in blood cardioplegia. Recovery of Systolic Function There was no statistically significant difference between the percent recovery of developed pressure between the two groups, indicating equivalent myocardial protection. This is in contrast to the results of other investigators using cold blood pinacidil cardioplegia [1]. These investigators noted superior myocardial protection with cold blood pinacidil (200 mol/l) cardioplegia when compared with hyperkalemic cardioplegia in an intact porcine model [1]. This difference may be related to a species difference in the response to myocardial ischemia, or perhaps to the protective effect of hypothermia in overcoming the increased oxygen consumption after ischemia with pinacidil. Recovery of Diastolic Function The hearts in the hyperkalemic cardioplegia group had evidence of a decrease in ventricular compliance, as the slopes of the diastolic pressure-volume relationships significantly increased after ischemia. The hearts in the pinacidil group also sustained a diastolic injury; however, this was not statistically significant. This suggests superior diastolic protection by the pinacidil group. Reperfusion Coronary Blood Flow and Myocardial Oxygen Consumption Upon reperfusion, hearts in both cardioplegia groups demonstrated an increase in coronary blood flow compared with baseline, consistent with a postischemic hyperemic response. After reperfusion, however, the coronary blood flow in the pinacidil group remained significantly higher than that in the hyperkalemic group for 25 minutes of reperfusion. This is similar to our previous findings with crystalloid cardioplegia, and to the findings of other investigators using PCOs [2, 18 20]. Pinacidil is a known vasodilator, and the increased coronary blood flow may represent a prolonged vasodilatory effect as the drug washes out of the coronary circulation during reperfusion. It is unlikely, however, that the drug remained in the coronary vasculature for 25 minutes at a flow rate of to ml/min. The increased coronary blood flow in the pinacidil group may be reflective of an increased sensitivity of the coronary resistance vessels to metabolic vasodilators or an increased production of vasodilator messengers, such as adenosine during ischemia [11]. This would, however, suggest less effective myocardial protection during ischemia. This would be supported by the elevated myocardial oxygen consumption during reperfusion in this group. An increased energy demand upon reperfusion would result in an increased supply (coronary blood flow). This, however, is not consistent with the equivalent recovery of developed pressure in the pinacidil group. The elevated reperfusion myocardial oxygen consumption in the pinacidil group, compared with the hyperkalemic group, is similar to our previous finding in crystalloid cardioplegia in this model [21]. This elevated myocardial oxygen demand suggests that a higher oxygen debt was incurred during ischemia in the pinacidil group that had to be repaid during reperfusion, to result in equivalent recovery of function [22]. The elevated myocardial oxygen consumption in the pinacidil group could also be explained by the Gregg phenomenon, in which the increased coronary blood flow resulted in a change in the myocardial fibers, thus affecting (increasing) their contractility and oxygen consumption [23]. In addition, it has been suggested that a decrease in extravascular compressive forces (such as edema) is associated with improved coronary blood flow rate [11]. However, this is not consistent with our findings, as both postischemic tissue water (myocardial edema) and coronary blood flow were elevated in the pinacidil group. It is more likely, however, that the increased coronary blood flow during reperfusion in the pinacidil group resulted in an elevated level of myocardial edema by increasing hydrostatic pressure, as has been proposed by other investigators [24]. Determining which came first (the increase in reperfusion coronary blood flow or the increase in percent tissue water) in this study was impos-

6 Ann Thorac Surg LAWTON ET AL 1998;66: PINACIDIL IS EFFECTIVE IN BLOOD CARDIOPLEGIA 773 sible to determine because the myocardial sample was obtained at the end of reperfusion. Further research is needed to delineate the etiology of the increased coronary blood flow and myocardial oxygen consumption during reperfusion associated with PCOs. Myocardial Tissue Water The elevated percent of tissue water in the pinacidil group suggests an increased level of myocardial edema after ischemia and reperfusion. This is consistent with previous work using the PCO aprikalim as a crystalloid cardioplegic agent in this model [3]. The possible reasons for this were discussed previously. Despite the increased percent of tissue water, pinacidil blood cardioplegia was able to provide systolic protection and diastolic protection comparable to hyperkalemic blood cardioplegia in this model. The difference in percent tissue water between the groups was small and did not appear to be functionally significant. Shortcomings of the Model The advantages and disadvantages of this model have been described previously [2]. An isolated heart model lacks the neurohumoral, structural, and vascular effects seen in intact preparations. The blood-perfused isolated rabbit heart Langendorff model has advantages over crystalloid perfusion, including a higher oncotic pressure, improved oxygen carrying and buffering capacity, free radical scavengers, and improved capillary flow distribution [8]. However, although this model offers a closer approximation of the clinical scenario than a crystalloid-perfused preparation, care should be taken in extrapolating these results to the clinical setting. In vivo studies are clearly needed to establish the feasibility of PCO cardioplegia. Summary Hyperpolarizing blood cardioplegia containing pinacidil (50 mol/l) provides equivalent systolic and diastolic myocardial protection to hyperkalemic blood cardioplegia during normothermic arrest in the isolated, bloodperfused rabbit heart model. However, the associated elevated tissue water, the prolonged mechanical and electrical activity, and the elevated myocardial oxygen demand upon reperfusion are worrisome and warrant further investigation. We gratefully acknowledge the help of Luke Wolfe for statistical analysis and Cynthia T. Allen and Nancy N. Lee for technical assistance, and the generous donation of pinacidil by Leo Pharmaceuticals, Denmark. This work has been supported by a National Institutes of Health National Research Service Award grant HL (J.S.L., R.J.D.) and National Institutes of Health R01 HL51032 (R.J.D.). References 1. Kirvaitis RJ, Krukenkamp IB, Bukhari EA, Gaudette GR, Miyatake T, Levitsky S. 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