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(Circulation. 1995;92:912-917.)
© 1995 American Heart Association, Inc.

Articles

Myocardial Protection by Na⁺-H⁺ Exchange Inhibition in Ischemic, Reperfused Porcine Hearts

Hermann H. Klein, MD; Sibylle Pich, PhD; Rainer M. Bohle, MD; Jutta Wollenweber; Klaus Nebendahl, VMD

From the Department of Cardiology, University of Marburg (H.H.K., S.P.); the Department of Experimental Animal Research, University of Göttingen (J.W., K.N.); and the Department of Pathology, University of Giessen (R.M.B.), Germany.

Correspondence to Prof Dr H.H. Klein, Abteilung für Kardiologie, Städt Krankenanstalten Idar-Oberstein GmbH, Dr Ottmar-Kohler-Str 2, 55743 Idar-Oberstein, Germany.

	Abstract

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Background Studies in isolated myocytes and isolated heart preparations have suggested that Na⁺-H⁺ exchange is an important mechanism for myocardial ischemia–reperfusion injury. This study was undertaken to determine whether inhibition of Na⁺-H⁺ exchange limits infarct size and improves regional systolic shortening in regional ischemia and reperfusion in intact pigs.

Methods and Results The left anterior descending coronary artery was occluded in 18 anesthetized and thoracotomized pigs for 45 minutes and then reperfused for 24 hours. As main end points of this study, regional systolic shortening (sonomicrometry) and infarct size (percentage of infarcted to ischemic myocardium) were determined at the end of the experiments. Infarcted myocardium was assessed by histochemistry (tetrazolium stain) and by quantitative histology of one heart slice. The Na⁺-H⁺ exchange inhibitor Hoe 694 was administered intravenously at a dose of 3 mg/kg in 6 pigs each either 10 minutes before ischemia (group A) or 10 minutes before the onset of reperfusion (group B). Six pigs served as controls (group C). Treatment with Hoe 694 before ischemia decreased histochemical infarct size from 65±18% (control group) to 13±8% (P<.01) and histological infarct size from 49±20% (control group) to 14±4% (P<.01). Histochemical (55±19%) and histological (42±15%) infarct sizes of group B were insignificantly reduced by 15%. Myocardial protection in group A was associated with an attenuated contracture after 10 minutes of reperfusion and an improved regional systolic shortening after 24 hours of reperfusion (group A, 25±12%; control group, 6±5%; P=.01). These parameters remained unaffected in group B.

Conclusions This study clearly demonstrates that Na⁺-H⁺ exchange is an important mechanism for cell death in myocardial ischemia and reperfusion in intact pigs; thus, inhibition of this exchange system may prove a promising new strategy in the clinical treatment of myocardial ischemia and reperfusion.

Key Words: reperfusion • ischemia • Na⁺-H⁺ exchange • infarction

	Introduction

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Reperfusion achieved by thrombolysis or percutaneous transluminal coronary angioplasty has become standard treatment for patients with acute myocardial infarction. A further improvement in the treatment of acute myocardial infarction can be expected by identification and prevention of the pathobiochemical mechanisms causally involved in the transition from reversible injury to myocardial necrosis. Several studies in isolated myocytes or isolated heart preparations^{1 2 3 4 5 6 7} suggest that Na⁺-H⁺ exchange is linked to intracellular sodium and calcium overload during ischemia and reperfusion, assumed key events for myocardial arrhythmias, contracture, and cell death (reviewed in Reference 8). Use of the specific Na⁺-H⁺ exchange inhibitor Hoe 694³ (3-methylsulfonyl-4-piperidinobenzoyl guanidine methane sulfonate) was tested to determine whether the described beneficial actions of Na⁺-H⁺ exchange inhibition result in a decreased infarct size and improved recovery of myocardial function in regionally ischemic, reperfused porcine hearts.

	Methods

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Anesthesia and Medication
Eighteen farm pigs (Department of Agriculture, University of Göttingen) of either sex weighing between 37 and 51.6 kg were used in this study. Each was premedicated with azaperon (2.5 mg/kg IM), metomidate hydrochloride (15 mg/kg IM), and atropine (0.5 mg IV). General anesthesia was maintained with continuous infusion of metomidate hydrochloride (20 mg/h) and injections of piritramide (3.5 mg/h IV). Pancuronium bromide was used as a neuromuscular blocking agent (12 mg/h IV). Artificial ventilation was performed with nitrous oxide and oxygen (4:1) by use of a Sulla 19 respirator (Dräger). Arterial blood gases were controlled frequently (ABL 300, Radiometer) and adjusted to physiological values. All pigs were anticoagulated with heparin (10 000 IU) before the first arterial catheter was introduced. No antiarrhythmic treatment was used in this study. In case of ventricular fibrillation, direct electrical countershocks (25 J) were applied to restore sinus rhythm as soon as possible. The countershocks were applied carefully outside the experimental territory. During the 24-hour reperfusion period, the pigs received 500 mL glucose solution (5%) and 1000 mL isotonic saline solution intravenously.

General Experimental Design
The general experimental setup was described in previous studies.^{9 10} All experiments were carried out under sterile conditions. A standard lead of the ECG and rectal body temperature were monitored throughout the experiments. The body temperature was kept constant between 37°C and 38.5°C by a temperature-controlled operating table. After a median thoracotomy, the left anterior descending coronary artery (LAD) was dissected free at the beginning of its distal third. Left ventricular pressure and its first derivative (dP/dt) were measured with a 5F Millar catheter-tipped manometer. Blood pressure was assessed in the aorta with a fluid-filled catheter connected to a Statham transducer. A 5F multipurpose catheter was placed through the coronary sinus into the ostium of the great cardiac vein. The LAD was occluded at the prepared site for 45 minutes and then reperfused for 24 hours. Forty-five minutes after the onset of reperfusion, the chest was closed in layers, and the pig was allowed to recover. The next day, the pig was reanesthetized, and the thoracotomy was repeated.

Measurement of Global Hemodynamics, Regional Systolic Shortening, and LAD Blood Flow
Global hemodynamic variables, including left ventricular peak pressure (LVPP), left ventricular end-diastolic pressure, diastolic pressure, maximum dP/dt (dP/dt_max), and heart rate, were recorded before treatment, before coronary artery occlusion, at 5-minute intervals during ischemia, during 45 minutes of reperfusion, and at the end of the experiment. To assess the increase in heart rate during early reperfusion, this parameter was determined immediately before and exactly after 1, 2, 3, 4, and 5 minutes of reperfusion. Heart rate during early reperfusion was expressed as the mean of these five determinations. Global left ventricular oxygen consumption was estimated from the hemodynamic data (LVPP, dP/dt_max, heart rate, systolic time interval, and ejection time interval) before treatment and before and during ischemia with the use of Bretschneider's equation.¹¹

Two pairs of ultrasonic crystals¹² were implanted in the subendocardial-midmyocardial layer of the heart and oriented parallel to the short axis.¹³ One pair was placed in the center of the ischemic, reperfused region at a distance of about 2 cm from the apex; the other pair was positioned in a segment perfused by the circumflex artery (control segment). The ultrasonic crystals were left in place until the end of the experiment. Regional systolic shortening was determined by the ultrasonic transit time method¹⁴ with a Triton 120 sonomicrometer (Triton Technology). Analogue tracings of the signals were obtained from direct written recordings. The end-diastolic distance (EDD) of the crystals was determined at the onset of ventricular systole. The end-systolic distance (ESD) was defined by peak negative dP/dt. Relative systolic shortening (SS%) was assessed as EDD minus ESD divided by EDD times 100. SS% was recorded before treatment, immediately before coronary artery occlusion, after 1 minutes of ischemia, immediately before reperfusion, after 45 minutes of reperfusion, and at the end of the experiment after the thoracotomy had been repeated.

Blood flow of the LAD (milliliters per minute) was measured with a 2-mm ultrasonic flow probe (T-106 Flowmeter, Transonic Systems Inc) before treatment, before ischemia, at 5-minute intervals during ischemia, and during 45 minutes of reperfusion. The flow probe was fitted around the coronary artery in close vicinity proximal or distal to the site of occlusion.

Plasma Concentrations of Hoe 694
In both treatment groups, plasma concentrations of Hoe 694 were determined before ischemia, before reperfusion, and after 45 minutes and 24 hours of reperfusion by Hoechst AG with the use of a high-performance liquid chromatography method.

Measurement of Infarct Size
After 24 hours of reperfusion, the heart was excised after occlusion of the coronary artery at exactly the same site and after administration of a central venous injection of 10 mL 10% fluorescein sodium solution to label the well-perfused myocardium with fluorescent green. After determination of left ventricular weight, the heart was cut into slices of about 5 mm parallel to the AV groove. All slices containing reperfused myocardium were weighed and photographed under UV light. The slices proximal to the inserted crystals (in general, four) were stained with nitroblue tetrazolium solution^{15 16} to assess the infarcted tissue and photographed once more, always at the same magnification. The areas at risk of necrosis and the corresponding infarcts were determined by planimetry of the heart slices with the use of enlarged photographs (13x18 cm). Infarct size was calculated as the ratio of infarcted myocardium divided by risk region times 100. The ischemic, reperfused regions and the well-perfused left ventricular myocardium of those slices that were not used for measurement of infarct size were also measured by planimetry to allow calculation of the total weight of the risk region.

Micromorphometric Evaluation of Infarct Size
The area at risk of necrosis was cut out from the most proximal slice toward the apex under fluorescent illumination. This tissue and the rest of the slice were mounted on a pap and were fixed with 4% Sörensen phosphate formalin. From each area at risk, a photograph and a photocopy of the slice were made to compare size and shape with the histological slides. The tissue was routinely embedded with paraffin, serially sectioned, stained with hematoxylin and eosin, and quantitatively analyzed by light microscopy. Only those sections that contained the entire risk region were used for micromorphometry. Because the necroses within the area at risk consisted mostly of many ill-defined zones, a reliable analysis could be performed only at x16 objective magnification. Morphometry was performed with a 20x20 field eyepiece grid (Zeiss) with a 10-mm-long edge. The number of necrotic and nonnecrotic myocardial fields within each area at risk was determined by a pathologist (R.M.B.) with 12 years of pathoanatomic experience. Infarct size was calculated as the sum of necrotic fields in relation to the entire risk region (sum of necrotic and nonnecrotic fields) and expressed as a percentage of the risk region.

Experimental Protocol
Three groups with 6 pigs in each were formed. The pigs in group A were treated with an injection of 3 mg/kg IV Hoe 694 10 minutes before ischemia; the pigs in group B received the same intravenous treatment 10 minutes before the onset of reperfusion. Hoe 694 was dissolved in 50 mL distilled water immediately before administration. The control pigs were treated with 50 mL distilled water 10 minutes before ischemia. The pigs of group A and the control group were randomly assigned to either treatment; the experiments of group B were performed later.

Statistics
All data are presented as mean±SD. Statistical comparisons among the three groups were performed with the Kruskal-Wallis H test. When this test indicated a significant difference, the data were analyzed further with the Mann-Whitney U test. The Wilcoxon test for paired data was used for comparisons within one group. The relations between histochemically and histologically determined infarct sizes were evaluated with linear regression analysis. Statistical significance was accepted at the 5% level (P<.05); in case of multiple (three) comparisons (Mann-Whitney U test), at the 2% level (P<.02).

	Results

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Plasma Concentrations of Hoe 694
In group A, plasma concentrations of Hoe 694 amounted to 1337±236 ng/mL before ischemia, 389±75 ng/mL before reperfusion, and 196±58 ng/mL after 45 minutes of reperfusion. In group B, plasma levels were 1576±436 ng/mL before reperfusion and 294±119 ng/mL after 45 minutes of reperfusion. In both groups, Hoe 694 was not detectable in plasma taken after 24 hours of reperfusion.

Rhythm Disturbances
Ventricular fibrillation during ischemia occurred in 1 pig in group A, 4 pigs in group B, and 4 control pigs. During early reperfusion, ventricular fibrillation took place in 3 pigs of group A and 1 pig of group B. This rhythm disturbance could always be terminated immediately by direct electric countershocks. All pigs survived until the end of the experiment. The heart rates immediately before the onset of reperfusion did not differ among the three groups (group A, 79±12 beats per minute [bpm]; group B, 82±8 bpm; control group, 88±13 bpm). The increase in heart rate during early reperfusion was significantly less (P<.01) in group A compared with the other two groups. The average heart rates determined exactly after 1, 2, 3, 4, and 5 minutes of reperfusion amounted to 96±14, 128±9, and 128±11 bpm (group A, group B, and control group, respectively). Thus, only treatment with the Na⁺-H⁺ exchange inhibitor before ischemia attenuated the increase in heart rate during early reperfusion.

Global Hemodynamic Variables
The only significant difference (Table 1) among the three groups was the heart rate during 45 minutes of reperfusion. At that time interval, the average heart rate was lower in group A compared with the other two groups (P<.02). Although pretreatment with Hoe 694 had only minor hemodynamic effects, left ventricular peak pressure and calculated global oxygen consumption had increased very slightly but significantly 10 minutes after the intravenous injection (P<.05). After 24 hours of reperfusion, dP/dt_max was equally depressed in all groups.

View this table: [in this window] [in a new window]   Table 1. Global Hemodynamic Variables

Regional Myocardial Function
The EDDs and regional systolic shortening of the control and LAD supplied segments (Table 2) did not differ among the groups before ischemia and after 1 and 45 minutes of ischemia. After 10 minutes of reperfusion, EDD of the reperfused myocardium in pigs in group A exhibited significantly less contracture (-1±4%, P<.01) than in pigs in groups B (-25±14%) and C (-32±8%) compared with that observed after 45 minutes of ischemia (Fig 2), whereas the EDD of the control segments of the three groups remained almost unaltered. Thus, treatment with Hoe 694 before ischemia abolished myocardial contracture during early reperfusion, whereas Hoe 694 administration before reperfusion had no significant effect. SS% of the ischemic, reperfused myocardium improved to a greater extent in group A after 24 hours of reperfusion (group A versus group B, P=.02; group A versus group C, P=.01). These parameters of regional myocardial function indicate that treatment with Hoe 694 before ischemia provided significant cardioprotection (prevention of contracture during early reperfusion, improved SS% after 24 hours of reperfusion). The same treatment administered before reperfusion was ineffective.

View this table: [in this window] [in a new window]   Table 2. Regional Systolic Shortening and End-Diastolic Distances

View larger version (20K): [in this window] [in a new window]   Figure 2. Bar graph showing the percentage of change in alterations of the end-diastolic distances of the control and the ischemic, reperfused segments after 10 minutes of reperfusion compared with 45 minutes of ischemia. In contrast to both other groups, treatment with Hoe 694 before ischemia (group A) significantly (*) attenuated myocardial contracture of the reperfused myocardium. Values are mean±SD.

Histochemical Infarct Size
The weights of the left ventricles (group A, 121±11 g; group B, 124±8 g; control group, 119±17 g) and of the risk regions (group A, 17±3 g; group B, 14±2 g; control group, 15±2 g) did not differ significantly among the three groups (Fig 3). Pretreatment with Hoe 694 reduced infarct size from 65±16% (control group) to 13±8% (P<.01). Hoe 694 administered before reperfusion limited infarct size insignificantly by 15% to 55±19%.

View larger version (11K): [in this window] [in a new window]   Figure 3. Bar graph showing histochemical infarct sizes after 45 minutes of ischemia and 24 hours of reperfusion. Treatment with Hoe 694 before ischemia (group A) significantly (*) decreased infarct size compared with both other groups.

Micromorphometric Infarct Size
The histochemical infarct sizes determined with the tetrazolium stain were confirmed by quantitative histology of one heart slice from each experiment. Histological infarct sizes amounted to 14±4% (group A), 42±15% (group B), and 49±20% (control group). Histochemical infarct sizes of the same heart slices were 9±5% (group A), 49±19% (group B), and 56±24% (control group). A linear regression analysis of the histochemical and histological infarct sizes of the evaluated 18 heart slices demonstrated a close relation (histochemical infarct size=-6.093+1.252xhistological infarct size, r=.95; Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 4. Plot showing the relation between histological and histochemical infarct sizes. Linear regression analysis revealed a close relation between histological and histochemical infarct sizes of 18 evaluated heart slices.

	Discussion

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Applied Methods
The effects of Hoe 694 were evaluated in regionally ischemic, reperfused porcine hearts, a preparation with negligible collateral blood flow.^{17 18} An ischemic period of 45 minutes was chosen to induce a mean histochemical infarct size of about 70% in the control group. Ischemia of 45 minutes is in the middle of the time range during which the speed of cell death is greatest.¹⁹ A disadvantage of the ischemic, reperfused porcine heart preparation is the relatively high incidence of ventricular fibrillation during ischemia. All pigs that required defibrillation could be resuscitated within 1 minute and were not discarded because we did not observe that electric counterschocks applied outside the ischemic territory affected infarct size.²⁰ Regional myocardial function was determined by measurement of circumferential SS% in the subendocardial-midmyocardial layer. Although this may underestimate SS% of the subepicardial layer,²¹ we preferred this technique to the measurement of wall thickening because of our good experience with the stability of the chronically implanted ultrasonic crystals. Infarct size was measured by histochemistry and histology to rule out the possibility that the staining mechanism of the tetrazolium stain was affected by Hoe 694. Because only one dose of Hoe 694 was tested, which resulted in plasma concentrations of at least 10^-6 mol/L at the onset of ischemia and reperfusion, no statement can be made on the dose-dependent effects of Hoe 694 in this preparation. The achieved plasma concentrations were shown to be high enough to suppress the amiloride-sensitive Na⁺ influx into rabbit erythrocytes and inhibit the Na⁺-H⁺ exchange in bovine aortic endothelial cells.³

Myocardial Protection by Na⁺-H⁺ Exchange Inhibition
Although this study clearly demonstrates that pretreatment with Hoe 694 is cardioprotective in regional ischemia and reperfusion in intact pigs, the determined parameters and the findings of this study do not allow an exact definition of the mechanisms involved in myocardial protection. One fundamental feature of myocardial ischemia is the intracellular generation of protons.²² The intracellular-extracellular pH gradient activates the Na⁺-H⁺ exchange system,²³ which results in an increase of intracellular Na⁺ (Na⁺_i). Na⁺_i can be exchanged with extracellular K⁺ (Na⁺, K⁺)-ATPase or extracellular Ca⁺,²⁴ which may ultimately lead to intracellular calcium overload. Whether myocardial injury resulting from intracellular calcium overload occurs predominantly during reperfusion or during ischemia has not yet been resolved. It appears that myocardial protection can be achieved best by inhibition of the Na⁺-H⁺ exchange system during ischemia.^{5 25} However, beneficial effects have also been observed by blocking this system during reperfusion.⁶

Myocardial Protection by Na⁺-H⁺ Exchange Inhibition in Regionally Ischemic, Reperfused Porcine Hearts
In this study, Hoe 694 has been used as a selective Na⁺-H⁺ exchange inhibitor (NHE). This agent, which is pharmacologically comparable to N-5–substituted amiloride substitutes, preferentially blocks the ubiquitously expressed amiloride-sensitive NHE₁ isoform.²⁶ Because there is growing evidence from studies in isolated myocytes and isolated heart preparations that Na⁺-H⁺ exchange is a key mechanism for ischemia-reperfusion injury (reviewed in Reference 27), we tested whether Hoe 694 prevents or attenuates cell death in a more clinically oriented regionally ischemic, reperfused heart preparation. The lack of a biologically significant effect of Hoe 694 on global hemodynamics before coronary artery occlusion excludes any observed protective actions related to a reduced myocardial oxygen consumption at the immediate onset of ischemia. Pretreatment with Hoe 694 (group A) reduced infarct size dramatically, prevented myocardial contracture during early reperfusion, attenuated the increase in heart rate during early reperfusion, and improved recovery of regional SS%. Because of the small number of pigs in each group, we could not determine whether Hoe 694 given before reperfusion (group B) provided no or only slight cardioprotection. Mean infarct sizes of group B determined by histology and histochemistry were insignificantly reduced by 15%, and recovery of SS% after 24 hours of reperfusion was only slightly improved compared with that in the control group. The increases in heart rate and myocardial contracture during early reperfusion were not affected in group B. These results strongly suggest that myocardial protection was achieved primarily by inhibition of Na⁺-H⁺ exchange during ischemia. Because the kinetics of Na⁺-H⁺ exchange inhibition by Hoe 694 in pig hearts is not known, it cannot be ruled out that at least some protection occurred at the moment of reperfusion. The experimental design of this study does not allow a definite conclusion on the potential antiarrhythmic actions of Hoe 694. Although ventricular fibrillation during ischemia occurred in 1 pig in group A and in 4 pigs in groups B and C each, it is possible that Na⁺-H⁺ exchange inhibition delays but does not reduce the incidence of ischemic ventricular fibrillation. On the other hand, 3 pigs in group A developed ventricular fibrillation during reperfusion in contrast to 1 pig in group B and no pigs in group C. Because in our experience ventricular fibrillation during reperfusion is more likely to occur in experiments with smaller infarcts, the incidence of ventricular fibrillation during reperfusion should be compared between treated and nontreated pigs with similar infarct sizes. Thus, additional studies are required to evaluate the potential antiarrhythmic effects of Na⁺-H⁺ exchange inhibition in regional ischemia and reperfusion in intact pigs.

Conclusions
With the use of the Na⁺-H⁺ exchange inhibitor Hoe 694, it was clearly demonstrated that Na⁺-H⁺ exchange is an important mechanism causally involved in cell death in myocardial ischemia and reperfusion. This new insight into the mechanism of myocardial cell death in ischemia and reperfusion may improve the clinical treatment in the setting of acute myocardial ischemia and reperfusion, such as acute myocardial infarction treated by reperfusion therapies, heart surgery with extracorporeal circulation, percutaneous transluminal coronary angioplasty, and heart transplantation.

View larger version (32K): [in this window] [in a new window]   Figure 1. Bar graph showing systolic shortening of the ischemic, reperfused segment. The only significant (*) difference among the three groups was observed after 24 hours of reperfusion when systolic shortening of the pretreated group (group A) had recovered to a greater extent (P=.02 vs group B; P=.01 vs control group). Values are mean±SD.

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Kl 724/1-4). Hoe 694 was kindly provided by Hoechst AG, Frankfurt/Main, Germany. We thank Shauna Kruse-Morgan, PhD, for her help in preparing the manuscript.

Received December 5, 1994; revision received February 13, 1995; accepted February 21, 1995.

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