Ann Thorac Surg 2003;76:1614-1622
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Optimal dose and mode of delivery of Na+/H+ exchange-1 inhibitor are critical for reducing postsurgical ischemia-reperfusion injury
Joel S. Corvera, MDa,
Zhi-Qing Zhao, MD, PhDa,
L. Susan Schmarkey, BSa,
Sara L. Katzmark, BSa,
Jason M. Budde, MDa,
Cullen D. Morris, MDa,
Thomas Ehring, PhDb,1,
Robert A. Guyton, MDa,
Jakob Vinten-Johansen, PhDa*
a Cardiothoracic Research Laboratory and Carlyle Fraser Heart Center, Emory University School of Medicine, Atlanta, Georgia, USA
b Merck KGaA, Darmstadt, Germany
Accepted for publication May 29, 2003.
* Address reprint requests to Dr Vinten-Johansen, Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center, 550 Peachtree St, NE, Atlanta, GA 30308, USA.
e-mail: jvinten{at}emory.edu
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Abstract
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BACKGROUND: In clinical trials, perioperative intravenous Na+/H+ exchange isoform-1 (NHE1) inhibitors were only moderately effective in high-risk patients undergoing surgical reperfusion (GUARDIAN trial). However, effective myocardial concentrations of NHE1 inhibitor may not have been achieved by parenteral administration alone. We tested the hypothesis that increasing doses of NHE1 inhibitor EMD 87580 ((2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine) delivered in blood cardioplegia (BCP) and by parenteral route at reperfusion reduce myocardial injury after surgical reperfusion of evolving infarction.
METHODS: Twenty-six anesthetized dogs underwent 75 minutes of left anterior descending coronary artery occlusion, followed by cardiopulmonary bypass and 60 minutes of arrest with multidose 10°C BCP. In the control group (n = 8), BCP was not supplemented. In the three EMD-BCP groups, BCP was supplemented with 10 µmol/L EMD 87580 (EMD-10, n = 5), 20 µmol/L EMD 87580 (EMD-20, n = 5), or 20 µmol/L EMD 87580 combined with an immediate reperfusion bolus (5 mg/kg intravenously) (EMD-20R, n = 8). The left anterior descending coronary artery occlusion was released just before the second infusion of BCP. Reperfusion continued for 120 minutes after discontinuation of cardiopulmonary bypass.
RESULTS: Postischemic systolic and diastolic function in the area at risk was dyskinetic in all groups. Infarct size (percentage of area at risk) was not significantly reduced in the EMD-10 (26.2% ± 3.6%) and EMD-20 (22.5% ± 2.4%) groups versus control (30.7% ± 2.4%); however, infarct size was significantly reduced in the EMD-20R group (16.1% ± 2.8%, p = 0.003). Edema in the area at risk in the EMD-10 (81.1% ± 0.5% water content), EMD-20 (81.7% ± 0.3%), and EMD-20R (81.9% ± 0.3%) groups was less than in controls (83.2% ± 0.2%), (p < 0.056). Neutrophil accumulation (myeloperoxidase activity) in postischemic area-at-risk myocardium was less in the EMD-20R group versus the control group (5.3 ± 0.7 versus 8.7 ± 1.4 absorbance units · min-1 · g-1; p = 0.05), which suggests an attenuated postischemic inflammatory response.
CONCLUSIONS: Optimal delivery of NHE1 inhibitor to the heart through combined cardioplegia and parenteral routes significantly attenuates myocardial injury after surgical reperfusion of regional ischemia. Timing, dose, and mode of delivery of NHE1 inhibitors are important to their efficacy.
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Introduction
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The sodium/hydrogen exchange isoform-1 (NHE1) is an ion exchange protein found in myocytes and other cell types that is associated with the maintenance of intracellular pH through an electroneutral exchange of an extracellular sodium ion for an intracellular hydrogen ion. During myocardial ischemia, the buildup of intracellular hydrogen ions stimulates increased activity of the NHE1 antiporter resulting in the accumulation of intracellular Na+. Accumulation of Na+ ions is further increased within the cardiac myocyte during ischemia in part because Na+ extrusion is limited by inactivation of the sodium-potassium ATPase pump [1]. The buildup of intracellular sodium ions may contribute to calcium ion influx through a reversal of the sodium/calcium (3 Na+/Ca2+) exchanger, resulting in accumulation of intracellular calcium that overwhelms other calcium-related homeostatic mechanisms [2, 3]. Accumulation of intracellular calcium has been linked to myocyte damage [4, 5]. The NHE1 may become inhibited during prolonged ischemia by extracellular acidosis [6]. However, a sufficient buildup of intracellular H+, as well as of hydrogen peroxide [7] and lysophosphatidylcholine [8] formed during ischemia, and 
-adrenergic agonists [9], among other stimuli, may continue to drive the exchanger even in the presence of extracellular acidosis.
During reperfusion, rapid normalization of extracellular pH removes one inhibitory stimulus on NHE1 and prompts increased exchanger activity, which leads to further intracellular sodium [10] and, in turn, calcium accumulation, with the potential for myocyte injury [11]. It is thought, however, that ischemia-induced intracellular sodium accumulation is the major contributor to myocardial injury, not reperfusion-related intracellular sodium influx [6].
Experimentally, the efficacy of NHE1 inhibitors administered before ischemia relative to administration at reperfusion has been borne out in several studies. Specific and potent inhibitors of NHE1 (HOE 694 [12, 13], HOE 642 or cariporide [14–17], EMD 85131 or eniporide [18]) have consistently reduced myocardial infarct size in models of ischemia and reperfusion using pigs, rabbits, and dogs when administered prior to the ischemic event. However, only three [15, 16, 18] of these seven studies reported a reduction in infarct size when the NHE1 inhibitor was given prior to reperfusion. Therefore, the consensus is that the most effective time to administer NHE1 inhibitors is before ischemia, whereas the efficacy of NHE1 inhibitor administration prior to reperfusion is controversial [19]. Preischemic treatment is often impossible in clinical scenarios of coronary occlusion, but it is applicable to cardiac surgical procedures in which cardiac arrest and global ischemia are electively initiated.
In a clinical study, Rupprecht and associates [20] found that administration of the NHE1 inhibitor cariporide intravenously prior to reperfusion through percutaneous transluminal coronary angioplasty was associated with greater myocardial function after the procedure and reduced myocardial enzyme elaboration in patients with acute myocardial infarction. These results were not reproduced in the ESCAMI trial using parenteral eniporide as an adjunct to either primary angioplasty or thrombolysis for acute myocardial infarction [21]. In agreement with laboratory studies showing inconsistent results using NHE1 inhibitors with reperfusion treatment, intravenous NHE1 inhibition as an adjunct to clinical reperfusion therapy through percutaneous transluminal coronary angioplasty or thrombolysis does not appear to consistently yield beneficial results.
Cariporide was used in a large phase II/III clinical trial (GUARDIAN) comprised of high-risk patients undergoing either percutaneous or surgical reperfusion [22]. Overall, there was no significant difference between treatment groups or dosage regimens. However, there was a significant reduction in nonfatal myocardial infarction and death in a subset of patients undergoing coronary artery bypass grafting and given the highest dosage regimen (120 mg three times daily beginning preoperatively for a total of 2 to 7 days). Although cariporide treatment was initiated prior to a scheduled ischemic event (elective cardiac arrest), optimal myocardial concentrations of NHE1 inhibitor may not have been achieved by parenteral administration during the treatment period, especially in the groups treated with the lower dosage. Hence, both the dosage regimen and the timing of delivery may not have been optimal in the GUARDIAN study to achieve optimal inhibition of NHE1.
Accordingly, we propose that short-term delivery of NHE1 inhibitor in blood cardioplegia (BCP) at appropriate concentrations combined with parenteral delivery at reperfusion attenuates ischemia-reperfusion injury. In the present study, we tested the hypothesis that increasing doses of a new NHE1 inhibitor EMD 87580 (2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine; Merck KGaA, Darmstadt, Germany) delivered in the BCP and by parenteral route at reperfusion reduce myocardial injury after surgical reperfusion of evolving infarction.
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Material and methods
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All animals received humane care in compliance with the Committee on Animal Welfare Act and Emory University Veterinary Policies and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and the National Research Council and published by the National Academy Press (revised 1996).
Twenty-six dogs were premedicated with morphine sulfate (4 mg/kg intramuscularly). General anesthesia was induced with Pentothal (sodium thiopental) (20 mg/kg intravenously) and maintained with a continuous infusion of fentanyl (0.4 µg · kg-1 · min-1) and diazepam (0.003 mg · kg-1 · min-1). Nembutal (sodium pentobarbital) injections (20 to 30 mg/kg intravenously) were administered as needed for supplemental anesthesia. Dogs were endotracheally intubated and mechanically ventilated to maintain a pH of 7.35 to 7.45, an oxygen tension higher than 100 mm Hg, and a carbon dioxide tension of 35 to 45 mm Hg. Intravenous normal saline solution was infused at 10 mL · kg-1 · L-1 and also was given in boluses to counteract intravascular volume depletion at the beginning of the procedure. Polyethylene catheters were inserted into the right femoral artery for monitoring systemic arterial pressure and into the right femoral vein for intravenous drug injection.
The chest was opened using a median sternotomy. Millar MPC-500 solid-state pressure catheters (Millar Instruments, Inc, Houston, TX) were placed into the left ventricle through an apical incision and into the aortic arch through the right internal thoracic artery to continuously measure left ventricular (LV) and aortic pressures, respectively. A proximal portion of the left anterior descending coronary artery (LAD) was dissected free from its surrounding tissue for reversible coronary occlusion with an atraumatic snare. A pair of 2-mm 10-MHz piezoelectric crystals (Sonometrics Corporation, London, ON, Canada) were placed in the subendocardium in the direction of circumferential shortening in the area of the left ventricle subtended by the LAD to measure segmental systolic contractile and diastolic characteristics.
Prior to LAD occlusion, heparin sodium (300 u/kg intravenously) was given for systemic anticoagulation and supplemented (150 mg/kg) thereafter at 90-minute intervals. The LAD was reversibly occluded for 75 minutes before cardiopulmonary bypass (CPB) was initiated. During the ischemic interval, the left subclavian artery was cannulated (20F; Baxter Healthcare Corporation, Midvale, UT), and the right atrium was cannulated with a two-stage cannula (36/46 F; United States Surgical Corporation, Norwalk, CT). A DLP cardioplegia cannula (Medtronic, Inc, Minneapolis, MN) with a pressure-monitoring port was inserted into the proximal ascending aorta for delivery of BCP.
After 75 minutes of total LAD occlusion, CPB was initiated in standard fashion using a membrane oxygenator (COBE Cardiovascular, Inc, Arvada, CO). Mean arterial blood pressure during CPB was maintained at 70 mm Hg by adjusting flow through the arterial cannula. Thermistor probes were placed in the anterior (ischemic-reperfused) and posterior (nonischemic) myocardium to monitor intramyocardial temperature continuously.
Multidose hypothermic, hyperkalemic BCP (66 parts blood to 1 part crystalloid solution, "mini-cardioplegia" [23, 24]) was delivered using the Myocardial Protection System console (Quest Medical, Inc, Allen, TX). The BCP was delivered using the cold induction modality (3-minute induction at 10°C, final concentration of 20 mEq/L K+). Subsequent intermittent antegrade infusions were made at 20 and 40 minutes of arrest and delivered at 10°C for 2 minutes each (10 mEq/L K+). The terminal delivery at 60 minutes of arrest was a modified "hot shot" in which temperature was ramped from 28°C to 37°C (3-minute infusion, 20 mEq/L K+) [25]. The coronary occlusion was released just prior to the 20-minute infusion of BCP, simulating surgical reperfusion through a bypass conduit. All cardioplegia deliveries were made at a pressure of 50 mm Hg. The total time of elective arrest was 60 minutes.
The animals were assigned to one of four groups on the basis of treatment. In the control group (n = 8), BCP was not supplemented. Three groups received EMD 87580 in the BCP. In the EMD-10 group (n = 5), BCP was supplemented with 10 µmol/L EMD 87580. In the EMD-20 group (n = 5), BCP was supplemented with 20 µmol/L EMD 87580. In the EMD-20R group (n = 8), BCP was supplemented with 20 µmol/L EMD 87580, and 5 mg/kg of EMD 87580 was infused systemically through the right femoral vein 5 minutes before the terminal "hot-shot" BCP delivery.
Immediately after completion of the terminal cardioplegia delivery, systemic blood pressure was reduced to 50 mm Hg, and the aortic cross-clamp was removed. After electromechanical reanimation, the perfusion pressure was increased to 80 mm Hg over a 3- to 5-minute period. If the heart was fibrillating on reanimation, it was cardioverted at 20 J using internal paddles. The heart was reperfused on CPB for 30 minutes, after which CPB was discontinued. Reperfusion was continued in the beating and working state off bypass for a total of 2 hours.
The animals were killed with an injection of sodium pentobarbital (100 mg/kg intravenously) at the end of the experiment. The heart was immediately excised and placed in 4°C Krebs-Henseleit buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, 12.5 mmol/L NaHCO3, and 10 mmol/L glucose at pH 7.4).
Data acquisition and analysis
Analog cardiodynamic and hemodynamic data were digitized and stored on a personal computer using SonoView cardiovascular acquisition software (Sonometrics Corporation). Data were collected (1) after instrumentation with the exception of placement of the atriocaval cannula for CPB (baseline), (2) after 60 of the 75 minutes of LAD occlusion (ischemia), and (3) after 60, 90, and 120 minutes of reperfusion off CPB (R60, R90, and R120, respectively). Data analysis was performed using SPECTRUM (Wake Forest University, Winston-Salem, NC).
Physiological end points
Contractile function in the ischemic-reperfused area at risk was described by systolic shortening and segmental work derived from ultrasonic crystals placed in the subendocardium of the area at risk. Segmental myocardial stiffness was determined by fitting the declining end-diastolic pressure–segment length points obtained during transient inferior vena cava occlusion to an exponential curve. The ß coefficient (modulus of diastolic stiffness) represents the degree of curvature of the exponential curve. Percent systolic shortening, segmental work, and segmental myocardial stiffness were determined as previously described [26].
Area at risk and infarct size, plasma creatine kinase (CK) activity, myocardial tissue edema, myocardial myeloperoxidase activity (a marker of neutrophil accumulation), and postexperimental coronary artery endothelial and smooth muscle function were determined as described previously [26].
Statistical analysis
All data are shown as the mean ± the standard error of the mean. Continuous variables between two groups were compared using the unpaired Student t test. A one-way analysis of variance and analysis of variance for repeated measures were used to compare single-point and time-dependent variables in multiple groups, respectively. A Student-Neuman-Keuls post hoc test was used to indentify the source of significant differences detected by analysis of variance. A p value of less than 0.05 was considered significant.
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Results
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Hemodynamic data
At baseline, LV systolic pressure and aortic systolic pressure were significantly greater in the EMD-10 group versus the EMD-20R group (Table 1).
During LAD occlusion, there were no significant differences between the four groups in any hemodynamic variable. Occlusion of the LAD was associated with a significant increase in LV end-diastolic pressure from baseline in the control and EMD-10 groups, but not in the EMD-20 and EMD-20R groups. At 60 minutes of reperfusion, LV systolic pressure and aortic systolic pressure were significantly greater in controls versus the EMD-20 group. At 90 minutes of reperfusion, aortic mean pressure was significantly lower in the EMD-20 group versus the EMD-20R dogs. At 120 minutes of reperfusion, LV systolic pressure, maximal value of the first derivative of LV peak systolic pressure with respect to time, and aortic systolic pressure were significantly greater in the EMD-10 group than in the EMD-20 group. Although these hemodynamic measures at reperfusion were significant, hemodynamic and cardiodynamic determinants of infarct size (ie, heart rate and LV systolic pressure) during ischemia were similar between groups. There were no other group differences in hemodynamics important to development of infarction that were significant (see Table 1).
Regional systolic function in the area at risk
Regional systolic shortening in the area-at-risk myocardium was similar in all groups at baseline (Table 2).
Segmental work, calculated as the area circumscribed by the pressure–segment length loop, was also comparable among groups. There was a significant reduction in segmental shortening and work during ischemia in all groups, with no significant group differences. After 2 hours of reperfusion, there was no significant recovery of systolic shortening or segmental work in any group compared with the dysfunction observed during LAD occlusion.
Segmental diastolic stiffness in the area at risk
The four groups were comparable at baseline in regard to segmental diastolic stiffness (see Table 2). Occlusion of the LAD was not associated with a significant increase in segmental stiffness. Reperfusion at 120 minutes was associated with a significant increase in segmental stiffness from baseline in all groups, with no significant group differences.
Blood cardioplegia
The volumes and characteristics of BCP were similar between groups except the magnesium concentration in select groups and the potassium concentration in the control group during the second and third infusions (Table 3).
The EMD-10 group had significantly greater magnesium content than the EMD-20R group for each delivery of BCP (p < 0.05). Magnesium was not added to the BCP in any group and was derived only from blood.
Infarct size
The area at risk was determined ex vivo after ligation of the LAD at the original location of reversible occlusion and injection of Unisperse blue dye into the coronary arteries by way of the coronary ostia (Fig 1).
The area at risk was comparable in all groups (p = 0.96) (Fig 2).
Infarct size in the EMD-10 (p = 0.29) and EMD-20 (p = 0.13) groups was not significantly smaller compared with the control group, the EMD-10 (p = 0.29) and EMD-20 (p = 0.13) groups did not have a significantly smaller infarct size (see Fig 2). In contrast, infarct size was significantly smaller in the EMD-20R group relative to the control group (p = 0.003).
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Fig 1. Determination of area at risk. The left anterior descending coronary artery (LAD) was ligated at the point of reversible occlusion. Unisperse blue dye was injected directly into right main coronary artery, LAD, and left circumflex coronary artery by way of the coronary ostia to outline area at risk (regions that are not blue).
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Fig 2. Infarct size. Area at risk (AAR) is expressed as percentage of left ventricular mass (LV). Infarct size as area of necrosis (NEC) is expressed as percentage of AAR. * = p = 0.003 versus control and p = 0.056 versus EMD-10. (Control = blood cardioplegia without supplementation; EMD-10 = blood cardioplegia supplemented with 10 µmol/L EMD 87580; EMD-20 = blood cardioplegia supplemented with 20 µmol/L EMD 87580; EMD-20R = blood cardioplegia supplemented with 20 µmol/L EMD 87580 plus infusion of 5 mg/kg of EMD 87580 5 minutes before terminal "hot-shot" cardioplegia.)
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Plasma creatine kinase activity
All groups showed comparable plasma CK activity at baseline (Fig 3).
There was no significant increase in CK activity during LAD occlusion. However, plasma CK activity increased significantly from baseline and ischemia after 60 minutes of reperfusion in all groups. At 120 minutes of reperfusion, there were no significant group differences in plasma CK activity (p = 0.18 by analysis of variance).
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Fig 3. Plasma creatine kinase (CK) activity. (Control = blood cardioplegia without supplementation; EMD-10 = blood cardioplegia supplemented with 10 µmol/L EMD 87580; EMD-20 = blood cardioplegia supplemented with 20 µmol/L EMD 87580; EMD-20R = blood cardioplegia supplemented with 20 µmol/L EMD 87580 plus infusion of 5 mg/kg of EMD 87580 5 minutes before terminal "hot-shot" cardioplegia; R1 = reperfusion 1 minute after aortic cross-clamp removal; R30 = reperfusion for 30 minutes with heart beating during cardiopulmonary bypass; R60, R90, R120 = minutes of reperfusion.)
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Myocardial tissue edema
There was significantly less tissue edema in ischemic-reperfused subendocardium and subepicardium in the EMD 87580–supplemented BCP groups versus comparable regions in controls (p < 0.05 for all comparisons except EMD-20 versus control subepicardial area at risk [p = 0.056]) (Fig 4).
However, there were no significant differences between the EMD-BCP groups.
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Fig 4. Myocardial tissue water content in nonischemic myocardium, ischemic-reperfused area at risk in subepicardium (AAR Epi), and ischemic-reperfused area at risk in subendocardium (AAR Endo). * = p < 0.05 versus control. (Control = blood cardioplegia without supplementation; EMD-10 = blood cardioplegia supplemented with 10 µmol/L EMD 87580; EMD-20 = blood cardioplegia supplemented with 20 µmol/L EMD 87580; EMD-20R = blood cardioplegia supplemented with 20 µmol/L EMD 87580 plus infusion of 5 mg/kg of EMD 87580 5 minutes before terminal "hot-shot" cardioplegia.)
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Myocardial myeloperoxidase activity
Myocardial myeloperoxidase activity was analyzed only in the control and EMD-20R groups. There was no difference in activity between the two groups in the nonischemic myocardium (4.9 ± 1.0 versus 5.1 ± 0.7 absorbance units · min-1 · g-1; p = 0.83). In the control group, myeloperoxidase activity in the transmural area-at-risk myocardium (8.7 ± 1.4 absorbance units · min-1 · g-1) was significantly greater than in nonischemic myocardium (p = 0.03). In the EMD-20R area at risk, activity was significantly less (5.3 ± 0.7 absorbance units · min-1 · g-1) compared with controls (p = 0.05).
Coronary vascular endothelial and smooth muscle function
Maximal relaxation responses to the endothelial-dependent vasodilator acetylcholine are shown in Table 4.
Endothelium-dependent vasorelaxation was significantly lower in the postischemic LAD than in the nonischemic left circumflex coronary artery in both the control group (p < 0.001) and the EMD-10 group (p = 0.004), suggesting postischemic endothelial dysfunction. In contrast, there was no significant difference in endothelial-dependent vasorelaxation between postischemic LAD and nonischemic left circumflex coronary vessels in the EMD-20 and EMD-20R groups, which suggests relatively preserved postischemic LAD endothelial function.
Maximal relaxation responses to sodium nitroprusside are shown in Table 5.
There was no significant difference between groups in the relaxation response of the ischemic-reperfused LAD to nitroprusside. In the control group, there was a significantly greater relaxation response in the left circumflex coronary artery compared with the ischemic-reperfused LAD (p = 0.03).
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Comment
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Inhibition of NHE1 during the period of cardioplegic arrest has been shown to improve postischemic recovery of LV function in isolated Langendorff preparations of the rat and rabbit [27–29]. In dogs, NHE 1 inhibitor in crystalloid cardioplegia decreases myocardial edema and preserves preload-recruitable stroke work after CPB in normal hearts [30, 31]. In the present in vivo canine study of evolving infarction, direct administration of NHE1 inhibitor to the heart during the period of arrest with BCP and early reperfusion was associated with a significant decrease in infarct size confirming less morphologic myocardial injury, a reduction in myocardial tissue edema, and a reduction in neutrophil accumulation in the area-at-risk myocardium suggesting a reduced inflammatory response to ischemia-reperfusion. Neither postischemic regional systolic nor diastolic function was improved with EMD 87580 treatment at any dose or modality. However, postischemic coronary endothelial dysfunction (assayed by the relaxation response to acetylcholine), observed in the control and EMD-10 groups, was attenuated in the EMD-20 and EMD-20R groups compared with the contralateral nonischemic left circumflex coronary artery. Therefore, dose and timing of administration were factors in the efficacy of EMD 87580 in reducing postischemic coronary artery endothelial dysfunction.
Hemodynamic profiles of the groups were similar except for lower postischemic LV and aortic pressures in the EMD-20 group. It is unclear if these hemodynamic differences in the higher-dose EMD-20 group were caused by drug-related vasodilatation, as hypotension was not observed in the highest-dose group (EMD-20R).
In the present study, the NHE1 inhibitor EMD 87580 did not significantly attenuate postischemic infarct size when given only as an adjunct to BCP at 10 and 20 µmol/L concentrations; the reductions in CK release were also not significant. The administration of EMD 87580 as an adjunct to BCP and early reperfusion (EMD-20R group) conferred greater myocardial protection than did the same concentration of EMD 87580 in the BCP but without a systemic reperfusion bolus (EMD-20 group). Therefore, the NHE1 inhibitor EMD 87580 had less effect on infarct size when administered directly to the heart during the period of cardioplegic arrest but provided optimal cardiac protection when sequentially administered directly to the heart in BCP and systemically during early reperfusion. The difference in cardiac protection between the EMD-20 and EMD-20R groups may be a dose-response issue, as the latter group received an additional treatment to the myocardium. It has been speculated that higher drug concentrations are needed when the drug is given during reperfusion to achieve the beneficial effect of the NHE1 inhibitor [32]. In addition, the timing of treatment, prior to reperfusion, may blunt the burst of sodium ion influx [10] and hence calcium entry during the initial moments of reperfusion in accordance with the hypothesis proposed by Lazdunski and co-workers [11].
Muraki and colleagues [26] from our laboratory reported a significant reduction in infarct size and coronary artery endothelial dysfunction with 10 µmol/L cariporide (HOE 642) administered only in hyperkalemic cold BCP using a similar model of acute regional myocardial ischemia and reperfusion. In the present study, EMD 87580 reduced myocardial injury after ischemia and reperfusion, but a systemically administered dose at reperfusion was needed to provide significant protection, which is different from the findings of Muraki and associates [26] using cariporide. Although EMD 87580 is a selective NHE1 inhibitor like cariporide, it is less potent than cariporide, having a 50% inhibitory concentration for NHE1 of 113 ± 6 nmol/L compared with 22 ± 2 nmol/L for cariporide (personal communication, T.E.). The need of a higher BCP concentration (20 µmol/L) and a reperfusion dose (5 mg/kg) with EMD 87580 to show significant myocardial protection may be related to its reduced potency.
The accumulation of neutrophils, which are major inflammatory mediators of myocardial injury in the early period of ischemia and reperfusion [33], was significantly reduced in the EMD-20R group compared with the control group in our study. Muraki and colleagues [26] similarly found that BCP supplemented by cariporide reduced neutrophil accumulation compared with unsupplemented BCP. In addition, using in vitro assays, Muraki and coauthors [26] found that the concentration of cariporide needed to directly attenuate neutrophil degranulation and superoxide production was 10-fold higher than that used to protect the myocyte. Because EMD 87580 is less potent than cariporide, EMD 87580 probably protected the myocyte rather than directly inhibiting neutrophils in the current study. The attenuated accumulation of neutrophils in ischemic-reperfused myocardium observed in this study, therefore, may not represent a direct inhibition of neutrophil function by EMD 87580 but rather an attenuated inflammatory response to reduced postischemic tissue injury.
A limitation of the present study is the relative clinical infrequency of surgical reperfusion therapy for an evolving infarct. Most evolving infarcts are treated by percutaneous reperfusion methods or thrombolysis. The canine model of acute regional ischemia, however, is a well-accepted model of severely injured myocardium that is vulnerable to ischemia-reperfusion injury manifested by infarction and endothelial dysfunction and that is amenable to interventions that attenuate ischemia-reperfusion injury.
Another limitation is the omission of retrograde cardioplegia. Only antegrade cardioplegia was delivered to ensure transmural distribution of EMD 87580 and BCP in the nonischemic myocardium while keeping the area at risk ischemic and "unprotected" until the second infusion of cardioplegia. This construct mimics the clinical situation of completing a distal anastomosis and infusing cardioplegia through the graft.
An additional limitation of the study is that the efficacy of higher concentrations of EMD 87580 in BCP was not determined. Future studies should also separate the effective concentration of EMD 87580 needed to target the myocyte compared with the neutrophil.
In summary, delivery of the new NHE1 inhibitor EMD 87580 as a supplement to hyperkalemic cold BCP with sequential administration at reperfusion in a canine model of surgical reperfusion of evolving infarction attenuated myocardial ischemia-reperfusion injury. The present study and that of Muraki and associates [26] provide evidence that administration of a NHE1 inhibitor directly into an ischemic heart during the period of cardioplegic arrest and on reperfusion is cardioprotective. Parenteral preischemic and postoperative administration of the NHE1 inhibitor, as administered in the GUARDIAN trial, was not necessary to show benefit in this model of surgical reperfusion of severe, acute regional ischemia. Optimal dose and optimal mode of delivery to the myocardium (intracoronary versus intravenous) may be critical factors in determining the efficacy of NHE1 inhibitors in clinically relevant models of myocardial ischemia and reperfusion.
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Acknowledgments
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We are indebted to Jill Robinson for her technical expertise and assistance during this study. We thank Laurie Berley for her help preparing this manuscript.
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Footnotes
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1 Doctor Ehring discloses that he has a financial relationship with Merck KGaA. 
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Abstract
Introduction
