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Ann Thorac Surg 1997;64:1735-1741
© 1997 The Society of Thoracic Surgeons
Departments of Cardiovascular Surgery and Biochemistry and Pathology, and INSERM U-127 and U-141, Hôpital Lariboisière, Paris, France
Accepted for publication June 17, 1997.
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Abstract |
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Methods. Thirty isolated, crystalloid-perfused rabbit hearts were subjected to 60 minutes of normothermic potassium arrest and 60 minutes of reperfusion. They were divided into three equal groups. The first group had no prearrest intervention and served as a control. In the second group, ischemic preconditioning was achieved with 5 minutes of zero-flow ischemia followed by 5 minutes of buffer reperfusion before arrest. In the third group, the hearts were first infused for 5 minutes with the nucleoside transport inhibitor draflazine (10-6 mol/L), the efficacy of which was demonstrated by reversal of the normally high inosine to adenosine ratio in the coronary effluent. These hearts subsequently were given 2 additional minutes of ischemic (zero-flow) preconditioning followed by 5 minutes of reperfusion before arrest. During reperfusion, function was measured serially under isovolumic conditions. Myocardial necrosis was estimated from the release of creatine kinase after the initial 5 minutes of reflow, and the planimetrically determined extent of infarction was determined by triphenyltetrazolium chloride staining.
Results. Baseline hemodynamic data were comparable among the three groups. Neither ischemic preconditioning alone nor ischemic preconditioning with draflazine-induced enhancement of endogenous adenosine levels improved postischemic recovery of function over that seen in control, untreated hearts. These results correlated with a minimal amount of infarction in the control group (on average, <10% of the left ventricle), which was not reduced further by either preconditioning regimen.
Conclusions. These data support the idea that, in the absence of substantial necrosis, ischemic preconditioning does not ameliorate postischemic stunning, which leads to the question of its usefulness in clinical cardiac operations. Although, in this model, protection was not potentiated by increasing endogenous concentrations of adenosine, it remains a worthwhile goal to identify the final effectors of the signaling pathway accounting for the otherwise demonstrated cardioprotective effects of preconditioning because of the potential for these mediators to act as effective antiischemic agents.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Retrograde aortic perfusion was initiated at a constant pressure of 100 cm H2O with filtered Krebs-Henseleit solution (in mmol/L: NaCl, 118; KCl, 4.7; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; CaCl2, 2.5; glucose, 11) bubbled with a mixture of 95% O2/5% CO2. Both the column and the heart chamber were water-jacketed to maintain myocardial temperature at 37°C throughout the experiment.
Functional Measurements
A latex balloon, connected through saline-filled polyethylene tubing to a pressure transducer (Gould Model P23 ID; Cleveland, OH), was inserted into the left ventricle. Output from the transducer was differentiated electronically (Gould Model 13-4615-71) to record the first derivative of left ventricular developed pressure (dP/dt). All hemodynamic data were displayed on a Schlumberger Model OM-4502 chart recorder (Enertec, St. Etienne, France). Coronary flow was measured by timed collection of the coronary venous effluent. Left ventricular pacing was maintained at a rate of 300 beats/min throughout the control and reperfusion periods.
Experimental Protocol
The hearts were allowed to stabilize for 20 minutes, during which the left ventricular balloon was filled with saline to produce a left ventricular end-diastolic pressure of approximately 10 mm Hg. Baseline measurements of peak systolic pressure, positive dP/dt, and coronary flow were made subsequently at this balloon volume.
All hearts then were subjected to 60 minutes of normothermic potassium arrest. Initial asystole was achieved with a single dose of potassium chloride added directly to the Krebs perfusate (to a final concentration of approximately 20 mEq/L), after which the hearts were kept globally ischemic at normothermia. During the ischemic period, the left ventricular balloon was deflated and the pacer was turned off. At the end of arrest, the hearts were reperfused for 60 minutes. The left ventricular balloon was then refilled to the preischemic control volume, and isovolumic measurements of function were obtained after 15, 30, 45, and 60 minutes of reflow. In addition, the coronary venous effluent was collected at the fifth minute of reperfusion and was assayed for creatine kinase activity. At the end of reperfusion, the hearts were removed from the column, sliced into 3-mm-thick sections (after elimination of the atria, right ventricle, and great vessels), and processed for measurement of infarct size by incubation at 37°C for 20 minutes in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 mol/L phosphate buffer, adjusted to pH 7.4. The slices were photographed, and projections of these slides were traced to determine the boundaries of the area at risk (which, because the hearts were made globally ischemic, was the entire left ventricle) and the area of necrosis. These areas were then quantified by computerized planimetry and summed for each heart. Thus, infarct size was expressed as the percentage of the left ventricle that stained negative for triphenyltetrazolium chloride. All these measurements were performed in a blinded fashion.
Experimental Groups
The hearts were assigned randomly to three groups (n = 10 per group) before cardioplegic arrest. The first group consisted of control hearts that received no manipulations during the preischemic period. In the second group, ischemic preconditioning was induced by 5 minutes of total global (zero-flow) ischemia followed by 5 minutes of Krebs buffer reperfusion before cardioplegic arrest. The third group was composed of hearts that were infused with the nucleoside transport inhibitor draflazine (10-6 mol/L) for 5 minutes, followed by 2 minutes of ischemic preconditioning and 5 minutes of reperfusion with normal, drug-free buffer before cardioplegic arrest. Draflazine was a generous gift from Janssen Pharmaceuticals (Bersee, Belgium). It was dissolved in Krebs buffer immediately before use and delivered into the aortic root at a pressure of 100 cm H2O through a separate column.
In the two groups of preconditioned hearts, additional functional measurements were obtained after the preconditioning intervention, immediately before the onset of arrest. In these two groups, aliquots of the effluent obtained after preconditioning and at the end of the 60-minute cardioplegic arrest were also processed for determination of adenine nucleosides using high performance liquid chromatography. These measurements were performed to check that the high inosine to low adenosine ratio normally found in the extravascular space was reversed in the drug-treated hearts, thereby confirming the efficacy of the selected dose. The experimental time course with the various end points is illustrated in Figure 1
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Ischemic preconditioning did not result in a significant decrease in left ventricular systolic function, but caused a twofold increase in diastolic pressure that was not reversed by the subsequent 5-minute period of intervening reperfusion (Table 1). In the drug-treated hearts, the patterns of changes in left ventricular systolic and diastolic function were similar to those yielded by ischemic preconditioning alone (see Table 1). Conversely, the inosine to adenosine ratio measured at the end of the preconditioning intervention was 5.3 ± 1.0 in ischemically preconditioned hearts, whereas the infusion of draflazine reversed this ratio to a value of 1.0 ± 0.2 (p < 0.003).
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and Table 2. Values for contractility indices (left ventricular systolic pressure and dP/dt), diastolic pressure, and coronary flow, pooled over the 60-minute reperfusion period, were not significantly different between the control hearts and the ischemically preconditioned hearts (at the 5-minute postarrest study point, left ventricular dP/dt was even significantly higher in control than in preconditioned hearts). Likewise, shortening of the preconditioning ischemia (from 5 to 2 minutes) and its combination with preischemic administration of the nucleoside transport inhibitor draflazine failed to improve recovery of function or flow over those seen in the two other groups. Five minutes after the onset of reperfusion, hearts preconditioned by draflazine and ischemia even had a significantly higher diastolic pressure and a significantly lower dP/dt than control, nonpreconditioned hearts. This result was concomitant with a sustained effect of the drug, as reflected by an inosine to adenosine ratio which, at the end of the 60-minute period of cardioplegia, was 38 ± 10 in control hearts, 21 ± 2 in ischemically preconditioned hearts, and 0.6 ± 0.1 in hearts pretreated with draflazine before preconditioning (p < 0.0001 versus the two other groups).
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, right panel). Although ischemic preconditioning tended to reduce infarct size further, the difference with the control group failed to achieve statistical significance (p = 0.36). Nor was there any significant difference between infarct size associated with the combination of draflazine infusion and ischemic preconditioning and that of the two other groups. In keeping with these data, there was no significant difference in early postischemic creatine kinase washout among the three groups (see Fig 4, left panel).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The present results support this tight relation between infarct size and postischemic function in the surgical setting of global ischemia. Control hearts that only had cardioplegic arrest had small infarcts at the completion of the experimental protocol, which probably made it difficult to demonstrate any additional infarct size–limiting effect that might have been expected from preconditioning. Notably, the lack of significant differences in the amount of necrotic tissue (as determined by creatine kinase leakage and triphenyltetrazolium chloride staining) between the groups was paralleled by the finding that hearts that had been exposed to preconditioning before arrest did not recover their systolic and diastolic functions to a significantly greater extent than the nonpreconditioned controls. The 5-minute ischemia/5-minute reperfusion prearrest sequence used in our experiments is representative of those that normally elicit a preconditioning response [2]. Thus, failure of these hearts to demonstrate any functional benefit during reperfusion is unlikely to reflect an absence of preconditioning, but rather an inability of preconditioning to relieve stunning in the absence of substantial infarction.
Overall, our results are consistent with those of Kolocassides and coworkers [6], who failed to show any difference in postischemic contractile function between control and preconditioned rat hearts subjected to 35 minutes of arrest. (Of note, this study also reported that preconditioning accelerated contracture, which is in keeping with our observation of increased prearrest diastolic pressures in ischemically preconditioned hearts.) Likewise, Bolling and associates [1] found, in a rabbit model subjected to 2 hours of cardioplegic arrest at 34°C, that 5 minutes of prearrest preconditioning did not improve postischemic function over that seen in control hearts. This observation was paralleled by a lack of histopathologic evidence of necrosis in either group. Conversely, Hendrikx and coworkers [4] reported that preconditioning allowed an improved recovery of function in isolated rabbit hearts exposed to 45 minutes of global ischemia. However, detailed analysis of the protocol used in this study shows that no protection was used during the ischemic period. Not unexpectedly, we have found, in additional experiments, that this resulted in a substantial amount of necrosis in control hearts (approximately 70% of the left ventricle), thereby offering much room for preconditioning to reduce infarct size and, consequently, improve function during reperfusion. For unclear reasons, the results of Hendrikx and associates [4] are at variance with those reported recently by Asimakis and coworkers [7], who found that single-cycle or triple-cycle preconditioning was unable to improve contractile function of rabbit hearts exposed to global unprotected ischemia (unfortunately, infarct size was not measured in this study either).
Overall, the experimental observations cited earlier are corroborated by the clinical findings made in patients undergoing coronary artery bypass grafting operations. Thus, Alkhulaifi and coworkers [8] reported that two cycles of ischemic preconditioning (each consisting of 3 minutes of aortic cross-clamping followed by 2 minutes of reperfusion) resulted in better preservation of myocardial levels of adenosine triphosphate (ATP) after a subsequent 10-minute period of normothermic ventricular fibrillation. However, this isolated biochemical result has limited clinical relevance, as it has long been demonstrated that myocardial ATP concentrations do not correlate readily with function. Conversely, we [9] have been unable to document any benefit of a single cycle of preconditioning before warm cardioplegic arrest. It is likely that, in the first case, preconditioning was beneficial because it slowed the rate of ATP depletion otherwise associated with ventricular fibrillation at normothermia. In the second scenario, however, this ATP-sparing effect of preconditioning, which is attributed to the opening of potassium channels and the subsequent cardioplegic effect of increased extracellular potassium accumulation [10], probably became redundant to that of cardioplegia.
The above considerations explain why the two experimental situations in which preconditioning has been shown to add protection to that already provided by cardioplegia are long ischemic times [11] and impaired distribution of antegradely delivered cardioplegic solution [12]. In these two settings, it is reasonable to hypothesize that myocardial protection may have been suboptimal, thereby resulting in some degree of myocardial necrosis, which was amenable to limitation by preconditioning. However, the fact that preconditioning may not exert protective effects on postischemic cardiac function in the absence of a discrete area of necrosis does not necessarily imply that it is of no interest in cardiac operations. Rather, it emphasizes the importance of identifying the pharmacologic mediators of this adaptive phenomenon with the hope of therapeutically exploiting them as antiischemic agents. Among these mediators, adenosine has received particular attention, especially in the rabbit heart [13, 14].
Adenosine and Preconditioning
The role of adenosine in mediating the cardioprotection provided by preconditioning has been suggested by the observations made in rabbits, dogs, and pigs that the administration of this nucleoside or of agonists of its A1 receptors could duplicate the effects of ischemic preconditioning, whereas these effects were abolished by adenosine receptor blockers [15]. More recently, however, this hypothesis has been challenged by some studies showing that the cardioprotective effects of adenosine did not fully overlap those of an ischemically induced preconditioning regimen [4, 5].
In the present study, we used the nucleoside transport inhibitor draflazine to increase the concentration of endogenous adenosine [16]. Although we did not measure adenosine levels in myocardial tissue directly, the adequacy of the dosage regimen was suggested by the reversal of the inosine to adenosine ratio in the coronary venous effluent of draflazine-treated hearts [17], both after preconditioning and at the end of the subsequent period of cardioplegic arrest. Namely, an increase in circulating levels of adenosine is thought to reflect the accumulation of this nucleoside in the interstitial space and its subsequent escape into the vascular lumen through clefts within the endothelial barrier, instead of being transported into endothelial cells where it would otherwise be degraded, in particular to inosine (hence the high inosine/low adenosine ratio normally seen in the coronary effluent in the absence of any pharmacologic blockade of adenosine transport into endothelial cells) [16].
A first hypothesis for explaining the failure of draflazine to improve functional recovery over that seen in the two other groups (recovery was even worse than in controls at the very beginning of reperfusion) is that the 2-minute period of ischemic preconditioning used after administration of the drug was not long enough to elicit an increase in endogenous adenosine, which was then expected to accumulate in the interstitial fluid because of the previous exposure to draflazine. The lack of benefits seen in the drug-treated hearts would then be consistent with the inability of increased adenosine levels to induce protection in the virgin, ie, nonpreconditioned heart [18]. However, this hypothesis is challenged somewhat by the results of microdialysis techniques showing that a 5-minute period of preconditioning ischemia was associated with an increase in interstitial adenosine levels that started from the onset of the preconditioning stimulus [19]. Thus, the failure of draflazine to potentiate the cardioprotective effects of ischemic preconditioning alternatively could suggest that, at least in our model, these effects are not directly dependent on increased concentrations of endogenous adenosine. This conclusion is consistent with the results of Hendrikx and coworkers [4] showing that adenosine A1 receptor activation was not the primary mechanism whereby preconditioning could improve functional recovery in rabbit hearts subjected to an episode of unprotected global ischemia. Our data are also consistent with the previous findings of Lasley and associates [5] who demonstrated, using the globally ischemic rabbit heart, that if pharmacologic activation of adenosine A1 receptors was terminated before ischemia (which corresponds to true preconditioning, as opposed to pretreatment, which would continue until arrest), it did not reduce infarct size or improve functional recovery. That adenosine does not completely mediate the cardioprotective effects of ischemic preconditioning is supported further by the disparate effects of preconditioning and adenosine on interstitial adenosine concentrations and on the time course of protection. Thus, during a period of regional ischemia, interstitial levels of adenosine decrease after ischemic preconditioning (which is consistent with the idea that preconditioning reduces the rate of adenine nucleotide catabolism), whereas they remain unchanged after adenosine preconditioning [19]. Furthermore, the adenosine-induced cardioprotection subsides much more rapidly than that elicited by ischemic preconditioning [20]. Taken together, these data may account for the failure of the draflazine-induced expected increase in endogenous levels of adenosine to enhance protection in our ischemically preconditioned hearts, as their functional recovery and ultimate infarct size were not significantly different from those seen in their nonpreconditioned counterparts. Nevertheless, it should be stressed that the above conclusions strictly pertain to the use of draflazine and cannot be extrapolated to the use of other nucleoside transport inhibitors [21]. Nor can we completely exclude that the use of draflazine in other experimental setting (eg, as a pretreatment continued until the onset of arrest or as an additive to cardioplegia) might yield more convincing cardioprotective effects.
Limitations and Implications of the Study
Several methodologic features of this study should make one cautious when extrapolating its results to the clinical setting. These include the use of an isolated heart model, the crystalloid nature of the perfusate, and the maintenance of strict normothermia during arrest. Species differences with regard to the response to preconditioning are an additional confounding factor [14]. Within a given species, the effects of preconditioning might even be different depending on whether the end point is reduction of infarct size, amelioration of stunning, or prevention of arrhythmias. These factors may account, at least in part, for the conflicting results regarding the role of adenosine as the main trigger of the phenomenon.
With these caveats in mind, however, we conclude that the results do not support the use of ischemic preconditioning before routine cardioplegia, a conclusion consistent with our previous clinical findings [9]. This observation does not dismiss the search for the pharmacologic mediators of preconditioning, with the ultimate goal of therapeutically exploiting their antiischemic properties. In this setting, our data raise some concerns about interventions targeted at increasing endogenous concentrations of adenosine. In fact, based on the literature [22, 23] and personal data [24, 25], we believe that potassium channel openers might turn out to be more effective for duplicating the cardioprotection attributed to preconditioning.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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