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Ann Thorac Surg 2004;77:1398-1407
© 2004 The Society of Thoracic Surgeons
a Cardiac Surgical Research and Cardiothoracic Surgery, The Rayne Institute, Guy's and St. Thomas' NHS Trust, St. Thomas' Hospital, London, United Kingdom
b Molecular Cardiology, The Rayne Institute, Guy's and St. Thomas' NHS Trust, St. Thomas' Hospital, London, United Kingdom
Accepted for publication September 15, 2003.
* Address reprint requests to Dr Chambers, Cardiac Surgical Research, Cardiothoracic Surgery, The Rayne Institute, Guy's and St Thomas' NHS Trust, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK
e-mail: david.chambers{at}kcl.ac.uk
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Abstract |
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METHODS: Isolated rat hearts were Langendorff-perfused (20 mins) with bicarbonate buffer and function (left ventricular developed pressure) measured. In each of three separate protocols that incorporated progressively longer ischemic durations, hearts were randomly allocated to one of three groups: group 1 was the control group with 40, 60, or 80 minutes of continuous global ischemia. Group 2 was the ICCF group with 4, 6, or 8 cycles of 10 minutes ICCF and 10 minutes of reperfusion in sinus rhythm. Group 3 was the ICCF plus cariporide group, which was the same as group 2, but also with 3 µmoles/L cariporide present in perfusate from 10 minutes before the ICCF cycles. Hearts were reperfused for 60 minutes with drug-free buffer and recovery (percentage of initial function) was measured. Hearts were maintained at 37°C throughout the protocols. In protocol 3 (80 minutes ischemia per 8 cycles of ICCF), creatine kinase leakage (myocardial injury) and triphenyl tetrazolium chloride staining (myocardial viability) were also measured. Protocols 1, 2, and 3 had n = 8 hearts, n = 6 hearts, and n = 6 hearts in each group, respectively.
RESULTS: In the three protocols, the recoveries of left ventricular developed pressure in the control group, the ICCF group, and the ICCF plus cariporide group, respectively, for protocol 1 were: 26% ± 3%, 70% ± 2% (p < 0.05 vs the control group) and 74% ± 2% (p < 0.05 vs the control group), respectively. For protocol 2 these were: 16% ± 2%, 55% ± 1% (p < 0.05 vs the control group), and 70% ± 3% (p < 0.05 vs the control and ICCF groups), respectively. For protocol 3 these were: 8% ± 2%, 41% ± 3% (p < 0.05 vs the control group), and 63% ± 2% (p < 0.05 vs the control and ICCF groups), respectively. Recovery of left ventricular end-diastolic pressure mirrored that of left ventricular developed pressure in all protocols. In protocol 3, total creatine kinase leakage (international units per gram wet weight) was 88 ± 12, 47 ± 4 (p < 0.05 vs the control group), and 17 ± 1 (p < 0.05 vs the control and ICCF groups), respectively, and triphenyl tetrazolium chloride staining (arbitrary units per gram wet weight) was 0.17 ± 0.04 in the control group, 0.39 ± 0.04 (p < 0.05 vs the control group) in the ICCF group, and 0.47 ± 0.08 (p < 0.05 vs the control group) in the ICCF plus cariporide group, respectively.
CONCLUSIONS: Sodium-hydrogen exchanger inhibition with cariporide enhances the myocardial protection afforded by ICCF, with the additive benefit becoming more apparent with increasing severity of the ischemic insult. Sodium-hydrogen exchanger inhibition may provide a significant protective reserve during ICCF, particularly when longer procedures are required.
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Introduction |
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Numerous studies have now shown that inhibition of the sodium-hydrogen exchanger (NHE) affords significant protection from myocardial injury during ischemia and reperfusion [10], most likely through the attenuation of intracellular sodium and consequently, calcium accumulation [11]. In the context of cardiac surgery, in experimental studies the specific NHE inhibitor cariporide (Aventis Pharma, Frankfurt, Germany) has been shown to provide enhanced protection when used as an additive or an adjunct to cardioplegia at a variety of temperatures [12]. Furthermore the benefit afforded by cariporide has been shown to be additive to that afforded by preconditioning [13]. In recent years, several clinical trials with NHE inhibitors also have been completed with preliminary evidence that cariporide may provide significant cardioprotective benefit in high-risk patients undergoing CABG surgery [14, 15]. In addition, studies [16] in the rat model of VF arrest and resuscitation have demonstrated increased spontaneous defibrillation and survival when treated with cariporide.
The individual cardioprotective efficacy of both ICCF and NHE inhibition is now well established, but there have been no reports in which the interaction between these two modalities has been studied. Therefore we conducted an experimental study to investigate whether cariporide used in combination with ICCF can enhance the protective effect of ICCF.
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Material and methods |
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Heart isolation and perfusion
The heart was excised from the anesthetized rat and immersed in cold (4°C) Krebs-Henseleit buffer. The aorta was then rapidly cannulated and the heart was perfused in the Langendorff mode with Krebs-Henseleit buffer at a constant pressure (75 mm Hg) and temperature (37°C) within 30 seconds of excision. The pulmonary artery was incised to allow free drainage of coronary effluent.
A deflated ultrathin intraventricular balloon, constructed from cling film (Saran Wrap [SC Johnson, Racine, WI]) over the tip of a 20-gauge cannula (made to match the internal dimensions of the left ventricle) was introduced through the mitral valve into the left ventricle. The balloon was attached to a pressure transducer from which the calibrated output was recorded on an Apple Macintosh computer (Apple Computer Inc, Cupertino, CA) using the PowerLab system (ADInstruments Ltd, Hastings, East Sussex, UK).
All hearts were subjected to an equilibration period of aerobic perfusion (37°C) for 20 minutes. At the beginning of the period, the intraventricular balloon was gradually inflated with water to give a stable left ventricular end-diastolic pressure (LVEDP) of 3.0 to 8.0 mm Hg, and this isovolumic state was maintained throughout the rest of the protocol. Baseline readings of left ventricular systolic pressure (mm Hg), LVEDP (mm Hg), heart rate (beats per minute), and coronary flow rate (mL per minute) were taken at the end of the 20-minute equilibration period. Left ventricular developed pressure (LVDP) was calculated as the left ventricular systolic pressure minus LVEDP. Coronary flow rate was measured by timed collections of the coronary effluent. The development of contracture during ischemia was recorded by means of the intraventricular balloon. After the baseline readings, the hearts were randomly assigned to one of three groups in each protocol (Fig 1) and were perfused with aerobic perfusion medium with or without added cariporide (3 µmol/L) for a further 10 minutes.
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Exclusion criteria
The experimental design required that hearts not fulfilling preassigned functional criteria at the time of the baseline readings should be excluded from the study. The acceptable values for LVDP, heart rate, and coronary flow rate were more than 100 mm Hg, more than 220 beats per minute, and 8 to 16 mL per minute, respectively. No hearts were excluded in this study.
Induction and termination of VF
VF was induced by an electrical fibrillator (model G570, Department of Bioengineering, St Thomas' Hospital, London, UK) by passing alternating current through two silver electrodes coated with silicone; one was attached to the apex of the ventricle and the other to the aortic cannula for grounding. The minimum current necessary to achieve an alternating current that maintained VF was usually within 2.5 mA. If VF occurred during reperfusion or did not terminate spontaneously after the fibrillator was turned off, it was terminated by the use of a defibrillator (model G434, Department of Bioengineering, St Thomas' Hospital, London, UK). The initial direct current shock energy was set at 40 mJ; if this failed to induce defibrillation, energy was progressively increased to 70, 100, 150, 200, 250, or 300 mJ. At the next requirement for defibrillation, the energy band immediately below the previous successful shock was tried (eg, if 70 mJ was successful, 40 mJ was chosen at the next challenge).
Drug administration
Cariporide (HOE-642) was a gift from Aventis Pharma (Frankfurt, Germany) and it was dissolved in de-ionized water to make a 3 mmol/L stock solution, which was stored at 4°C for a maximum of 7 days. The stock solution was diluted in perfusion solution to obtain a final drug concentration of 3 µmol/L immediately before use. This concentration was chosen because it is sufficient to fully inhibit sarcolemmal NHE activity in rat ventricular myocytes [17].
Experimental protocols
Three protocols were conducted, each of which comprised three groups (the control group, ICCF group, and intermittent cross-clamp fibrillation plus cariporide [ICCF plus Car] group; see Fig 1) that were subjected to an identical cumulative duration of global ischemia. In the control groups, hearts were subjected to 40, 60, or 80 minutes of global ischemia, respectively. In the ICCF groups, hearts were subjected to 4, 6, or 8 cycles of 10 minutes of global ischemia with VF followed by 10 minutes of reperfusion in sinus rhythm. In the ICCF plus Car groups, hearts were again subjected to 4, 6, or 8 cycles of 10 minutes of global ischemia with VF followed by 10 minutes of reperfusion in sinus rhythm; however, this time cariporide (3 µmol/L) was included in the perfusion medium from 10 minutes before the start of the first cycle to the start of the last cycle. Hearts in all groups were then subjected to 60 minutes of reperfusion during which recovery of myocardial function (LVDP, LVEDP and coronary flow rate) was monitored. Hearts were maintained at 37°C throughout the protocols. In protocol 3, which incorporated the most severe ischemic insult, additional indices of myocardial injury (creatine kinase leakage) and viability (triphenyl tetrazolium chloride staining) were also measured after reperfusion. In protocols 1, 2, and 3 there were 8 hearts, 6 hearts, and another 6 hearts in each group, respectively.
Contractile function
Postischemic recovery of LVDP was expressed as a percentage of the baseline value at the end of the initial 20-minute equilibration period. Left ventricular end-diastolic pressure was expressed in mm Hg.
Creatine kinase leakage
Total creatine kinase leakage (expressed as IU/g heart wet weight) was assessed using a commercially available kit (Sigma-Aldrich Diagnostic kits, Poole, Dorset) by spectrophotometric analysis of enzyme activity in the coronary effluent collected during the reperfusion periods.
Triphenyl tetrazolium chloride staining
Hearts were immediately frozen in liquid nitrogen at the end of the 60-minute reperfusion period and stored for later analysis. For analysis, using a modification of the method by Ferrera and colleagues [18], the hearts were placed into a conical tube, minced into small pieces in 20 mL of phosphate buffer solution containing triphenyl tetrazolium chloride (1 mmol/L at final concentration) and incubated for 90 minutes at 37°C. The buffer was decanted and the heart tissue was homogenized in 5 mL dimethyl sulfoxide and was agitated at a shaking table for 90 minutes. The homogenate was then centrifuged at 10,000 rpm for 1 minute, the absorbance of the supernatant was measured at 480 nm, and the results were expressed as arbitrary units per gram heart wet weight.
Statistics
Statistical analysis was performed with StatView (Abacus Concepts Inc, Berkeley, CA) on an Apple Macintosh computer (Apple Computer, Cupertino, CA). All data are reported as mean ± standard error of the mean. Comparisons between groups were assessed for significance by one-way analysis of variance with post hoc analysis by means of Fisher's protected least significant difference test, which allowed for multiple comparisons. The Student's unpaired t test (or when appropriate, the Mann–Whitney U test) was used to make comparisons between the two groups. A value of p less than 0.05 was considered statistically significant.
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Results |
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Controls hearts in protocol 3 (Fig 2C) showed a similar pattern of recovery to those in protocol 2, with no recovery for 30 minutes, but slight recovery over the subsequent 30 minutes of reperfusion. As with the previous protocols, hearts subjected to ICCF and ICCF plus Car recovered rapidly to a plateau value by 5 minutes of reperfusion. Recovery in hearts in the ICCF plus Car group was significantly higher than that in the ICCF group throughout reperfusion.
The final recovery at 60 minutes of reperfusion in all groups of hearts in the three protocols (shown in Fig 3) clearly indicates that increasing durations of ischemia, either continuous or intermittent, lead to a considerable decrease in postischemic function. However, the presence of cariporide attenuates this decrease.
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Similar changes were observed in protocol 3 (Fig 5C), with LVEDP in control hearts peaking at 4 minutes and gradually declining to around 90 mm Hg. In ICCF hearts, LVEDP had a significant secondary increase and remained elevated at 30 mm Hg, significantly higher than the ICCF plus Car hearts, which again declined to plateau values that were not different to baseline. These differences are seen more clearly in Fig 6, which show the final LVEDP values after 60 minutes of reperfusion in all groups of hearts in the three protocols. Increasing durations of ischemia or increasing episodes of ICCF caused LVEDP levels to increase. In contrast, cariporide was able to prevent the increased LVEDP and maintain values at levels similar to baseline.
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Creatine kinase leakage
In protocol 3, which was associated with the most severe ischemic insult, we also measured creatine kinase leakage as an index of myocardial injury. Total creatine kinase leakage was significantly reduced in ICCF or ICCF plus Car hearts (Fig 7A).
Interestingly the addition of cariporide significantly reduced creatine kinase leakage compared with ICCF alone. Cariporide also significantly reduced creatine kinase leakage at each reperfusion time during ICCF cycles compared with ICCF alone (Fig 7B). Thus, intergroup differences in recovery of LVDP were reflected by differences in creatine kinase leakage.
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Comment |
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The safety and efficacy of noncardioplegic methods of myocardial protection (such as VF with or without intermittent aortic cross clamping) have been previously demonstrated in elective primary and reoperative CABG patients, assessed by clinical outcome [2, 5, 8], cardiac-specific enzymes [3, 4, 8], free radical activity [6] and from preoperative and postoperative changes in the electrocardiogram [3, 6, 8] as well as in higher risk patients [1, 19]. Casthely and colleagues [20] found that diastolic function impairment in the immediate perioperative period was minimal when ICCF was used compared with cold blood cardioplegia. In a recent clinical study, Antunes and coworkers [19] reported that the use of intermittent VF without aortic occlusion afforded good myocardial protection and operating conditions with excellent applicability, even in patients with severe left ventricular dysfunction. In comparative studies of ICCF and cardioplegic protection, the cumulative ischemic period for ICCF was generally significantly shorter than the ischemic period when cardioplegic protection was used; this could account for comparative or improved protection by inducing less ischemic injury. However, in an experimental study in which the same cumulative duration of ischemia was used, Bessho and Chambers [9] demonstrated similar levels of myocardial protection with either multidose cardioplegia or intermittent cross clamping (with or without VF) indicating intrinsic protection by ICCF. Interestingly, they showed that the technique of VF with maintained coronary perfusion was detrimental. Their results confirmed the safety and efficacy of using ICCF during CABG surgery.
Sunderdiek and coworkers [21] recently suggested that myocardial protection with the technique of ICCF may be suboptimal when the total ischemic duration was longer than 40 minutes. Our data are consistent with these results [21], because we showed that longer cumulative ischemic periods resulted in significantly worse recovery of LVDP and maintained elevation of LVEDP in the ICCF groups. However, the present study suggests that concomitant administration of the NHE inhibitor cariporide may be a means of maintaining effective myocardial protection with ICCF even when longer periods of ischemia become necessary.
At the present time it remains unclear whether ICCF protects the myocardium in a manner similar to the phenomenon of ischemic preconditioning [22]. In a cardiopulmonary bypass study in dogs, Abd-Elfattah and colleagues [23] used multiple intermittent cross-clamping episodes and showed that ATP levels were maintained after the second episode of ischemia and reperfusion and were significantly higher at the end of the intermittent protocol than after a comparative period of sustained ischemia. Thus, because myocardial high energy phosphates are preserved by ischemic preconditioning [22, 24], it was suggested that intermittent cross-clamping episodes may also be acting through an endogenous protective mechanism analogous to preconditioning. In contrast, controversial results exist in human myocardium. The technique of intermittent ischemia with fibrillation was shown to be less effective for cardioprotection than when a preconditioning stimulus was used before ICCF and perioperative troponin T was measured as the endpoint [25]. However, Jenkins and coworkers [26] reported that in the human heart there was no cumulative ATP depletion after the initial 10-minute ischemic episode during intermittent cross clamping in the absence of preconditioning stimuli. Although the question of whether ICCF acts through a preconditioning mechanism is of considerable interest, it was not the objective of this study. Our aim was to determine whether cariporide could enhance the protective efficacy of a myocardial protective technique used by some surgeons during myocardial revascularization surgery, and the protocol defined in this study for ICCF is based on this clinical use in our institution.
The cardiac sarcolemmal NHE is a major regulator of intracellular pH and is activated by ischemia-induced intracellular acidosis (see recent review: [11]). The activated exchanger extrudes intracellular H+ in exchange for Na+ in an attempt to restore intracellular pH. The consequent accumulation of intracellular Na+ in turn leads to an increase in intracellular Ca2+ due to reduced Ca2+ efflux or increased Ca2+ influx through the Na+/Ca2+ exchanger, or both. This rise in intracellular Ca2+ (Ca2+ overload) is believed to be a major contributor to cardiac arrhythmia, myocardial stunning, and irreversible cell injury. Indeed, there have been many studies in which a marked cardioprotective effect against cardiac arrhythmias and myocardial stunning and infarction has been demonstrated by NHE inhibitors [10, 11]. In this study, we used cariporide, which is a specific inhibitor of NHE isoform 1 [27] (the molecular homolog of the cardiac sarcolemmal NHE) and has been shown to be efficacious for cardioprotection in numerous animal models of myocardial ischemia and reperfusion [10, 11]. Furthermore, there is suggestive clinical evidence that cariporide may provide significant myocardial protection in high-risk patients undergoing CABG surgery [14, 15], and the efficacy of cariporide in such patients is being tested in a phase III clinical trial [15].
The majority of studies that have shown significant myocardial protection with NHE inhibitors in animal models have used prolonged periods of ischemia. Nevertheless, there is evidence of significant intracellular accumulation of Na+ [28] and Ca2+ [29], even during brief periods of global ischemia such as those used in our study. Interestingly, in these studies the intracellular accumulation of Na+ and Ca2+ during the first 10 minutes of ischemia was significantly blunted by cariporide treatment [28, 29], suggesting that this occurred through NHE-mediated mechanisms. Indeed, there is now evidence from multiple laboratories that myocardial stunning arising from repeated brief episodes (5 to 10 minutes) of ischemia and subsequent reperfusion may be attenuated by NHE inhibition with cariporide [30] or a closely related compound [31]. Such amelioration of stunning is likely to contribute to the additive benefit afforded by cariporide in the present study, in hearts subjected to a moderate cumulative ischemic insult (4 to 6 cycles of ICCF). However, with a more severe insult it is likely that a limitation of irreversible injury (infarction) contributes to the additive benefit of cariporide. In support of this, our data show that in hearts that are subjected to 8 cycles of ICCF, cariporide not only improves the postischemic recovery of systolic and diastolic left ventricular function, but it also limits myocardial injury as reflected by creatine kinase release. In addition, cariporide induced a tendency toward improved myocardial viability as reflected by triphenyl tetrazolium chloride staining. Our inability to detect a significant difference from ICCF hearts may reflect a discrepancy in the sensitivity of this method as a marker of viability, relative to that of creatine kinase release as a marker of injury. Our study also showed that treatment with cariporide significantly reduced the cumulative energy of direct current shocks required to restore sinus rhythm after episodes of ICCF. This is consistent with recent data that NHE inhibition promotes spontaneous defibrillation in rat models of resuscitation [16, 32] and our earlier work that NHE inhibition suppresses reperfusion-induced VF [33].
In the clinical setting, cardioprotection by additional NHE inhibition during ICCF could provide an additional element of safety in situations in which more ischemia-reperfusion cycles may be required (eg, for increased numbers of anastomoses for complete revascularization or reanastomosis for bleeding complications).
Limitations of the study
In the present study, our findings were obtained in the normal rat heart. Therefore we are unable to extend our findings to hearts with extensive coronary artery disease in which heterogeneous distribution of cariporide may affect its efficacy. During cardiac surgery, the ICCF procedure is used only for myocardial revascularization; the fixed protocol durations of ICCF and reperfusion described in this experimental study are more rigorous than would occur during surgery. Clinically, the ICCF period correlates to the distal anastomosis of the graft onto the heart and is unlikely to exceed 12 to 13 minutes (but maybe shorter); similarly, the proximal anastomosis of the graft to the aorta correlates to reperfusion. Another possible limitation of the present study may be the use of an isolated heart preparation that does not possess a collateral circulation.
Conclusion
In conclusion, in an experimental preparation of isolated rat hearts, the use of the specific NHE inhibitor cariporide in combination with ICCF provided a significant improvement in myocardial protection, especially under conditions of extended ischemia-reperfusion cycles. Therefore this agent may be of benefit when used in combination with ICCF for myocardial preservation during coronary artery bypass surgery.
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Acknowledgments |
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This article has been cited by other articles:
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  D. J. Chambers and M. Fujii Reply to Mishra Eur J Cardiothorac Surg, May 1, 2006; 29(5): 861 - 861. [Full Text] [PDF]
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= control group; 
= intermittent cross-clamp fibrillation group; 
= intermittent cross-clamp fibrillation plus cariporide group. Values are mean ± standard error of the mean of either 8 hearts per group in protocol 1 or 6 hearts per group in protocols 2 and 3. *p less than 0.05 versus the control group; 
p less than 0.05 versus the control and intermittent cross-clamp fibrillation groups.
= ICCF during ischemia; 
= ICCF+Car during reperfusion. (I = ischemia; LVEDP = left ventricular end-diastolic pressure during reperfusion phases; LVP = left ventricular pressure during ischemic phases; R = reperfusion.)
