Ann Thorac Surg 2003;76:1264-1269
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Cariporide is cardioprotective after iatrogenic ventricular fibrillation in the intact swine heart
David G. Rabkin, MDa,
Santos E. Cabreriza, mBAa,
Faisal H. Cheema, MD,
Adam A. Hill, BAa,
Lauren J. Curtis, BAa,
Robert R. Sciacca, EngScib,
Ralph S. Mosca, MDa,
Henry M. Spotnitz, MDa*
a Departments of DEPARTMENT OF Surgery, New York, New York, USA
b DEPARTMENT OF Biostatistics, Columbia College of Physicians and Surgeons, New York, New York, USA
* Address reprint requests to Dr Spotnitz, Department of Surgery, Columbia College of Physicians and Surgeons, 622 West 168th St, PH 1422, New York, NY 10032, USA.
e-mail: hms2{at}columbia.edu
Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2003.
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Abstract
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BACKGROUND: We sought to introduce sodium–hydrogen exchange inhibition as prophylaxis against the development of ventricular dysfunction in the setting of implantable cardioverter defibrillator insertion in high-risk patients. Cariporide, shown to be safe in humans, was used to reproduce previous results in our laboratory that demonstrated that sodium–hydrogen exchange inhibition preserves left ventricular (LV) function after ventricular fibrillation (VF) and reperfusion.
METHODS: Twelve pigs (weight, 35 to 55 kg) were divided into two groups of six. Baseline ventricular function studies were based on echocardiography, conductance, aortic flow, and LV pressure. Animals were given vehicle (control) or cariporide (3 mg/kg intravenously). Ten minutes later, hearts underwent 80 seconds of VF. After reperfusion for 40 minutes, function studies were repeated.
RESULTS: Postmortem examination included measuring passive pressure-volume curves and myocardial water content. Systolic indices, including preload recruitable stroke work and ejection fraction, were significantly depressed from baseline after VF and reperfusion for control animals (preload recruitable stroke work, 30.13 ± 0.59 [standard error of the mean] versus 43.85 ± 2.60 mm Hg; ejection fraction, 25.7% ± 2.4% versus 33.5% ± 3.0%) but not for those in the cariporide group (preload recruitable stroke work, 38.36 ± 1.87 versus 40.86 ± 1.45 mm Hg; ejection fraction, 33.9% ± 3.5% versus 32.8% ± 3.9%). In vivo diastolic indices demonstrated trends toward diminished ventricular compliance in control animals but not in the cariporide group after VF and reperfusion. Control animals had significantly increased postmortem LV stiffness, myocardial water content, and normalized LV mass.
CONCLUSIONS: Cariporide preserves LV function after 80 seconds of VF and 40 minutes of reperfusion. Cariporide may prove useful in patients with severe LV dysfunction undergoing VF for implantable cardioverter defibrillator testing.
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Introduction
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Implantable cardioverter defibrillator (ICD) insertion requires induction of ventricular fibrillation (VF) to measure defibrillation thresholds. Implantable cardioverter defibrillator recipients often suffer from markedly depressed ventricular function as a result of cardiomyopathy, infarct-related scars, or left ventricular (LV) aneurysms [1]. Defibrillation threshold testing in some of these patients is considered too dangerous and is often deferred. This dilemma prompted us to search for a pharmacologic safety net for patients with severe ventricular dysfunction undergoing ICD insertion.
Preliminary results in our laboratory demonstrated that sodium–hydrogen exchange (NHE) inhibition attenuates ventricular dysfunction after a brief episode of VF followed by 40 minutes of reperfusion in an open-chest porcine model designed to simulate ICD testing [2]. Although many studies have demonstrated beneficial effects of NHE inhibition, our study showed that this approach was able to prevent changes in ventricular function after iatrogenic VF and reperfusion in an in vivo model. In that study we used an experimental NHE inhibitor called BIIB-513 (benzamide-N-(aminoiminomethyl)-4-[4-(2-furanylcarbonyl)-1-piperazinyl]-3-(methylsulfonyl) methanesulfonate), which is a potent inhibitor of the NHE-1 isoform (the prevailing cardiovascular subtype). This drug, however, has not been tested in a clinical trial, and its safety profile is unknown. Consequently, we sought to reproduce our results using a drug that has since been demonstrated to be well tolerated by humans in several large multicenter clinical trials [3, 4]. Cariporide differs from BIIB-513 in that it is eight times less potent, and it is less specific for the NHE-1 isoform [5].
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Material and methods
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Overview
All animals received humane care in compliance with "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 National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985). Reagents were obtained from Aventis (formerly Hoescht-Marion-Rousel) (Franfurt, Germany).
Nineteen male domestic pigs weighing 35 to 55 kg were anesthetized with atropine sulfate (1 to 2 mg intramuscularly), ketamine hydrochloride (20 mg/kg intramuscularly), and xylazine (0.5 mg/kg intramuscularly). They were intubated and mechanically ventilated, and anesthesia was maintained with isoflurane (1.5% to 2.5%) mixed with 100% oxygen. A heating pad was used to maintain body temperature. Peripheral blood pressure and electrocardiographic signals were monitored. Arterial blood gases and serum electrolytes were periodically checked to monitor oxygenation and optimize ventilation. During the experiments 0.9% saline solution was administered through an 18-gauge angiocatheter in the femoral vein or an ear vein at 10 mL · kg-1 · h-1 for the first hour and then decreased to 5 mL · kg-1 · h-1 for the duration of the study.
Animals were instrumented, and hemodynamic indices based on echocardiography, conductance, LV manometry, and aortic flow were determined before and after 80 seconds of VF and 40 minutes of reperfusion. Ten minutes before induction of VF, animals (in random order) were given 50 mL of 0.9% saline with (cariporide) or without (control) 3 mg/kg cariporide. One minute before induction of VF, a 10-mL sample of blood was drawn directly from the right ventricle for serum drug level analysis. Ventricular fibrillation was induced for approximately 80 seconds using a 13-V, 800-mA alternating current through a transformer (Archer AC adapter; Radio Shack, Fort Worth, TX). After 70 seconds, timed with a stopwatch, cardioversion was attempted with 50 J using the internal paddles of a defibrillator module (Hewlett Packard, Palo Alto, CA). If the initial shock was unsuccessful additional shocks were given until hearts successfully underwent cardioversion. Duration of ischemia (from initiation of VF to resumption of organized contraction) for control and cariporide groups averaged 81.67 ± 15.29 (standard error of the mean) and 77.50 ± 2.96 seconds, respectively (p = 0.68, Student's t test). If cardioversion resulted in asystole animals were paced (Medtronic 5375; Medtronics, Minneapolis, MN) at 60 beats/min until hearts regained an independent, organized rhythm. This occurred in 3 animals (2 control animals, 1 receiving cariporide). After cardioversion, animals were allowed to recover for 40 minutes, and hemodynamic data and echocardiograms were again collected. Animals were humanely sacrificed, and hearts were arrested with cold University of Wisconsin solution and explanted. Postmortem studies included generation of passive pressure-volume curves and determination of myocardial water content as previously described [2]. We used similar data acquisition and statistical analysis techniques as in our previous study [2].
Exclusion criteria
Of the 19 animals, 7 animals were excluded from the study. One animal was excluded for technical reasons during the instrumentation phase. In another 6 animals (evenly distributed between the two groups), we were unable to defibrillate the hearts after inducing VF. These hearts developed a fine VF that was unresponsive to our attempts to defibrillate or pace. Consequently, 12 animals (control, 6; cariporide, 6) remained in which we were able to successfully complete the experiment.
Assay procedure for cariporide
Serum samples were express-mailed on dry ice to Aventis Pharmaceuticals in Germany where the cariporide content was analyzed using high-performance liquid chromatography with ultraviolet detection. The working range of the assay is 0.050 to 50.000 µg Cariporide/mL serum or plasma. High-performance liquid chromatography was carried out isocratically on a C-8 reversed-phase column using a mixture of perchloric acid and acetonitrile, to which one bottle (20 mL) of PIC-B7 reagent per liter of mobile phase was added. The analyte was quantified by measuring peak height or area ratios using ultraviolet detection at a wavelength of 250 nm. One hundred microliters of the remaining sulfuric acid phase was injected into the high-performance liquid chromatography.
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Results
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Data for changes in preload recruitable stroke work are shown in Table 1
and Figure 1. Preload recruitable stroke work was significantly depressed from baseline after VF and reperfusion for the control group but not for the cariporide group. The x intercept of the preload recruitable stroke work relation, Vx, did not change significantly within or between groups before or after VF and reperfusion. End-systolic pressure-volume relations are reported in Table 1;
baseline values for control and cariporide groups are not significantly different from postischemic values, although trends suggested a decrease in end-systolic pressure-volume relations in the control group but not the cariporide group. Ejection fraction, reported in Table 1, was significantly depressed from baseline after VF and reperfusion for the control group but not for the cariporide group. Postischemic time to recovery of baseline systolic blood pressure was significantly longer for the control group when compared with the cariporide group (Table 1).
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Fig 1. Effect of cariporide on contractility after ventricular fibrillation and reperfusion. Effect of cariporide on preload recruitable stroke work (PRSW) after ventricular fibrillation and reperfusion. Standard errors are represented by brackets. Differences in preload recruitable stroke work for control animals after 40 minutes of reperfusion are significantly different from control baseline levels and cariporide animals after 40 minutes of reperfusion (p < 0.002). *p = 0.0019 versus control pre paired Student t test; **p = 0.0018 versus control post unpaired Student t test. Solid squares = control (n = 6); open squares = cariporide (n = 6). (POST = values after 40 minutes of reperfusion; PRE = baseline levels.)
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In vivo end-diastolic LV pressure-volume relations were unchanged before and after VF and reperfusion in both groups, although the control group demonstrated trends toward diminished postischemic compliance (Fig 2A).
Similar results were obtained when measuring in vivo end-diastolic LV pressure-area relations (Fig 2B). Postmortem passive end-diastolic LV pressure-volume relations demonstrated significant decreases in LV compliance for controls (Fig 3).
Slopes for in vivo and postmortem compliance curves are reported in the figure legends. Differences in myocardial water content and normalized LV mass (Table 1) between the control and cariporide groups were significant. Results for serum levels of cariporide are also shown in Table 1.
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Fig 2. Effect of cariporide on in vivo ventricular compliance after ventricular fibrillation and reperfusion. Effect of cariporide on in vivo ventricular compliance (pressure-volume measured by conductance [A], pressure-area measured by echocardiography [B]) after ventricular fibrillation and reperfusion. Black icons represent control animals; white icons represent cariporide animals. Squares represent baseline compliance; circles represent compliance after ventricular fibrillation and reperfusion. A logarithmic transformation of all pressures was performed to generate exponentials of the data and provide the best fit. Formulas are given for each group with slopes reported ± standard error of the mean. Differences within groups before and after ventricular fibrillation and reperfusion in both A and B were not statistically significant. (DHW = dry heart weight, LV = left ventricular.)
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Fig 3. Effect of cariporide on postmortem passive pressure-volume relation after ventricular fibrillation and reperfusion. Black circles represent control animals, white circles represent cariporide animals. Formulas for the cubic polynomial functions are reported for each group. The difference in slopes is statistically significant. Solid circles = control: p = -.253 + .173vol + .0023vol2-(2x10-6)vol3; open circles = cariporide: p = .640 + .046vol + .0023vol2-(2x10-6)vol3. (DHW = dry heart weight; LV = left ventricular.)
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Comment
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This study confirms previous work in our laboratory indicating that NHE inhibition preserves systolic function and attenuates diastolic dysfunction after 80 seconds of VF and 40 minutes of reperfusion. This study advances that work by using a drug previously demonstrated to be safe in humans, thus facilitating application to the clinical arena. The protection afforded by cariporide in this experiment is reflected by statistically significant differences in preload recruitable stroke work, ejection fraction, time to recovery of baseline systolic blood pressure, postmortem ventricular compliance, myocardial water content, and normalized LV mass. It is also supported by statistically insignificant trends in end-systolic pressure-volume relations, in vivo ventricular compliance, systolic blood pressure, and cardiac output after 40 minutes of reperfusion, which all favor improved performance by the cariporide group. These results suggest that cariporide could prove valuable for the prevention of ventricular dysfunction during ICD insertion in high-risk patients, particularly inasmuch as the results in our experiment were achieved in animals with normal baseline ventricular function.
In addition to ischemia–reperfusion, other potential injuries were present in our model, including myocardial stunning and shock. Ventricular fibrillation alone causes pathologic changes, including increases in wall stress, ventricular dilatation, and changes in extracellular ionic concentrations. Shock can lead to the elaboration of a circulating myocardial depressant, although this has been more commonly described in septic shock, not hypovolemic shock [6]. Thus the injury in the current experiment was likely multifactorial. Regardless, ICD implantation incurs the same type of injury; therefore a study of pure ischemia–reperfusion injury would be less relevant.
Our results are consistent with previous reports of the use of NHE inhibition during ischemia–reperfusion injury to attenuate systolic dysfunction [7, 8]. Although ischemia and reperfusion injury generally results in impaired diastolic function in the intact heart [9, 10], some contradictory evidence has been described in isolated hearts and trabeculae [11, 12]. A recent study in the isolated rat heart demonstrated that NHE inhibition ameliorated ischemic contracture, prevented postresuscitation diastolic dysfunction, and favored earlier return of contractile function after 25 minutes of iatrogenic VF [13]. Previous studies in pigs have suggested that diastolic dysfunction is worse immediately after resuscitation and improves throughout the reperfusion period [10]. Ischemic contracture has been reported as a cause of reduced diastolic compliance, but the duration of ischemia in our study is too short to support this mechanism.
Although in vivo differences in compliance were not statistically significant, a similar relationship in end-diastolic pressure-volume relations between the control animals and animals pretreated with cariporide was evident in the postmortem studies compared with the in vivo postischemic studies. This suggests that postmortem end-diastolic pressure-volume relations accurately reflect in vivo changes in compliance. In our experience, an advantage of using postmortem passive pressure-volume relations to reflect in vivo ventricular compliance is that it is considerably easier to generate meaningful pressure-volume relations by injecting aliquots of volume directly into the LV while measuring pressure throughout the entire compliance curve than to use a caval occlusion in which it can be difficult to get a broad range of pressures because the starting end-diastolic pressure is low. This is particularly vexing as differences in compliance are difficult to demonstrate at lower end-diastolic pressures. One solution, which we have tried with limited success in previous experiments, is to attempt to volume-load the animals before caval occlusion.
The current results are also consistent with our preliminary data using a different NHE inhibitor, although in that study differences in in vivo LV compliance before and after VF and reperfusion for control animals were statistically significant [2]. Together, these studies are consistent and represent the first evidence that NHE inhibition preserves ventricular function after limited VF and reperfusion.
A few potentially confounding factors deserve consideration. First, although electrical defibrillation can cause direct myocardial cell injury, leading to postischemic myocardial dysfunction, significant injury only occurs at energy levels far exceeding those used to reverse VF in the current study [14]. Negligible or no injury has been reported after single countershocks at energy levels comparable to those used in our study [15, 16]. Second, because animals were subjected to repeated episodes of limited ischemia during preload reduction from caval occlusions, it is worth considering the possibility of a role for ischemic preconditioning or myocardial stunning in our experiment. However, significant differences between animals protected by NHE inhibition and control animals suggest that the drug itself played an important role in these differences. Third, the duration of VF used in the present study is longer than that usually used during ICD insertion, but not outside the range previously reported [17]. Most patients undergoing ICD insertion will not need to endure 80 seconds of VF; however, the concern that patients with severe ventricular dysfunction will not tolerate a limited period of VF is what motivated our study. Finally, a significant number of animals (37%) were excluded from our study, one for technical misadventure, but the others because of inability to undergo successful cardioversion. Clearly this is a concern; however, the animals were evenly distributed between the two groups, suggesting the problem is with the model, not with the drug. Although pigs have notoriously irritable myocardium, owing presumably to poor noncoronary collaterals, our previous use of this model [2] yielded a much lower rate of failure to cardioversion (8%). There were no intentional differences in methods of instrumentation, blood gases, defibrillator equipment, or animal vendor used between the two experiments. Further studies are required to determine whether cariporide affects defibrillation thresholds. Despite these concerns, however, we believe our data supply ample motivation to further investigate the use of cariporide in the setting of ICD insertion.
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Acknowledgments
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The authors thank Dana DeBarr for audiovisual assistance, T. Alexander Quinn for technical assistance, Brianne Blumenthal, and Ivelisse Cruz, May Deutsch, and Monica Castro for clerical assistance. Supported in part by the National Heart, Lung and Blood Institute of the National Institutes of Health (NRSA F32 HL69641-01 to DGR and R01 HL48109 to HMS) and by a start-up grant from the Department of Surgery, Columbia University, College of Physicians and Surgeons.
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Discussion
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DR MARSHALL L. JACOBS (Philadelphia, PA): I would like to compliment you on the lovely presentation and elegantly designed experiment and ask you the following question.
You highlighted in the abstract and in your conclusion the relevance of your model to the situation where you simulate implantable defibrillator insertion and testing. Number one, have you any data as to the potential cardioprotective effect of cariporide in abnormal hearts, in myopathic or chronically ischemic hearts? Number two, are you introducing us to an area where we should be thinking about this mechanism in relation to mitigating reperfusion injury to hearts and other organs in general?
DR RABKIN: Thank you for your kind comments, Dr Jacobs.
In answer to your first question, the relevance of our study in normal pig hearts to human hearts with ischemic cardiomyopathy is uncertain. My speculation, for what it is worth, is that, if anything, benefits of cariporide would be more dramatic in dysfunctional hearts than in healthy hearts, although that would be the subject of a subsequent study.
What was your second question?
DR JACOBS: Whether you are suggesting a biochemical alteration of a cellular mechanism in reperfusion injury that might have much broader implications than in this setting of iatrogenically induced ventricular fibrillation.
DR RABKIN: We are not proposing that on the basis of our results from this study. However, I think that this is a remarkable drug with great potential for legion clinical scenarios, one of which clearly is ischemia–reperfusion injury in a variety of settings.
DR PEDRO J. DEL NIDO (Boston, MA): The use of sodium–proton inhibitors in ischemia–reperfusion injury has gained wide acceptance. My question relates to why you think it works in this particular model. Do you think that you have a significant degree of intracellular acidosis or proton accumulation with 75 seconds of ventricular fibrillation? Is this a regional phenomenon or is it global, or do you think this might more simply affect endothelial cells more than cardiac myocytes?
DR RABKIN: Thank you for your question, Dr Del Nido.
The only difference between these two groups is that one of them had the drug and one of them did not, because the mechanism of the drug is fairly well established. Working from the results backwards, it is clear to me that there must be an intracellular acidotic environment even after a fairly limited amount of ventricular fibrillation, and that must explain the difference in ventricular function after the 45-minute period of reperfusion. However, whether the mechanism of protection in our experiment relates to calicium overload, attenuation of either endothelial injury or oxygen-derived radical injury, or preservation of endothelin and nitric oxide balance is unclear because we did not perform molecular studies or histologic analyses.
DR DEL NIDO: So cariporide has no systemic effects on acid-base balance after an arrest period? In other words, you did not have any changes in acid-base balance in the treatment group versus the control group as far as your blood gases or any of that?
DR RABKIN: After 45 minutes of reperfusion, the blood gases were similar.
DR DEL NIDO: I am talking about during the period of arrest.
DR RABKIN: We did not measure the myocardial pH during the period of ventricular fibrillation.
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References
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Abstract
Introduction
