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J Thorac Cardiovasc Surg 1995;110:1083-1095
© 1995 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Richmond, Va.
Supported by National Institutes of Health National Research Service Award grant 08929-01 (S.M., R.J.D.), American Heart Association grant VA-93-G-12 (R.J.D.), and an American College of Surgeons Faculty Fellowship (R.J.D.).
Received for publication March 20, 1995. Accepted for publication April 12, 1995. Address for reprints: Ralph J. Damiano, Jr., MD, Chief, Surgical Electrophysiology, Division of Cardiothoracic Surgery, Medical College of Virginia, 1200 E. Broad St., P.O. Box 980068, Richmond, VA 23298-0068.
Abstract
Cardioplegic solutions that arrest the heart at or near the resting membrane potential may provide better myocardial protection than standard depolarizing hyperkalemic cardioplegia by reducing both metabolic demand and harmful transmembrane ion fluxes. This hypothesis was investigated in an isolated, blood-perfused, rabbit heart Langendorff model during 30 minutes of normothermic global ischemia. Hyperpolarized cardiac arrest induced by aprikalim, an opener of adenosine triphosphate–dependent potassium channels, was compared with hyperkalemic depolarized arrest and with unprotected global ischemia. Left ventricular pressure was recorded over a wide range of balloon volumes before ischemia and 30 minutes after reperfusion. End-diastolic pressure versus balloon volume data were fitted to a two-coefficient exponential relationship. Changes in the diastolic compliance of the left ventricle were assessed by comparison of preischemic and postischemic coefficients within each cardioplegia group. Postischemic recovery of developed pressure was used to assess changes in left ventricular systolic function. The tissue water content of each heart was also determined. Myocardial protection with aprikalim resulted in better postischemic recovery of developed pressure (90%±9%) than either protection with hyperkalemic cardioplegia (73%±11%) or no protection (62%±9%). Myocardial tissue water content in hearts protected with hyperkalemic cardioplegia (77.4%±1.4%) was less than the tissue water content of either unprotected hearts (79.4%±1.2%) or hearts protected with aprikalim (78.7%±0.9%). Despite these differences, neither hyperkalemic cardioplegia (p = 0.15) nor aprikalim cardioplegia (p = 0.30) was associated with a significant postischemic decrease in ventricular compliance. By contrast, unprotected global ischemia was associated with a significant decrease in ventricular compliance (p <0.001). (J THORAC CARDIOVASC SURG 1995;110:1083-95)
The goal of myocardial protection strategies used during elective cardiac arrest is the rapid induction of electromechanical asystole and the maintenance of the myocardium in a state of minimal metabolic requirement. 
1 Most current techniques of myocardial preservation use hyperkalemic solutions to achieve a rapid electromechanical standstill. 1 However, numerous detrimental consequences are associated with the membrane depolarization caused by hyperkalemic cardioplegia. 2-4 Abnormal intracellular accumulation of specific ions at depolarized membrane potentials can have deleterious physiologic effects. Progressive membrane depolarization results in a spontaneous influx of sodium that is manifested as the sodium "window current." 5,6 Intracellular calcium accumulation results from the exchange of intracellular sodium for extracellular calcium via the Na+ -Ca+2 exchanger. 5,6 There also is a concomitant influx of calcium through the calcium "window current" and leakage of calcium from the sarcoplasmic reticulum. 6,7 The resultant myocardial calcium overload has been implicated in ischemia/reperfusion injury and myocardial stunning, particularly after surgical global ischemia during depolarized cardiac arrest. 2-4 These ion fluxes also impose a metabolic burden on the myocyte by providing substrate for the activation of the energy-dependent ion pumps, including the sarcolemmal sodium-potassium and calcium adenosinetriphosphatases (ATPases). 3
At the normal resting membrane potential of the myocyte netintracellular ion flux is minimal because the transmembrane ion concentration gradients are largely balanced and few voltage-gated ion channels are open. 8 Consequently, energy-dependent ion pump activity, and thus metabolic demand, is minimal. The feasibility of maintaining the membrane potential of the myocyte at or near the normal resting membrane potential during global ischemia (termed hyperpolarized cardiac arrest) has been documented previously. 9-11 Hyperpolarized cardiac arrest may be more cardioprotective than depolarized arrest, both by optimizing the metabolic state of the myocardium and by avoiding the injurious ion fluxes that occur during depolarized arrest. 12,13
Previously, our laboratory has demonstrated the feasibility of hyperpolarized cardiac arrest induced by aprikalim, an agent that selectively opens ATP-dependent potassium channels (KATP channels). 14 The opening of myocardial KATP channels activates an outward potassium current that results in cell membrane hyperpolarization and a dramatic shortening of the action potential duration. 15 This decrease in the action potential duration and the accompanying decrease in the plateau phase of the action potential lead to a reduction in calcium influx and ultimately to contractile failure. 14,16,17 Numerous studies have documented the cardioprotective effects of potassium-channel openers, under both globally 14,17 andregionally 18,19 ischemic myocardial conditions. The administration of potassium-channel openers has also been shown to ameliorate myocardial stunning. 20,21 Furthermore,opening of the KATP channel has been implicated in the mechanism of ischemic preconditioning. 22,23
Previous work from our laboratory has used pharmacologic dosages of aprikalim (RP 52891, Rhone-Poulenc Rorer, Antony, France) to produce hyperpolarized cardiac arrest during normothermic global ischemia in a Krebs-Henseleit solution–perfused rabbit heart Langendorff model. 14 In this model, postischemic recovery of developed pressure in hearts protected with aprikalim was greater than recovery in hearts protected with hyperkalemic cardioplegia. 14 However, crystalloid perfusate has significant drawbacks that make it a less suitable medium for the investigation of ischemia/reperfusion injury. Specific adverse effects attributed to crystalloid perfusate include increased myocardial edema formation, 24 adecreased capacity to buffer myocardial tissue pH, 25-27 exacerbation of ischemia/reperfusion injury, 28 andpotentially adverse patterns of perfusate rheology. 27,29 These problems raise questions about the clinical relevance of crystalloid-perfused models of ischemia/reperfusion injury. To address this issue, the abilities of hyperpolarizing aprikalim cardioplegia and hyperkalemic, depolarizing cardioplegia to protect the myocardium during normothermic, global ischemia were compared in a more clinically relevant, blood-perfused, parabiotic rabbit heart Langendorff model.
METHODS
Adult New Zealand White rabbits of either sex, weighing 3 to 4 kg, were used in this study. All animals received humane care 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 No. 86-23, revised 1985).
Experimental preparation
Preparation of the support animal
The support rabbit was anesthetized by intramuscular injection of acepromazine (1 mg/kg), xylazine (5 mg/kg), and ketamine (40 mg/kg). The level of anesthesia was monitored continuously and was supplemented as needed.
A tracheostomy was performed, an endotracheal tube inserted, and mechanical ventilation begun with 100% oxygen (ventilator model 683, Harvard Apparatus, Dover, Mass.). Serial arterial blood gas determinations were made and ventilator settings were adjusted to maintain arterial pH between 7.3 and 7.5, carbon dioxide tension between 35 and 45 torr, and oxygen tension greater than 200 torr. Heparin (2500 U) was given through an ear vein. The right femoral artery was cannulated and continuous invasive monitoring of blood pressure was begun. A mean arterial pressure of 60 mm Hg was maintained by transfusion of blood or infusion of Plasma-Lyte solution (in millimoles per liter: Na 140, K 5, Mg 3, Cl 98, acetate 27, gluconate 23) as dictated by the results of serial hematocrit determinations. Blood for transfusion was obtained at the time of cardiectomy from the thoracic cavity of the rabbit providing the isolated heart.
The left internal jugular vein and the left carotid artery were cannulated. An extracorporeal circuit was established by continuous withdrawal of blood from the carotid artery cannula (Fig. 1). The blood was pumped to an 80 cm high modified Langendorff perfusion column. Effluent blood from the Langendorff apparatus was reinfused into the support rabbit via the venous cannula, thus completing the circuit. Indomethacin (1 mg/kg) was administered to the support rabbit to maintain stability of the preparation. 30
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The rabbit providing the isolated heart was anesthetized, intubated, placed on ventilator support, and heparinized in an identical manner to that used in the support rabbit. The heart was excised through a median sternotomy and the blood that drained into the thoracic cavity was collected for transfusion into the support rabbit. The aorta was cannulated and the heart was suspended from a modified Langendorff apparatus (Fig. 1). Retrograde aortic perfusion with support-animal blood from the column was instituted. Perfusate, bath, and myocardial temperatures were monitored continuously with thermocouples (model DP29-0031-001A, Shiley, Inc., Irvine, Calif.). The column and bath water-jacket temperatures were adjusted to maintain myocardial temperature between 37.0° C and 37.5° C (model 9010, Fisher Scientific, Pittsburgh, Pa.).
A latex balloon was placed through the mitral valve into the left ventricle. The balloon was secured in place with a purse-string suture in the mitral valve anulus. The balloon was connected via fluid-filled polyethylene tubing to a pressure transducer (model P231D, Gould, Cleveland, Ohio) and amplifier (Gould model 13-4615-50). The zero pressure reference was set at the level of the aortic valve. The pressure waveform was digitized on-line at a sampling rate of 1000 Hz (AT-CODAS, DATAQ Instruments, Akron, Ohio) and displayed on a personal computer (Z-386/20, Zenith, Glenview, Ill.).
Two needle electrodes (Grass Instruments, Quincy, Mass.) were fixed to the right atrial appendage to atrially pace the heart using a pulse generator (model 5320, Medtronic, Inc., Minneapolis, Minn.). Two additional Grass electrodes were applied to the epicardium of the left ventricular free wall to monitor a bipolar ventricular electrogram. The electrogram signal was boosted with an isolated preamplifier (Gould model 11-G5407-58), which was connected to a universal amplifier (Gould model 13-4615-58) and filtered with a low pass frequency of 0.05 Hz and a high pass frequency of 1 kHz. The electrograms were digitized at a sampling rate of 1000 Hz and displayed in real time on the Zenith computer.
Experimental protocol
After instrumentation, the heart was allowed to recover until stable end-diastolic and peak systolic left ventricular pressures were established (20 to 30 minutes). Control data were acquired. Intracavitary left ventricular pressure waveforms and left ventricular bipolar electrograms were recorded over a range of nine balloon volumes corresponding to nine randomly ordered left ventricular end-diastolic pressures (0, 2.5, 5, 7.5, 10, 12.5, 15, 20, and 25 mm Hg) at a fixed, paced rate of 150 to 180 beats/min. The analog signals were recorded on VHS videotape with a tape recorder (TEAC model XR-70, Tokyo, Japan) and also were digitized on-line for subsequent analysis.
After control data acquisition, hearts were randomized to receive one of three types of myocardial protection concomitant with the onset of a 30-minute period of normothermic, global, surgical ischemia: (1) no cardioplegia (control group), (2) depolarizing cardioplegia, or (3) hyperpolarizing cardioplegia. The retrograde perfusion column was clamped and either 40 ml of normothermic cardioplegic solution was delivered into the aortic root via a separate 80 cm perfusion column (hyperpolarizing or depolarizing cardioplegia groups) or no cardioplegic solution was delivered (control group). Depolarizing cardioplegic solution was made by adding a sufficient quantity of potassium chloride to Krebs-Henseleit solution (in millimoles per liter: NaCl 118.5, NaHCO3 25, KCl 3.2, KH2PO4 1.2, MgSO4 1.2, CaCl22.5, and glucose 5.5) to obtain a final potassium ion concentration of 20 mmol/L. Hyperpolarizing cardioplegic solution was made by adding aprikalim (RP 52891) to the Krebs-Henseleit solution. The aprikalim concentration used was 100 µmol/L. This was previously shown to be the concentration that maximized the percentage recovery of developed pressure after 20 minutes of global ischemia in a Krebs-Henseleit solution–perfused isolated rabbit heart Langendorff model. 14 The difference in osmolality between the cardioplegic solutions was eliminated by the addition of a sufficient quantity of sucrose to the hyperpolarizing cardioplegic solution to bring its osmolality into the 305 to 310 mOsm/kg range as measured by a freezing-point depression osmometer (Osmette A model 5002, Precision Systems, Inc., Natick, Mass.).
After 30 minutes of global ischemia, the perfusion column was unclamped and the heart was reperfused with support animal blood. Postreperfusion data were collected after 30 minutes of reperfusion. Intracavitary left ventricular pressure waveforms and left ventricular bipolar electrograms were recorded over a range of nine randomly ordered balloon volumes at the identical paced rate used during control data acquisition. Balloon volumes were identical to those used during control data acquisition when feasible. When left ventricular compliance changes precluded this, the number of matching volumes examined was maximized. However, a left ventricular end-diastolic pressure of 40 mm Hg was not exceeded. After postreperfusion data were acquired, a portion of the left ventricular myocardium was excised, weighed, and then dried in a 70° C oven until a constant dry weight was reached. Tissue water percentage was expressed as the difference between wet and dry weights of the sample divided by the wet weight of the sample.
Data analysis
Computer software was developed to standardize the analysis of the digitized pressure waveforms.
End-systolic pressure
The end-systolic pressure (ESP) of a beat was defined as the maximum of the three-point moving average of the digitized pressure waveform. The average ESP was calculated by averaging the ESP values of 10 consecutive beats. Average ESP values were obtained in this manner for each of the 18 (9 control and 9 postreperfusion) balloon volumes (Vol). The ESP versus balloon volume data were fitted to a linear ESP-volume relationship (ESPVR) (equation 1), with a least-squares linear regression algorithm.
ESP = Emax x Vol + K (1)
where Emax is the slope of the linear ESPVR and K is the Y-axis intercept of the linear ESPVR. Control slope (Emax-c) and intercept (Kc) were obtained from the control ESP-volume data. Postreperfusion slope (Emax-pr) and intercept (Kpr) were obtained from the postreperfusion ESP-volume data.
End-diastolic pressure. The end-diastolic pressure (EDP) of a beat was defined as the pressure at which the first derivative of the digitized pressure waveform with respect to time first exceeded 0.25 mm Hg/msec. The instantaneous first derivative of the digitized pressure waveform with respect to time was approximated from the pressure waveforms digitized at a sampling rate of 1 kHz. Its value at any particular time was defined as the slope of the line obtained by a linear least-squares regression of five consecutive pressures centered about that particular time. The average EDP was calculated by averaging the EDP values of 10 consecutive beats. Average EDP values were obtained in this manner for each of the 18 (9 control and 9 postreperfusion) balloon volumes (Vol). The EDP versus balloon volume data were fitted to a simple exponential EDP-volume relationship (EDPVR) 31 (equation 2), with a least-squares nonlinear regression algorithm.
EDP = 
x eß x Vol (2)
where and ßare the nonlinear coefficients of the exponential EDP-volume relationship and e is the base of natural logarithms. Control (c) and ß(ßc) were obtained from the control EDP-volume data. Postreperfusion (pr) and ß(ßpr) were obtained from the postreperfusion EDP-volume data.
Developed pressure. Developed pressure was defined as the difference between the ESP and EDP. The average developed pressure was calculated by averaging the developed pressure values of 10 consecutive beats. Average developed pressure values were obtained in this manner for each of the 18 (9 control and 9 postreperfusion) balloon volumes (Vol). The developed pressure (DP) versus balloon volume data were fitted to a nonlinear developed pressure–volume relationship (equation 3), obtained by subtracting equation 2 from equation 1.
DP = ESP - EDP = Emax x Vol + K - x eß x Vol (3)
Control developed pressure–volume data were fitted using the values of Emax-c and Kc (determined by linear regression of the control ESP-volume data) and c and ßc (determined by nonlinear regression of the control EDP-volume data) to equation 4. Postreperfusion developed pressure–volume data were fitted using the values of Emax-pr and Kpr and pr and ßpr to equation 5.
DP = Emax-c x Vol + Kc -c x eßc xVol (4)
DP = Emax-pr x Vol +Kpr - pr x eßpr x Vol (5)
Postischemic recovery of developed pressure. The postischemic recovery of developed pressure (%Recov) was calculated as the ratio of the postreperfusion average developed pressure to the control average developed pressure at the same balloon volume, expressed as a percentage. Percentage recoveries were obtained in this manner for each of the nine postreperfusion balloon volumes that had an identically matching control balloon volume. The percentage recovery of developed pressure versus balloon volume data were fitted to a nonlinear relationship (equation 6), obtained by dividing equation 5 by equation 4 using the previously determined values of Emax-c, Emax-pr, Kc, Kpr, c, pr, ßc, and ßpr.
The average postischemic percentage recovery of developed pressure (Avg % Recov) was defined as the average value of the postischemic recovery of developed pressure function (equation 6) over the relevant balloon volume range and was formally calculated by equation 7.
where Vb is the maximum postreperfusion balloon volume and Va is the minimum postreperfusion balloon volume. Because the integrand in equation 7 cannot be formally integrated, the value of the definite integral was approximated by a numeric method (trapezoidal rule).
Statistical analysis
All cumulative results are expressed as the mean plus or minus the standard deviation. Analysis of variance was used for multiple comparisons. When either the equal variance criterion or normality criterion was not satisfied, the Kruskal-Wallis analysis of variance on ranks was used as a nonparametric alternative. When a significant F value was obtained, comparison between groups was made by a Student-Newman-Keuls post test. Preischemic and ßcoefficients of the exponential left ventricular EDP-balloon volume relationship were compared with the postischemic coefficients within each group by a method of multivariate analysis (Hotelling t2 test) to determine whether ventricular compliance had changed. The 
2 test was used to compare proportions among multiple groups.
A difference was considered statistically significant when p < 0.05.
RESULTS
Temporal aspects of the development of electromechanical quiescence
Eight hearts were subjected to 30 minutes of unprotected, normothermic global ischemia. Developed pressure fell to 10% and 0% of its prearrest value at 161 ± 40 and 387 ± 113 seconds, respectively, after the onset of ischemia (Table I). Electrical activity persisted throughout the 30-minute ischemic period in six of the eight hearts.
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Table I). With hyperkalemic cardioplegia, mechanical quiescence occurred six times faster and electrical quiescence occurred 17 times faster than with aprikalim cardioplegia. Delivery time for the hyperkalemic cardioplegic solution was not significantly different from the delivery time for aprikalim cardioplegic solution (p = 0.834).
Reperfusion ventricular arrythmias
Three hearts protected with hyperpolarizing cardioplegia and a single unprotected heart sustained ventricular fibrillation on reperfusion and were electrically converted to sinus rhythm (Table I). The remaining 20 hearts spontaneously achieved sinus rhythm after reperfusion. There was not a statistically significant difference in the occurrence of postreperfusion ventricular fibrillation among the three groups (p = 0.122).
Postischemic diastolic properties
Retrograde aortic perfusion was restored after 30 minutes of normothermic, global ischemia in all hearts. Balloon pressure rose from its preischemic value of 5 mm Hg in all hearts to 9 ± 5 mm Hg in hearts protected with depolarizing cardioplegia, to 7 ± 2 mm Hg in hearts protected with hyperpolarizing cardioplegia, and to 14 ± 4 mm Hg just before reperfusion in unprotected hearts (p < 0.05 versus depolarizing, hyperpolarizing cardioplegia).
After 30 minutes of reperfusion, postischemic and ßcoefficients of the exponential EDPVR (equation 2) were determined for each heart. Fig. 2, A,shows control and postischemic EDPVR curves from a single unprotected heart. The relatively large leftward displacement of the postischemic curve with respect to the preischemic curve seen in this heart was typical of all hearts of this group. Fig. 2, B,shows control and postischemic EDPVR curves from a single heart protected with hyperpolarizing cardioplegia. Neither hyperpolarizing cardioplegia nor depolarizing cardioplegia resulted in significant displacement of the postischemic EDPVR curve.
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and ßcoefficients demonstrated that the postischemic coefficients were significantly higher (p < 0.001) than the preischemic coefficients in unprotected hearts, which indicated the occurrence of a significant postischemic decrease in ventricular compliance in this group. Ventricular compliance did not change significantly in hearts protected with either depolarizing cardioplegia (p = 0.15) or hyperpolarizing cardioplegia (p = 0.30).
Postischemic systolic function
After 30 minutes of normothermic, global ischemia and 30 minutes of reperfusion, ventricular systolic function was assessed in each heart. Postischemic Emax and K coefficients of the linear ESPVR (equation 1) were determined for each heart in each group. Fig. 3, A, shows the control and postischemic ESPVR of a single unprotected heart.
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and ßcoefficients determined from the EDPVR and the Emax and K coefficients determined from the ESPVR. Fig. 3, B, shows the control and postischemic developed pressure–volume relationship of a single unprotected heart.
Percentage recovery of developed pressure versus balloon volume data from each heart were fitted to a relationship of the form of equation 6 using the previously determined values of , ß, Emax, and K. Fig. 3, C, shows the postischemic percentage recovery of developed pressure versus volume relationship of a single unprotected heart.
An average percentage recovery of developed pressure was computed for each heart using equation 7. The average percentage recoveries were 90% ± 9% in hearts protected with hyperpolarizing cardioplegia, 73% ± 11% in hearts protected with depolarizing cardioplegia, and 61% ± 9% in unprotected hearts (Fig. 4). Hyperpolarizing cardioplegia was significantly better than both depolarizing cardioplegia and unprotected global ischemia (p < 0.05).
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The cellular adaptation to ischemia
After the fast upstroke of the normal cardiac action potential, there is a delay before the activation of the delayed rectifier current, an outward potassium current that repolarizes the myocyte during the third phase of the cardiac action potential. 8 The length of the delay is largely responsible for the duration of the plateau phase and gives the cardiac action potential its characteristic shape and relatively long duration. 8 Profound shortening of the action potential duration occurs rapidly during myocardial ischemia, 32 anoxia, 33 and metabolic blockade. 34,35 An increase in a time-independent outward potassium current is responsible for initiating and maintaining action potential collapse under these conditions of impaired energy metabolism. 15,35-37 Collapse of the plateau phase of the cardiac action potential shortens the length of time that voltage-gated calcium channels are open and therefore decreases calcium influx, ultimately resulting in contractile failure. 33,36,37 This cascade of events is beneficial during ischemia because suspension of contractile activity decreases metabolic demand. 1 In addition, the reduction of calcium influx ameliorates the sequela of myocyte calcium overload, which has been implicated in the pathogenesis of ischemia/reperfusion injury. 2-4
A specific population of potassium channels inactivated by intracellular ATP (KATP channels) is responsible for the time-independent outward potassium current that shortens the action potential duration during ischemia. 15,16,38 These channels open during metabolic blockade, 34,35 anoxia, 33 and ischemia 32 and on exposure to specific potassium channel openers. 38,39
KATP channels are found in abundance on the cell membrane of the myocyte. 40 Open channels have a relativelyhigh single-channel conductance of 25 pS. 40,41 The kinetics of channel opening in myocytes is such that half-maximal closure of channels occurs at intracellular ATP concentrations in the range of 20 to 100 µmol/L. 35,40,42 However, the normal range of intracellular ATP concentrations is 5 to 10 mmol/L. Thus, under normal circumstances, virtually all myocyte KATP channels are closed. Because a high percentage of KATP channels remain closed even at submillimolar ATP levels, there was initially difficulty reconciling the role of KATP channels in action potential collapse, which occurs almost concomitantly with the onset of ischemia and before the development of a significant decline in the level of intracellular ATP. 43 However, because of the high membrane density ofKATPchannels, the action potential duration is exquisitely sensitive to channel opening and demonstrable action potential shortening will occur with nominal decreases in intracellular ATP levels. 40-42,44,45 Experimental evidence has verified theoretic considerations that predict that opening of less than 1% of all available channels would result in a 50% reduction of action potential duration. 40-42,44,45
Mechanism of potassium-channel opener–induced hyperpolarized arrest
Potassium-channel openers comprise a diverse group of chemical agents and include pinacidil, nicorandil, aprikalim, and cromakalim. 17,38-40 The interaction between potassium-channel openers, ATP, and KATP channels has been delineated in cardiac myocytes with use of the Hill model of channel opening. 36,37 Potassium-channel openers promote channelopening by causing an order of magnitude increase in ki, the ATP concentration resulting in half maximal closure of KATP channels, without changing the steepness of the channel opening versus ATP concentration relationship. 36 These observations suggest that potassium-channel openers competitively inhibit binding of ATP at the ATP binding site of the channel. 37
The putative mechanism of potassium-channel opener–induced hyperpolarized arrest is that pharmacologic dosages of potassium-channel openers (twice the maximal effective dose in single cell preparations) shift the ki for channel binding of ATP into the normal physiologic range of intracellular ATP concentrations. 14 This simulates a decrease in the effective ATP concentration at the intracellular face of the KATP channel and results in channel opening at normal intracellular ATP levels. This causes progressive shortening of the action potential duration and, ultimately, action potential collapse. Inexcitability and electromechanical quiescence result as the membrane voltage is stabilized at or near the reversal potential for potassium, as a direct consequence of the large increase in membrane permeability to potassium. Potassium-channel opener binding is reversible and on reperfusion the potassium-channel opener is washed out of the coronary circulation.
The feasibility of potassium-channel opener hyperpolarized arrest
Our laboratory has previously demonstrated the feasibility of myocardial protection with hyperpolarizing cardioplegic solution containing the potassium-channel opener, aprikalim. 14 These studies were performed in an isovolumic, Krebs-Henseleit solution–perfused, rabbit heart Langendorff model during a 20-minute interval of normothermic, global ischemia. In hearts protected with aprikalim cardioplegia, electromechanical standstill developed significantly more slowly than in hearts protected with standard, hyperkalemic, depolarizing cardioplegia. Nevertheless, postischemic recovery of developed pressure in hearts protected with aprikalim cardioplegia was superior to recovery in hearts protected with hyperkalemic cardioplegia. Furthermore, in contrast to results with hyperkalemic cardioplegia, use of aprikalim cardioplegia did not result in a postischemic increase in EDP. These results suggested that aprikalim could ameliorate ischemic/reperfusion injury. However, because of the disparity between the crystalloid-perfused model and the clinical situation, the clinical relevance of these findings remained uncertain. This present study was undertaken to verify these findings in a blood-perfused preparation.
Preservation of systolic function with potassium-channel opener–induced hyperpolarized arrest versus hyperkalemic depolarized arrest
Recovery of developed pressure after 30 minutes of global, normothermic ischemia in a parabiotic, blood-perfused Langendorff model was significantly better in hearts protected with hyperpolarizing cardioplegic solution containing aprikalim than in either unprotected hearts or hearts protected with hyperkalemic depolarizing cardioplegia. These data confirm our earlier results 14 and demonstrate that aprikalim cardioplegia is cardioprotective in a blood-perfused Langendorff model.
Preservation of diastolic function with potassium-channel opener–induced hyperpolarized arrest versus hyperkalemic depolarized arrest
Postischemic and ßcoefficients of the exponential EDP–balloon volume relationship did not differ significantly from their respective preischemic values either in hearts protected with hyperpolarizing cardioplegia or in hearts protected with depolarizing cardioplegia. Thus both types of cardioplegia were able to preserve left ventricular diastolic function despite the apparently small reduction of myocardial edema with hyperkalemic, depolarizing cardioplegia. As expected, a significant decrease in ventricular compliance occurred in unprotected hearts subjected to normothermic, global ischemia.
Cell swelling
Use of hyperkalemic cardioplegic solution was associated with a lower tissue water content (77.4%) than use of either unprotected ischemia (79.4%) or aprikalim cardioplegic solution (78.7%).
Although cell swelling is a recognized complication of hyperkalemic cardioplegia, 46 there was slightly more myocardial edema formation with aprikalim cardioplegia. The vasodilatory properties of potassium-channel openers 38 may play a role in increasing myocardial edema. Vasodilation of the resistance vessels of the coronary vasculature under conditions of a fixed perfusion column height will effect an increase in coronary flow rate, capillary hydrostatic pressure, and, consequently, myocardial edema formation. 47 Although delivery times of cardioplegic solutions did not differ between groups in this experiment, subsequent, unpublished data demonstrate that postreperfusion coronary flow rates (measured by an ultrasonic flow probe) in hearts protected with hyperpolarizing cardioplegia exceed the flow rates in hearts protected with hyperkalemic cardioplegia. Therefore aprikalim cardioplegia may predispose to the formation of myocardial edema during reperfusion.
Alternatively, the low tissue water content of hearts protected with hyperkalemic cardioplegia may be artifactual. The membrane depolarization associated with hyperkalemic cardioplegia results in incomplete sodium channel inactivation and intracellular sodium accumulation via the sodium "window current" during ischemia. 5,6 On reperfusion, the accumulated sodium is expelled by the Na+-K+-ATPase. The electrogenic nature of this pump causes a net efflux of water and results in a transient contraction of cell volume to less than the original control cell volume during reperfusion. This transient, ouabain-sensitive cell volume contraction on transfer from hyperkalemic cardioplegic solution to normokalemic solution has been documented in single rabbit ventricular myocytes. 48 Because there theoretically is less sodium accumulation during ischemia in hearts protected with hyperpolarizing cardioplegia, the rebound cell volume contraction on reperfusion may be smaller.
Because the left ventricular tissue water biopsy samples in this experiment were obtained after the acquisition of the postreperfusion data, it is conceivable that the observed tissue water content of hearts protected with hyperkalemic cardioplegia reflects the transient volume contracted state, rather than actual edema formation. Left ventricular biopsy samples obtained soon after reperfusion would more accurately reflect actual edema formation during ischemia. However, because of the small size of the rabbit heart, this was not feasible. Further work in a large-animal model will be needed to clarify this phenomenon.
Regardless of whether differences in myocardial edema formation between cardioplegia groups in this study were actual or artifactual, the small increase in observed tissue water content was not sufficient to alter the postischemic diastolic properties of the left ventricle in hearts protected with hyperpolarizing cardioplegia.
Temporal aspects of the development of electromechanical arrest with hyperpolarizing versus depolarizing cardioplegia
Ongoing electrical and mechanical activity accelerates the rate of energy consumption in the globally ischemic heart. 1 Therefore traditional cardioplegic solutions have been designed to rapidly induce electromechanical asystole to optimize myocardial protection. 1 In these experiments, mechanical and electrical quiescence developed more slowly in hearts protected with aprikalim cardioplegia than in hearts protected with hyperkalemic cardioplegia, despite comparable delivery times of the cardioplegic solutions.
In hyperkalemic, depolarized arrest, the cardioplegic solution raises the potassium ion concentration of the extracellular fluid, which leads to membrane depolarization, membrane inexcitability, and electrical and mechanical arrest. 1 Arrest is rapid because potassium ions must only permeate the extracellular space. By contrast, in potassium-channel opener–induced arrest, extreme shortening of the action potential duration as a consequence of KATP channel opening reduces calcium influx and results in contractile failure. 14 However, spikelike action potentials persist for several minutes after the development of mechanical arrest. 14,33-36
Despite the prolongation of mechanical and electrical activity, potassium-channel opener–induced arrest afforded significantly better preservation of left ventricular systolic function than protection with hyperkalemic cardioplegia in both our Krebs-Henseleit solution–perfused 14 and blood-perfused models. This suggests that the cardioprotective properties of potassium-channel openers are sufficient to offset the energy consumption associated with the electrical and mechanical work that occurs before quiescence in hearts protected with hyperpolarizing cardioplegia. Evidence obtained in an isolated rat heart model supports this hypothesis. 13 Basal myocardial oxygen consumption during nonischemic depolarized arrest has been shown to be 50% greater than the oxygen consumption during nonischemic hyperpolarized arrest in rat hearts. 13 This difference was attributed to increased myocardial wall tension caused by the increased intracellular calcium concentration in hearts undergoing depolarized arrest. The large decrease in basal energy consumption associated with nondepolarized arrest could easily compensate for the excess energy consumption associated with the persistent electrical activity, because the generation of electrical activity comprises less than 1% of the total basal oxygen consumption of the heart. 49
Proarrhythmic sequelae of potassium-channel openers
Potassium-channel openers increase the dispersion of refractoriness and shorten refractoriness in myocardial tissue. 50 They have been shown to be proarrhythmic for reentrant arrhythmias, which commonly occur in the setting of ischemia/reperfusion. 50 However, in this experiment, there was no statistically significant difference in the rates of occurrence of ventricular fibrillation on reperfusion among the three groups. Furthermore, arrhythmias occurred only on reperfusion, were easily cardioverted, and did not recur after initial cardioversion. Thus they did not present a significant experimental problem.
Vasodilatory effects of potassium-channel openers
Clinical use of potassium-channel opener–induced hyperpolarized arrest may be limited by the profound systemic vasodilation and hypotension that develop when pharmacologic dosages of potassium-channel openers are administered systemically. 38 This effect is a result of the presence of KATP channels in the smooth muscle of the arterial vasculature. 38 In this experiment, measures were taken to avoid circulation of the spent cardioplegic solution to the support rabbit. Similar precautions would need to be taken in the clinical setting.
Unfortunately, currently available potassium-channel openers are more potent openers of vascular smooth muscle KATP channels than of myocyte KATP channels. 38 This suggests the existence of tissue-specific differences in KATP channel structure. Once KATP channels are cloned and their primary, secondary, and tertiary structures elucidated, the differences between cardiac and vascular smooth muscle channels can potentially be exploited to develop cardioselective potassium-channel openers.
Advantages and disadvantages of the parabiotic, blood-perfused model
The main disadvantage of the parabiotic model is the loss of the ability to exert absolute control over the perfusate composition, as is possible in a crystalloid-perfused Langendorff model. In the parabiotic model, only a relative degree of control over the pH, hematocrit value, osmolality, and ion concentration is possible. Furthermore, temporal changes in circulating catecholamine levels in the support animal can confound evaluation of the postischemic functional recovery of the isolated heart. However, continuous, careful monitoring of the level of support-animal anesthesia will minimize fluctuations in support-animal catecholamine levels.
The more physiologic nature of the parabiotic, blood-perfused model favors its use over the crystalloid-perfused model. The blood-perfused model more closely resembles the clinical situation. Therefore results obtained from this model will assume greater clinical relevance than results obtained in a crystalloid-perfused model.
The absence of hemoglobin greatly limits the oxygen-carrying capacity of crystalloid solutions and necessitates supranormal coronary flow rates in crystalloid-perfused models to support normal cellular respiration. 24,51 As a consequence of the higher flow rates with crystalloid perfusate, capillary hydrostatic pressure will be elevated. 24,51 Furthermore, owing to an absence of plasma proteins, crystalloid perfusate exerts a lower oncotic pressure than blood perfusate. 52 The combination of a lower perfusate oncotic pressure and elevated capillary hydrostatic pressure will shift the normal equilibrium of Starling forces and lead to increased edema formation with a crystalloid perfusate. 24
The buffering capacity of blood is far superior to that of crystalloid perfusate because of the presence of a large number of hydrogen ion–binding histidine residues in hemoglobin and plasma proteins. 25-27 Furthermore, the action of erythrocyte carbonic anhydrase permits the erythrocyte to extract hydrogen ions from the tissues to buffer tissue pH at the expense of erythrocyte pH. 27 Finally, use of crystalloid perfusate may have unanticipated rheologic implications, such as the alteration of capillary flow distribution because of shunting of flow. 27,29 All of these factors are important in the investigation of ischemia/reperfusion phenomena and make the blood-perfused model preferable.
SUMMARY
These experiments have demonstrated that myocardial protection with hyperpolarizing cardioplegic solution containing aprikalim is superior to standard hyperkalemic, depolarized arrest in a blood-perfused isolated heart model. Despite the prolonged electrical activity associated with its use, aprikalim cardioplegia was superior to hyperkalemic cardioplegia at preserving ventricular systolic function during normothermic global ischemia. Furthermore, our data suggest that use of aprikalim cardioplegia is not associated with a postischemic decrease in left ventricular compliance even though there was a slight increase in myocardial edema formation. Our data support the hypothesis that hyperpolarized cardiac arrest is a more advantageous form of myocardial preservation than traditional depolarized arrest. To determine the clinical feasibility of potassium-channel opener–induced arrest, future studies are needed in an intact animal model.
Acknowledgments
We would like to acknowledge the technical assistance of Cynthia Allen, BA.
References
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