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Ann Thorac Surg 2000;69:1363-1368
© 2000 The Society of Thoracic Surgeons
a Section of Cardiothoracic and Vascular Surgery, Department of Surgery, The Milton S. Hershey Medical Center, Penn State Geisinger Health System, Hershey, Pennsylvania, USA
Address reprint requests to Dr Damiano, Division of Cardiothoracic Surgery, Washington University Medical Center, 1 Barnes-Jewish Hospital Plaza, Suite 3108 Queeny Tower, St. Louis, MO 63110
e-mail: damianor{at}msnotes.wustl.edu
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Abstract |
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Methods. In a parabiotic rabbit Langendorff model, 36 hearts underwent global normothermic ischemia (1 hour) followed by reperfusion (30 minutes). Cardioplegia (50 mL, every 20 minutes) consisted of: StT; PinPT/StT, where Pin PT preceded StT arrest; Pin alone; Pin in StT (Pin/StT); and Pin in low potassium StT. Systolic function after reperfusion (percent recovery of developed pressure) and compliance (diastolic slope from pressure–volume relationship) were measured.
Results. There was no significant difference between StT and PinPT/StT in percent recovery of developed pressure (51.54% ± 3.5%, 42.17% ± 4.0%, respectively) or compliance. Likewise, no significant differences occurred between Pin, StT, Pin/StT, and Pin in low potassium StT in percent recovery of developed pressure (58.99% ± 4.8%, 51.54% ± 3.5%, 53.09% ± 3.2%, 66.43% ± 7.3%, respectively) or compliance.
Conclusions. Pin is as effective a cardioplegic agent as StT; however, its use as a pretreatment or additive to traditional and Pin in low potassium StT provided no additional benefit in functional recovery.
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Introduction |
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Recent work in our laboratory has involved the investigation of hyperpolarized arrest using potassium channel openers (PCOs). These agents arrest the heart at hyperpolarized potentials, close to the natural resting state of the cell membrane, and thus theoretically may avoid many of the detrimental aspects of depolarized arrest [2]. The PCOs also have the advantage of being inherently cardioprotective during both regional and global ischemia and have been shown to play a critical role in ischemic preconditioning [4, 5]. Myocardial protection with the PCO pinacidil (Pin) was comparable to traditional hyperkalemic St. Thomas’ solution (StT) in studies using a blood-perfused, rabbit heart Langendorff model [6]. However, the role of these drugs as pretreatment or preconditioning agents or as additives to potassium cardioplegia remains controversial.
The objectives of this study were twofold. Our first objective was to examine the effect of Pin on myocardial protection when given as a pretreatment agent before arrest with StT. The second objective was to investigate the effect of Pin on myocardial protection when given as an additive to: (1) traditional, hyperkalemic (16 mmol/L) StT, and (2) low potassium (10 mmol/L) StT.
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Material and methods |
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Experimental preparation
Preparation of the support animal
Each support animal was anesthetized with an intramuscular injection of acepromazine (1 mg/kg) and xylazine (17.5 mg/kg), followed by ketamine (62.5 mg/kg). The animal was heparinized through an ear vein and the left femoral artery was cannulated. Arterial pressure was transduced (model 041-500-503A; Argon Division, Maxxim Medical, Athens, TX) and continuously monitored. A systolic blood pressure was maintained above 70 mm Hg throughout the study by transfusion of donor animal blood or Plasma-Lyte (Baxter Healthcare Corp, Deerfield, IL). Serial hematocrits were obtained.
A tracheotomy was performed and mechanical ventilation (model 683; Harvard Apparatus, S. Natick, MA) begun with an endotracheal tube. Arterial blood was pumped to perfuse a modified Langendorff apparatus, previously described [7].
Preparation of the donor animal and isolated heart
The donor animal was anesthetized, heparinized, intubated, and ventilated in the same manner as the support animal. A rapid cardiectomy was performed through a median sternotomy and blood for support animal transfusion collected from the thoracic cavity. The aorta was cannulated and the heart suspended from the Langendorff apparatus. Blood perfusion was begun.
A fluid-filled latex balloon was inserted into the left ventricle and secured in place with a pursestring suture around the mitral valve annulus. The intraventricular balloon was connected with polyethylene tubing to a pressure transducer (model P23ID; Gould Instrument Systems, Inc, Valley View, OH) and amplifier (model 20-4615-50; Gould). The zero pressure reference was set at the level of the aortic valve. Two-needle electrodes were placed on the epicardium of the left ventricle to monitor the bipolar electrogram. The signal was filtered between 0.05 and 1000 Hz with an isolated preamplifier (model 11-G5407-58; Gould) and a universal amplifier (model 20-4615-58; Gould). Two other electrodes were secured to the right atrial appendage and connected to a pacemaker (model 5320; Medtronic, Inc, Minneapolis, MN). Heart rate was maintained at a constant rate for each experiment. The pressure and electrogram tracings were displayed continuously and digitized in real time (at a sampling rate of 1,000 Hz) using a data acquisition system (WinDAQ; DATAQ Instruments, Akron, OH). A needle probe was placed in the right ventricle and myocardial temperature was monitored continuously (model TM147T; Electromedics, Inc, Parker, CO). The heart was enclosed in a water-jacketed beaker and myocardial temperature was maintained at 37°C by adjusting the water bath (model 800; Fisher Scientific, Pittsburgh, PA).
Experimental protocol
Hearts that did not generate a systolic pressure of 80 mm Hg at an end-diastolic pressure of 10 mm Hg were excluded from the study. After cardiectomy and instrumentation, each heart was given a 30-minute equilibration period and then preischemic data were obtained. Intracavitary left ventricular (LV) pressure and the bipolar LV electrogram were recorded over seven balloon volumes, each corresponding to a fixed, intracavitary LV end-diastolic pressure (0, 2.5, 5, 10, 15, 20, and 25 mm Hg). After baseline data acquisition, the amount of fluid in the latex balloon was adjusted to obtain an end-diastolic pressure of 2.5 mm Hg.
Hearts were randomized to receive 50 mL of cardioplegia every 20 minutes, starting at the onset of a 1-hour period of global normothermic (37°C) ischemia. The cardioplegic solutions examined were: (1) StT (n = 7); (2) Pin pretreatment (Pin PT), where 5 minutes of 50 µmol/L Pin in Krebs-Henseleit solution followed by 5 minutes of drug-free blood perfusion preceded StT arrest (n = 7); (3) 50 µmol/L Pin in Krebs-Henseleit solution (n = 7) (Pin); (4) 50 µmol/L Pin in StT (n = 8) (Pin/StT); and (5) 50 µmol/L Pin in low potassium (10 mmol/L) StT (Pin/LoKStT).
Krebs-Henseleit solution consisted of (in mmol/L distilled water): NaCl, 118.5; NaHCO3, 25.0; KCl, 3.2; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; and glucose, 5.5. Pinacidil was provided by Leo Pharmaceutical Products, Ltd, Ballerup, Denmark. A concentrated stock solution was made by dissolving Pin in dimethyl sulfoxide. The appropriate amount of Pin stock solution was then added to either StT or Krebs-Henseleit solution before cardioplegia infusion. The 50 µmol/L Pin dose was found to be optimum in previous studies using this model [8]. Procaine (5 mmol/L) was added to the initial dose of Pin in Krebs-Henseleit solution cardioplegia [9]. St. Thomas’ solution (Plegisol, Abbott Laboratories, N. Chicago, IL) consisted of (in mmol/L): NaCl, 110.0; CaCl2, 1.6; MgCl2, 16.0; and KCl, 16.0. Sodium bicarbonate (8.4%) was added to StT solution (0.5 mL/50 mL) to adjust its pH to 7.6 to 7.8.
At the start of the ischemic period, the Langendorff perfusion column was clamped and 50 mL of normothermic cardioplegia was infused with a separate column. The cardioplegia effluent was collected and discarded. The time until both mechanical and electrical arrest were recorded. After 60 minutes of global ischemia, the Langendorff column was unclamped and the heart underwent 30 minutes of reperfusion. Intracavitary LV pressure waveforms and electrograms were recorded over the same range of balloon volumes recorded during preischemic data acquisition, after determination of the postischemic 0 mm Hg end-diastolic pressure balloon volume. At the conclusion of the study, a portion of the LV apex was excised, blotted, weighed, and then dried until a constant dry weight was reached. Myocardial edema was expressed as percent tissue water (%Tis H2O) according to the equation: 
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Data analysis
Digitized pressure waveforms were analyzed using a commercial software program (Spectrum version 2.0, Triton Technology, Inc, San Diego, CA).
End-systolic pressure
The end-systolic pressure (ESP) of a beat was defined as the maximum point of the digitized pressure waveform. Mean ESP (average of 10 to 20 beats) was calculated for each preischemia and postreperfusion balloon volume (V). The ESP versus V data were fitted to a linear ESP-V relationship (ESPVR) with a least-squares linear regression as previously described [6].
End-diastolic pressure
The end-diastolic pressure (EDP) of a beat was defined as the point at which the slope of the pressure waveform exceeded 0.5 mm Hg/ms. Mean EDP was calculated for each preischemia and postreperfusion balloon volume. The EDP versus V data were fitted to a linear EDP-V relationship (EDPVR) with a least-squares linear regression: 
, where m was the slope of the EDPVR and V0 was the balloon volume at which EDP was zero, or the x-axis intercept of the EDPVR. A linear representation of the EDPVR in this model has been shown to be appropriate across the range of balloon volumes examined [8].
Developed pressure
Developed pressure (DP) in the left ventricle was defined as the difference between ESP and EDP for a given beat. The DP versus V data were fitted to a linear pressure–volume relationship, as described previously [8].
Recovery of developed pressure
The recovery of developed pressure, expressed as a percentage (%Rec DP), was calculated as the ratio of the postreperfusion DP to the preischemia DP at each matched balloon volume. The average %Rec DP was determined from the integrated values, and approximated using the trapezoidal rule [8].
Statistical analysis
Statistical analysis was performed using Sigma Stat (version 2.0; Jandel Corp, San Rafael, CA). Results are expressed as the mean ± standard error of the mean. A Student’s t test was used to compare means between two groups, while a one-way analysis of variance was used for multiple comparisons. Individual comparisons between groups were made using a Student-Newman-Keuls posttest. When appropriate, the Mann-Whitney rank sum test or the Kruskal-Wallis analysis of variance on ranks was used as a nonparametric alternative. A one-way repeated measures analysis of variance was used for comparisons involving sequential, time-based measurements. When appropriate, the Wilcoxon signed rank test or the Friedman repeated measures analysis of variance on ranks was used. A p value of less than 0.05 was considered statistically significant.
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Results |
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Cardioplegia delivery
There were no differences in mean time required for the first, second, or third cardioplegia (50 mL) infusion within any treatment group. There also were no differences in time of cardioplegia delivery for each of the three infusions between any of the groups.
Electromechanical arrest
There were no differences in time to either mechanical or electrical arrest between the StT and the Pin PT groups (Table 1). However, there were significant differences in both time to mechanical and electrical arrest between the Pin group and both StT and Pin in StT groups (Table 2). Time to mechanical arrest was significantly longer in the Pin group compared to all others (p < 0.05). The Pin in LoKStT group also had a significantly longer time to mechanical arrest than either the StT or Pin in StT groups (p < 0.05). For electrical arrest, times were significantly longer in the Pin and Pin in LoKStT groups than in either the StT or Pin in StT groups (p < 0.05).
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Postischemic diastolic properties
Postreperfusion changes in diastolic compliance were quantified by determining the slope of the LV EDPVR. The Pin, Pin in StT, and Pin in LoKStT groups demonstrated significantly decreased compliance after reperfusion compared to before ischemia. However, there were no differences in slope between the StT and Pin PT groups at baseline or after reperfusion. In hearts protected with Pin, StT, Pin in StT, and Pin in LoKStT, diastolic slope was not significantly different between groups (Table 3).
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Myocardial tissue water
There were no significant differences in percent tissue water between any of the groups (Tables 1 and 2).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Our laboratory has shown in numerous studies that PCOs are either better than or comparable to traditional hyperkalemic arrest in both crystalloid- and blood-perfused models [6–8]. However, the use of PCOs in conjunction with potassium cardioplegia, either as pretreatment or as additives, remains controversial.
Pinacidil pretreatment
Ischemic preconditioning has been established as one of the most potent means of myocardial protection [14, 15]. Furthermore, the KATP channels have been shown to play an important role in preconditioning in some species [4, 5, 16]. Using the PCO nicorandil, Menasché and coworkers [17] demonstrated that pharmacologically preconditioned isolated rat hearts recovered systolic and diastolic function to a significantly greater extent than controls with no prearrest intervention. Contrary to the study by Menasché and colleagues, our findings revealed that pharmacologic pretreatment with PCOs was not effective in improving myocardial protection of either systolic function of the diastolic properties of the left ventricle. There may be several explanations for these discrepant results.
Our blood-perfused Langendorff model is more physiologic than crystalloid perfusion and considered to be more appropriate for the study of ischemia/reperfusion injury [8, 18]. The attendant benefits of blood perfusion may have ameliorated the ischemia/reperfusion injury in the StT group, resulting in recovery that was comparable to the Pin PT group. An additional reason for the differences in our findings may have been due to species variations. Asimakis and coworkers [19] could not find evidence that rabbit hearts had the capacity to be ischemically preconditioned against myocardial stunning, despite single and multiple brief preconditioning periods.
Another explanation for the contrast between our work and that of Menasché may lie in potential differences between the two PCOs. Both compounds share a common pyridine moiety, but their overall structures differ considerably [20]. Subtle structural differences between these PCOs may result in different modes of action and pharmacologic effectiveness. In addition, the equilibration period after initial perfusion of the heart varied between studies. Menasché and colleagues used a 15-minute period, whereas in this study 30 minutes were used. Asimakis and associates [19] have shown that hearts undergoing 15 minutes of stabilization clearly demonstrated a preconditioning effect, whereas those with 30 minutes showed no protection with respect to LV developed pressure.
Pinacidil as an additive
Recently, it has been demonstrated that PCOs prevent the intracellular calcium loading associated with use of potassium cardioplegia [11]. Dorman and colleagues [10] demonstrated that the addition of a PCO to potassium cardioplegic solutions prevented the deleterious effects of depolarized arrest on both myocyte contractile function and ionic homeostasis in isolated myocytes. Aprikalim added to 24 mEq/L potassium and to 12 mEq/L potassium cardioplegia resulted in improved contractile function when compared to hyperkalemic cardioplegia alone. Aprikalim added to 24 mEq/L potassium cardioplegia attenuated intracellular calcium accumulation, whereas the addition to 12 mEq/L potassium cardioplegia prevented this process. Our laboratory has recently confirmed these findings and has shown that 50 µmol/L Pin completely blocked the calcium overload seen with StT solution [21].
Pignac and coworkers [22] studied aprikalim added to StT in an isolated, crystalloid-perfused rabbit heart Langendorff model. Their data revealed that postischemic myocardial function was as good as preischemic values when aprikalim was added to cold StT; but with StT used alone, a significant decrease in postischemic myocardial function occurred in comparison to its preischemic values.
In the present study, no difference in functional recovery was found when Pin was added to either standard (16 mmol/L) or to low potassium (10 mmol/L) StT. It is unclear if the differences in temperature or experimental models between this and the other studies impacted on postischemic recovery. However, other investigators have similarly documented the inefficacy of PCOs as additives to potassium cardioplegia [23, 24]. These findings are in agreement with the fact that when a PCO is combined with potassium cardioplegia (even at the 10 mmol/L concentration), depolarization will still occur. As a result, the protective effects of PCOs on myocardial function may be attenuated or lost.
Electromechanical arrest
When Pin was used as a pretreatment, there were no differences in time to electromechanical arrest compared to arrest with StT alone. When Pin was used as an additive, significant differences in both time to mechanical and electrical arrest occurred. The Pin group had a significantly prolonged time to electromechanical arrest versus the StT group. This is consistent with our previous findings [6].
Despite the differences in electromechanical arrest times, the myocardial protection afforded by each group was similar. Previous work from our laboratory has demonstrated that persistent electrical activity did not adversely influence recovery of function, nor did it result in significant depletion of high energy nucleotides after ischemia [9]. This is consistent with the fact that mechanical work has been shown to represent more than 99% of total basal myocardial oxygen consumption, whereas electrical activity accounts for less than 1% of oxygen consumption [25]. The delay in mechanical arrest appears to be of little consequence in this model as the Pin groups provided protection comparable to that of StT. However, it is obvious that mechanical activity is energy consuming in a globally ischemic heart. This suggests that the beneficial effects of Pin were able to compensate for the additional energy expenditure imposed by the persistent mechanical activity.
Coronary flow
When Pin was added to StT, reperfusion coronary blood flow at 10 and 15 minutes was significantly greater than Pin alone. It is not certain whether this finding represents an augmented vasodilatory effect in the additive group or a lack of vasodilation extended to these time points in the Pin group. Because the Pin group values in this study correlate with those of earlier studies [6, 8], the former explanation seems most likely. One reason for this difference may be that the presence of traditional StT along with Pin potentiates the vasodilatory effects of Pin, more so than Pin in low potassium StT. However, all groups had comparable blood flow by 20 minutes of reperfusion.
In conclusion, although the PCO Pin is as effective a cardioplegic agent as StT, the use of Pin either as a pretreatment or as an additive to traditional or modified (10 mmol/L potassium) StT provided no additional benefit in functional recovery in this model. This may result from the inability of Pin to exploit the cardioprotective potassium channels during the depolarized arrest established by StT. Another possibility is that the mitochondrial KATP channel may play a more important role than the sarcolemmal channel opened by Pin [26]. Future studies with mitochondrial-specific PCOs, such as diazoxide, will be needed to define this issue.
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Acknowledgments |
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References |
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