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Ann Thorac Surg 1999;68:67-74
© 1999 The Society of Thoracic Surgeons
a Section of Cardiothoracic and Vascular Surgery, The Milton S. Hershey Medical Center, Pennsylvania State Geisinger Health System, Hershey, Pennsylvania, USA
Address reprint requests to Dr Damiano, Section of Cardiothoracic Surgery, The Milton S. Hershey Medical Center, Pennsylvania State Geisinger Health System, PO Box 850, Hershey, PA 17033
e-mail: damiano{at}psghs.edu
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Abstract |
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Methods. Sixteen pigs were placed on full cardiopulmonary bypass. Hearts underwent 2 hours of global ischemia (10° to 15°C). Either St. Thomas’ or 100 µmol/L pinacidil was administered every 20 minutes (10 mL/kg). Preischemic and postreperfusion slopes of the preload-recruitable stroke work relationship were determined. Changes in myocardial adenine nucleotide levels and cellular ultrastructure were analyzed.
Results. Pinacidil cardioplegia resulted in an insignificant change in the slope of the preload-recruitable stroke work relationship (40.6 ± 2.1 mm Hg/mm before ischemia and 36.5 ± 3.7 mm Hg/mm after ischemia; p = 0.466). In contrast, St. Thomas’ solution resulted in a significant decrease in the slope after reperfusion (34.3 ± 5.5 mm Hg/mm and 13.5 ± 2.3 mm Hg/mm; p = 0.003). Adenine nucleotide levels, myocardial tissue water, and ultrastructural changes were similar between groups.
Conclusions. Pinacidil ameliorated myocardial stunning associated with traditional hyperkalemic cardioplegia without causing significant differences in cellular metabolism.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Adenosine triphosphate–sensitive potassium channels open during cellular ischemia, and this results in a net potassium efflux and membrane hyperpolarization [8]. This causes action potential shortening, decreased calcium influx, and contractile failure, all of which constitute an intrinsic energy-sparing mechanism during ischemia [9]. The adenosine triphosphate–sensitive potassium channels are activated by a diverse group of pharmacologic agents collectively called potassium-channel openers (PCOs). Previous work in our laboratory [10, 11] has shown PCOs to be effective cardioplegic agents, as they adequately protect the myocardium during global ischemia. However, the efficacy of hyperpolarizing cardioplegia has not been studied in the more clinically relevant intact cardiopulmonary bypass model. This study tested the hypothesis that myocardial protection with the PCO pinacidil in the porcine model is superior to that obtained with the traditional hyperkalemic St. Thomas’ Hospital cardioplegic solution.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Adult pigs weighing 40 to 50 kg were anesthetized with intramuscular tiletamine and zolazepam (5 mg/kg) and intravenous sodium pentobarbital (30 mg/kg) and maintained on an intravenous pentobarbital infusion of 0.5 mg · kg-1 · min-1 throughout the study. Anesthesia was monitored carefully throughout the experiment and was supplemented as needed. A tracheostomy was performed, an endotracheal tube was inserted, and mechanical ventilation was begun (model BV-511; Bourns Medical Systems, Inc, Riverside, CA). Ventilator settings were adjusted to maintain arterial blood gases within the physiologic range (pH 7.35 to 7.45; carbon dioxide tension, 35 to 45 mm Hg; and oxygen tension, 150 to 250 mm Hg).
Animals underwent a standard median sternotomy, pericardiotomy, and placement of caval occlusion tapes. For preischemic and postischemic functional data acquisition, body temperature was maintained between 37.0° and 38.0°C by a heating blanket. During ischemia, animals were systemically cooled to 28°C.
Femoral artery blood pressure, electrocardiogram, and heart rate were displayed continuously (Sirecust 404-1A; Siemens Medical Systems, Inc, Danvers, MA.) The following variables were measured at regular intervals and maintained within the physiologic range: arterial blood gases, hematocrit (30% to 40%), and serum levels of sodium, potassium, and ionized calcium (in millimoles per liter: Na+, 135 to 145; K+, 3.5 to 5.0; and Ca2+, 0.8 to 1.2).
Instrumentation of heart
Pressure–dimension relationships were generated using ultrasonic dimension transducers and left ventricular manometry. Piezoelectric omnidirectional crystals (outer diameter, 2 mm) (No. Xtal-42-C; Sonometrics Corp, London, ON, Canada) were oriented across the minor-axis diameter of the left ventricular epicardium. A sonomicrometer (model 6TRX; Sonometrics Corp) was used to measure the distance between the transducers with a standard resolution of 0.024 mm. A micromanometer (model SPC-360; Millar Instruments, Inc, Houston, TX) was balanced and calibrated before each study and passed through the apex into the left ventricle. Epicardial electrogram leads were placed on the left ventricle, and the signal was amplified (preamp model 7P4 and amplifier model 7DA; Grass Instrument Co, Quincy, MA) and relayed to an oscillograph (model 79WU; Grass Instrument Co). Pacing wires were attached to the right atrial appendage.
Cardiopulmonary bypass and cardioplegia delivery
A standard perfusion circuit was assembled (pump model 5000; Sarns 3M, Ann Arbor, MI) and flushed with carbon dioxide. Oxygenators and reservoirs were provided by Cobe Cardiovascular (Arvada, CO), Avecor Cardiovascular (Plymouth MN), and Baxter Healthcare Corporation (Irvine, CA).
A blood prime consisting of 5 to 6 units of fresh (< 7 days old) citrate-phosphate-dextrose porcine blood and 2,000 units of heparin sodium per unit was circulated. Physiologic arterial blood gas and electrolyte values in the prime were achieved prior to cardiopulmonary bypass. Intravenous heparin (300 U/kg) was administered prior to cannulation for cardiopulmonary bypass. Activated clotting times were measured at regular intervals and maintained at higher than 450 seconds (coagulation timer model 801 Hemocron; International Technidyne Corp, Edison, NJ). The left internal carotid artery was cannulated with a metal-tipped catheter (inner diameter, 4 mm) for arterial inflow. After preischemic data collection, the superior and inferior venae cavae were cannulated. Cardiopulmonary bypass was then instituted at perfusion flow rates ranging from 1.8 to 2.6 L · min-1 · m-2. Mean arterial pressure was maintained between 50 and 70 mm Hg by transfusion of blood collected from a donor animal or Plasma-Lyte (Baxter Healthcare Corp, Deerfield, IL) or by administration of intravenous phenylephrine hydrochloride or mannitol.
Experimental protocol
Vents were placed into the right atrium and the left ventricle after initiation of bypass. A cardioplegia catheter was placed in the proximal ascending aorta (model 24009; DLP, Inc, Grand Rapids, MI). The aorta was cross-clamped, and antegrade cardioplegia was delivered. Vents were switched to wall suction during each cardioplegia infusion. Myocardial septal temperature was monitored with a needle probe (22 gauge (G), 18-mm length; Medtronic Inc, Parker, CO) and a thermistor (model TM-2100; Electromedics Inc, Englewood, CO) and was kept lower than 15°C with the aid of topical hypothermia. Mean cardioplegia delivery pressure was maintained between 60 and 90 mm Hg. Six doses of cardioplegia (4° to 8°C, 10 mL/kg, and 3- to 3.5-minute infusion) were infused at 20-minute intervals during a total cross-clamp time of 2 hours.
Cardioplegia consisted of either St. Thomas’ solution (Plegisol; Abbott Laboratories, Chicago, IL) (in milliequivalents per liter: Na, 110; Cl, 160; K, 16; Ca, 2.4; and Mg, 32) or 100 µmol/L pinacidil in Krebs-Henseleit buffer (in millimoles per liter: NaCl, 118.5; NaHCO3, 25; KCl, 3.2; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; and glucose, 5.5.) Pinacidil was provided by Leo Pharmaceuticals, Billup, Denmark.
Compared with St. Thomas’ solution, prolonged electromechanical activity with PCO cardioplegia has been documented in our laboratory [10–12]. Previous studies [12, 13] have shown that sodium-channel blockade using 5 mmol/L procaine hydrochloride eliminates this persistent electromechanical activity without influencing myocardial recovery. Therefore, the initial pinacidil infusion contained 5 mmol/L procaine. There was no return of electromechanical activity with subsequent cardioplegic infusions.
After 2 hours of ischemia, the cross-clamp was removed and the animal was rewarmed. The heart was defibrillated when necessary (model 9760; Medtronic, Inc, Minneapolis, MN). The animal remained on full cardiopulmonary bypass for 1 hour of cardiac reperfusion, during which time the heart was allowed to recover unpaced and vented. After 1 hour of reperfusion, the animal was weaned from bypass, and postreperfusion data were collected as previously described.
Data acquisition and analysis
Functional data
For each animal, functional data were acquired before ischemia and after 1 hour of reperfusion. Caval occlusions were conducted with the animal off cardiopulmonary bypass. All hearts were paced at a constant rate (110 to 140 bpm) at twice diastolic threshold (pacemaker models 5345, Medtronic, Inc, Minneapolis, MN, and 780-4 Sanborn Co, Waltham, MA). A bipolar left ventricular electrogram was recorded, and atrioventricular activation time was measured as the average P-R interval of five consecutive paced beats. Digitized left ventricular pressure–dimension waveforms were recorded at 424 Hz by a microcomputer during three consecutive vena caval occlusions (PC model P133-16-1.70; Sonometrics Corp). Between occlusions, sufficient time was allowed to permit hemodynamic variables to return to baseline. Digitized pressure–dimension data were analyzed using commercially available software (Spectrum version 2.0; Triton Technology, Inc, San Diego, CA).
End-diastolic pressure–segment length relationship
End-diastole was defined 20 ms before the first derivative of the digitized pressure waveform first exceeded 0.2 mm Hg/ms for each beat. The end-diastolic pressure (EDP) versus dimension data were subjected to nonlinear regression analysis over all loops in the series using the equation 
where 
and ß are the nonlinear coefficients of the exponential EDP–volume (segment length) relationship,e is the base of natural logarithms,and V is the left ventricular minor-axis diameter [14, 15]. Beat point definitions were checked videographically on all data.
Preload-recruitable stroke work relationship
Global net stroke work (SW) was calculated as the integral of left ventricular intracavitary pressure (P) and minor-axis diameter (V) over each cardiac cycle as described by the equation 
Individual vena caval occlusions were used to determine linear preload-recruitable stroke work where load was described by the left ventricular minor-axis diameter. Minor-axis dimension has been shown to adequately represent intracavitary volume when used in the preload-recruitable stroke work relationship [16]. Data collection for the analysis began three to five beats before the onset of occlusion and extended to steady-state maximal vena caval occlusion. Each occlusion was subjected to linear regression analysis over all loops in the series, yielding a slope, Mw, and an x-intercept, Lw. The Mw has been shown to be an accurate measure of intrinsic myocardial performance independent of loading, geometry, and heart rate [16]. Correlation coefficients (r) were determined. Pressure–volume loops were checked videographically on all data.
Metabolic data
Anteroapical left ventricular biopsy specimens were acquired before ischemia and after 1 hour of reperfusion. Specimens were taken using a 14G Tru-cut biopsy needle (20-mm specimen notch; Baxter Healthcare Corp) and frozen immediately in liquid nitrogen. Samples were stored at -70°C and extracted in perchloric acid. The acid extracts were neutralized by the addition of potassium hydroxide.
Reverse-phase high-performance liquid chromatography was used to determine high-energy nucleotide levels and has been described previously [17]. Aliquots (100 µL) of the neutralized perchloric acid extracts were assayed for adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, hypoxanthine, adenosine, inosine, and NAD+ (nicotinamide adenine dinucleotide [onidized form]). Separation was performed by a 30-minute gradient elution using a Hibar Lichrosphere 100 RP-18/5 column (Merck, Darmstadt, Germany). The linear gradient program for the mobile phase was as follows: 0 minutes, 100% buffer A; 0.1 to 4.1 minutes, 75% buffer A; 4.1 to 22 minutes, 0% buffer A; and 22 to 30 minutes, 50% buffer A. The composition of buffer A was 150 mmol/L ammonium phosphate (pH 5.80), and the composition of buffer B was 150 mmol/L ammonium phosphate with 20% methanol and 2% acetonitrile (vol/vol, pH 5.45). Separation was effected at room temperature with a flow of 0.8 mL/min. The detection wavelength was 254 nm. Peak identification was performed by comparison of retention times with known standards. Nucleotide quantity was determined by peak area.
Tissue water
Tru-cut needle biopsy specimens for tissue water content were obtained before ischemia, after 2 hours of ischemia, and after 1 hour of reperfusion. A sample of the anteroapical left ventricle was excised, blotted, weighed, and dried until a constant dry weight was reached. Myocardial edema was expressed as the percent tissue water in the following equation: 
Histologic data
At the conclusion of each experiment, the heart was preserved in situ for electron microscopic study. The method for fixation and sectioning of tissue samples has been described previously [18, 19]. The heart was fixed by vascular perfusion with Karnovsky’s fixative [18] in sodium cacodylate buffer containing 4% dextran. An aortic cross-clamp was applied, and the heart was perfused with fixative through the aortic root cannula. After fixation, three specimens of the anteroapical left ventricle were obtained using a Tru-cut needle. Specimens were immersed in Karnovsky’s fixative, washed, resuspended, and stored in sodium cacodylate buffer. Specimens were combined, and three sections, selected at random, were washed and postfixed in 1% OsO4/0.1 mol/L sodium cacodylate.
The sections were then placed in propylene oxide and embedded in Spurr’s resin. Semithin sections were cut at 1 to 2 µm and stained [19]. Thin sections were cut with diamond knives, mounted on 200-mesh copper grids, stained with lead citrate and uranyl acetate, and examined using a Philips 400 electron microscope (Philips Electronics NV, Eindhoven, the Netherlands). The central areas of each grid were photographed at 4,600x, 7,700x, and 12,500x the original magnification for each of the three sections per animal. Photomicrographs were evaluated semiquantitatively for myocyte ischemic damage to glycogen bodies, mitochondria, nuclei, sarcolemma, and myofibrils using the grading system shown in Table 1. The pathologist was blinded to the specific cardioplegic solution.
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2 analysis of contingency tables was used to compare mutually exclusive, categoric data where appropriate. A multivariate analysis of variance was used to analyze histologic data. |
Results |
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Electrophysiology
Pinacidil cardioplegia produced a rapid electromechanical arrest (43.8 ± 4.3 seconds) that was comparable to that of St. Thomas’ solution (39.9 ± 3.5 seconds). There were no significant differences in times to electromechanical arrest between PCO and St. Thomas’ solutions. During ischemia and cardioplegia, all hearts remained electromechanically quiescent.
Pacing threshold increased significantly after ischemia compared with preischemic values in both groups: St. Thomas’, 1.6 ± 0.7 mV to 38.0 ± 7.8 mV (p = 0.003), and pinacidil, 3.7 ± 1.5 mV to 29.0 ± 8.2 mV (p = 0.015). No significant differences were apparent between groups.
Both groups demonstrated significant prolongation of the P-R interval compared with preischemic values: St. Thomas’, 94 ± 5 ms to 138 ± 13 ms (p = 0.003), and pinacidil, 98 ± 8 ms to 128 ± 12 ms (p = 0.013). However, there were again no significant differences between groups.
Contractile recovery
The relationship between global net stroke work and minor-axis left ventricular chamber dimension was expressed as the linear preload-recruitable stroke work relationship. This relationship was highly linear in each group: St. Thomas’, mean r = 0.97 ± 0.02 at baseline and 0.95 ± 0.03 after reperfusion, and pinacidil, mean r = 0.98 ± 0.01 at baseline and 0.98 ± 0.03 after reperfusion. The preload-recruitable stroke work relationship was quantified by its slope, or Mw, and x-intercept, or Lw. St. Thomas’ solution resulted in a significant decrease in Mw after ischemia and reperfusion (34.3 ± 5.5 mm Hg/mm and 13.5 ± 2.3 mm Hg/mm, respectively; p = 0.003) (Fig 1A). This represented a 61% decrease. In contrast, myocardial protection with pinacidil cardioplegia resulted in preservation of contractile function, as assessed by no change in Mw (40.6 ± 2.1 mm Hg/mm and 36.5 ± 3.7 mm Hg/mm after ischemia (p = 0.466) (Fig 1B). Postischemic Mw was significantly decreased in the St. Thomas’ group compared with the pinacidil group (p < 0.001). The x-intercept changed significantly in both groups.
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and ß coefficients of the exponential end-diastolic pressure–dimension relationship were determined for each heart (Fig 2). Both groups demonstrated a leftward displacement of the curve, indicating decreased ventricular compliance. The changes were not significant between the two cardioplegia groups.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Alternatively, pinacidil cardioplegia may have ameliorated cell swelling, which has been linked to myocardial stunning with standard hyperkalemic solutions [23]. Cell swelling with hyperkalemic cardioplegia has been documented in isolated myocytes and is due to the high KCl product and resultant hypotonicity of the solution [1]. Our laboratory [1, 23] has shown that lowering the KCl product to produce an isotonic hyperkalemic solution results in improved contractile function in intact hearts. Pinacidil cardioplegia, which is isotonic, does not result in cell swelling, and this effect may have ameliorated myocardial stunning after reperfusion. It is not surprising that our biopsy specimens did not demonstrate differences in tissue water between pinacidil and St. Thomas’ solution (see Fig 3). Data from our laboratory [1, 23] and other groups [22] suggest that the cell swelling associated with potassium cardioplegia occurs only during ischemia, dissipates almost immediately on reperfusion, and is indistinguishable from interstitial edema on gross biopsy samples. Therefore, although tissue water in this study was not different between groups, intracellular volume cannot be excluded as a factor in the pathogenesis of injury associated with traditional hyperkalemic cardioplegia.
The apparent superiority of hyperpolarized arrest with pinacidil in this study must be qualified with two observations. First, there was no long-term evaluation of left ventricular function. Second, although systolic function was better preserved with pinacidil, there was no difference in myocardial chamber compliance between groups (see Fig 2). The decreased compliance may have been the result of ischemic injury, but the lack of metabolic, functional, or histologic evidence of ischemic injury in the pinacidil group argues against this hypothesis. The most likely explanation for the compliance loss is the myocardial edema seen in both groups after reperfusion. This is likely a result of the experimental preparation and the time on cardiopulmonary bypass, rather than a sequela of ischemia. Edema has clearly been shown to cause decreased ventricular compliance both clinically and experimentally [24].
The finding that there was no correlation between contractile recovery and high-energy nucleotide levels is similar to work by others [25, 26] in which adenosine triphosphate levels failed to predict recovery of contractile function (see Table 3). This reflects not only the fact that both cardioplegic solutions were able to similarly reduce metabolic demand during ischemia, but also the finding that both solutions equally preserved mitochondrial membrane capabilities [27]. This is consistent with our histologic data showing well-preserved mitochondria in each group. Notably, these findings refute the hypothesis that PCO cardiac protection is related to a state of minimal metabolic demand compared with myocardial protection with hyperkalemic cardioplegia.
Although a significant difference was demonstrated in sarcolemmal grade, both cardioplegic groups manifested only minimal ischemic changes in the sarcolemma and low histologic grades overall (see Table 3). It is unlikely that the mechanism of myocardial protection by pinacidil is related to sarcolemmal integrity.
In summary, this study demonstrated the feasibility of hyperpolarized arrest in the more clinically relevant intact animal. The superior systolic protection afforded during hyperpolarized arrest may be due to ionic factors not metabolic factors. The precise mechanisms of PCO–mediated myocardial protection in vivo remain to be elucidated. Further investigations are needed to support these hypotheses, as the limitations of an intact animal model precluded direct measurements of intracellular water, calcium, and membrane potential. However, this study strongly suggests that hyperpolarized arrest with PCOs represents an attractive alternative to depolarized arrest with conventional hyperkalemic solutions. Clinical trials will be needed to fully determine their efficacy.
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Acknowledgments |
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We gratefully acknowledge the technical assistance of Roland Myers, BS (Department of Comparative Medicine), Michael Bellamy, BS (Department of Perfusion), Baiyang Xu, PhD (Department of Physiology), The Milton S. Hershey Medical Center, and Luke Wolfe, PhD (Department of Statistics, Medical College of Virginia Hospitals).
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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