Cardiovascular Research

Cardiovascular Research 1997 35(1):80-89; doi:10.1016/S0008-6363(97)00106-5
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Prevention of ischemic rigor contracture during coronary occlusion by inhibition of Na⁺–H⁺ exchange

David Garcia-Dorado^a,*, Miguel A González^a, José A Barrabés^a, Marisol Ruiz-Meana^a, Julia Solares^c, Rosa-Maria Lidon^a, José Blanco^a, Yolanda Puigfel^a, H.Michael Piper^b and J Soler-Soler^a

^aServicio de Cardiologéa, Hospital General Universitari Vall d'Hebron, Pg. Vall d'Hebron 119–129, 08035 Barcelona, Spain
^bPhysiologisches Institut, Justus Liebig Universität, Giessen, Germany
^cServicio de Anatoméa Patológica, Hospital San Pedro de Alcántara, Cáceres, Spain

^* Corresponding author. Tel.: +34 (3) 4272000, ext. 4948; fax: +34 (3) 4284301; e-mail: dgdorado@ar.vhebron.es

Received 15 October 1996; accepted 15 April 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Objective: To determine the effect of Na⁺–H⁺ exchange blockade on ischemic rigor contracture and reperfusion-induced hypercontracture. Methods: Thirty-six pigs were submitted to 55 min of coronary occlusion and 5 h reperfusion. Myocardial segment length analysis with ultrasonic microcrystals was used to detect ischemic rigor (reduction in passive segment length change) and hypercontracture (reduction in end-diastolic length). Results: Pretreatment with the new, highly selective Na⁺–H⁺ exchange inhibitor HOE642 before occlusion reduced ischemic rigor (P<0.05), attenuated segment shrinkage (P<0.05) during subsequent reperfusion, dramatically reduced infarct size (P<0.0001) and attenuated arrhythmias (P<0.01). Inhibition of Na⁺–H⁺ exchange only during reperfusion by means of direct intracoronary infusion of HOE642 into the area at risk prevented reperfusion arrhythmias but had no effect on final infarct size, while treatment with intravenous HOE642 immediately before reperfusion had no detectable effects. Conclusion: These results indicate that inhibition of Na⁺–H⁺ exchange during ischemia is necessary to limit myocardial necrosis secondary to transient coronary occlusion, and that this action could be mediated by a protective effect against ischemic contracture. Inhibition of Na⁺–H⁺ exchange only during reperfusion has a partial and transient beneficial effect, but only when the inhibitor reaches the area at risk before reflow.

KEYWORDS Myocardial infarction; Ischemia; Reperfusion injury; Hypercontracture; Functional studies; Reperfusion arrhythmias; Infarct size

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Increased cytoplasmic calcium concentration plays a crucial role in the genesis of myocyte hypercontracture and death during reoxygenation and reperfusion [1]. While it has been clearly shown that intracellular Ca²⁺ rises during ischemia, anoxia or metabolic inhibition [2, 3], there is increasing evidence that reoxygenation and reperfusion may result in further Ca²⁺ gain [3, 4]. Na⁺–H⁺ exchange has been identified as a potentially relevant mechanism leading to Ca²⁺ gain during reperfusion by inducing Na⁺ gain and Na⁺–Ca²⁺ exchange [3, 4]. In addition, cellular acidosis is a potent contractile inhibitor [5], and it has been suggested that its rapid correction during reperfusion may favor the occurrence of hypercontracture [6].

Blockade of Na⁺–H⁺ exchange has been proposed as a potentially useful therapeutic approach to limit Ca²⁺ influx, hypercontracture, and cell death during reperfusion [7–12]. Previous studies have shown that pretreatment with amiloride or certain amiloride derivatives exhibiting Na⁺–H⁺ exchange blocking activity reduces reperfusion arrhythmias [9, 13, 14], stunning [8, 10, 15]and cell necrosis [14, 16]after transient myocardial ischemia or coronary occlusion. However, amiloride derivatives are not selective blockers of Na⁺–H⁺ exchange [8], and in many studies administration of the drug before coronary occlusion did not allow one to distinguish whether its beneficial effect was due to its action during reperfusion, as first hypothesized, or during coronary occlusion. Recent studies using the more selective inhibitors of Na⁺–H⁺ exchange derived from benzoylguanidine have demonstrated a marked protective effect of these substances against ischemia-reperfusion injury [7, 12, 17, 18]. Studies in isolated myocytes submitted to anoxia and acidosis have shown that highly selective Na⁺–H⁺ exchange blockade reduces calcium gain and hypercontracture during exposure to oxygenated media and normalization of pH [19].

Several studies suggest that Na⁺–H⁺ exchange blockers are less beneficial, or even not beneficial at all, when given during or just prior to reperfusion instead of before coronary occlusion [7, 10, 15–17]. Moreover, recent studies indicate that Na⁺–H⁺ inhibition delays or prevents anoxic rigor contracture in isolated myocytes [20]. However, the failure of Na⁺–H⁺ exchange blockers during reperfusion could be due to a too late action of the drug on reperfused myocytes. Hypercontracture may start almost immediately after reoxygenation, and the effective inhibition of Na⁺–H⁺ exchange in reperfused myocardium could take place after hypercontracture has already occurred when the inhibitors are administered intravenously.

The purpose of this study was to evaluate the ability of a highly selective Na⁺–H⁺ exchange inhibitor (HOE642) to limit rigor contracture secondary to transient coronary occlusion and hypercontracture secondary to reperfusion, and to determine the relative contribution of Na⁺–H⁺ exchange during coronary reperfusion to myocardial injury. A pig model with negligible collaterals was used and the drug was administered at different times during ischemia-reperfusion either intravenously or by selective intracoronary infusion into the area at risk.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
2.1 Animal preparation
Forty-two Large-White farm pigs (20–30 kg) were used in this study. Animals were premedicated with 10 mg/kg azaperone i.m. (Stressnil, Janssen Pharmaceutical), anesthetized with 30 mg/kg thiopental i.v., intubated and connected to a ventilator supplying room air (Monaghan 228 Ventilator, Littleton, CO, USA). Anaesthesia was maintained with a continuous infusion of thiopental. The right abdominal vein was catheterized with a 19G cannula for intravenous delivery of drugs and the right femoral artery catheterized with a 5F introducer. A mid-line sternotomy was performed and the pericardium opened.

The left anterior descending coronary artery (LAD) was dissected free as close as possible to the midpoint of its total length and surrounded by an elastic snare as previously described [21]. An electromagnetic flow probe (MDL 1401, Skalar, Netherlands) was placed around the dissected segment. Two pairs of ultrasonic crystals 1 mm in diameter were inserted into the inner third of the left ventricular wall. The crystals of each pair were placed 1–2 cm apart along a plane perpendicular to the long axis of the left ventricle. One pair was implanted in the myocardium to be made ischemic and the other pair in the lateral wall of the left ventricle. In a group of animals the left main coronary artery was catheterized with a Judkins 8F guiding catheter introduced via the right carotid artery, and a 2.5F catheter for intracoronary artery infusion (Cordis, Miami, FL, USA) was advanced into the distal LAD immediately before coronary occlusion according to a previously described method [20].

2.2 Study groups and protocol
All animals underwent 55 min of coronary occlusion followed by 5 h of reperfusion. This duration of coronary occlusion was selected to obtain an infarct size of 60% of the area at risk in the control group according to a previous regression analysis of infarct size over occlusion time [22]. The animals were allocated to one of 4 groups of treatment receiving either HOE642 or placebo as shown in Fig. 1. A group received 3 mg/kg of HOE642 i.v. over 1 min, starting 10 min before coronary occlusion (HOE preCO). In another group the same dose of HOE642 was administered 10 min before reperfusion (HOE preR). A third group received the same treatment as the previous group (3 mg/kg of HOE642 10 min before reperfusion), but additionally it received an intracoronary perfusion into the distal LAD during the last 5 min of occlusion HOE preR+IC. The perfusate consisted of a Ringer solution containing HOE642 6.7 µM, the peak plasma concentration observed 5 min after a bolus injection of 3 mg/kg. This solution was equilibrated with 100% argon and infused at 37°C through the intracoronary catheter at 3 times basal coronary blood flow with the aid of a programmable infusion pump (Model 2400-003, Harvard Apparatus, Southnatick, MA, USA). The fourth group received only the vehicle (10 ml of saline) with no active drug 10 min before coronary occlusion (Control). In a first series of experiments 13 animals were allocated to the Control group and 7 to each of the other 3 groups. In order to analyze the possible effect of HOE642 on collateral flow during coronary occlusion, 8 additional animals were allocated to either the HOE preCO (n = 4) or the Control group (n = 4) in a second series of experiments. Six animals had to be excluded for different reasons: 2 animals (from control and HOE preR groups) presented reocclusion of the LAD during the reperfusion period, 1 animal from the HOE preCO group suffered accidental extubation and another presented LAD bleeding upon release of the coronary occluder, and 2 animals from the HOE preR+IC group presented, respectively, occlusion of the intracoronary catheter by thrombotic material and intractable ventricular fibrillation. There thus remained 36 valid experiments—9 in the HOE preCO group, 6 in the HOE preR group, 5 in the HOE preR+IC group, and 16 in the Control group.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Study design. Animals were allocated to 4 groups of treatment. Three groups received the active drug as a bolus injection either 10 min before coronary occlusion (HOE preCO) or 10 min before reperfusion (HOE preR). The last of these 3 groups received in addition an intracoronary infusion of the drug into the area at risk during the last 5 min of coronary occlusion (HOE preR+IC).

 
2.3 Study monitoring
Arterial pH, partial pressure of O₂ and partial pressure of CO₂ were closely monitored, and the ventilatory parameters were adjusted to maintain normal blood gases. Hematocrit, platelet count, partial thromboplastin time, and plasma concentration of glucose, creatinine, Na⁺, and K⁺ were determined at the beginning and at the end of the experiment, and creatine kinase activity was determined at the end of the experiment. In 8 animals these determinations were repeated 5 min before and 5 min after the intravenous injection of HOE642 (n = 4) or placebo (n = 4). Aortic blood pressure was continuously monitored with a crystal quartz transducer (Micron Instruments, Simi Valley, CA, USA). This signal, along with lead II of the electrocardiogram, and instantaneous coronary blood flow was conditioned in a Modular Instrument System (Coulbourn Instruments) and digitized by a digitization card (Tecfen ISC-16E/CR, RC Electronics, Goleta, CA, USA), at a sampling rate of 100 Hz per channel. Digital signals were stored on a hard disk. The conditioned signals were continuously recorded in a thermic pad recorder (MT-9500, Astro-Med, West Warwick, RI, USA) using a sampling rate of 200 kHz.

2.4 Plasma HOE642 concentrations
Venous blood samples (10 ml) were withdrawn, before and 5, 30, 90, and 240 min after administration of HOE642. Plasma from these samples was separated, frozen, and stored at –80°C (Forma Scientific freezer, Marietta, OH, USA) until plasma HOE642 concentration was determined. Plasma samples were prepurified by solid phase extraction using 100 mg Varian Bond Elut CBA as sorbent, and adjusted to pH 5.5 by adding 0.5 mol/l citrate buffer; 1 µg/ml of S90 2604 was added as internal standard. This solution was clarified by centrifugation and passed over a CBA column. Elution was accomplished with 300 µl of 1% TFA in 50% methanol. The eluate was diluted 1:1 in water and analyzed by high-performance liquid chromatography (Lichrospher 60 RP-select B, Merck) using a gradient of acetonitrile in 5 mM heptane sulfonic acid (PIC B-7, Waters), and a flow rate of 0.6 ml/min. UV detection was set to 250 nm.

2.5 Injection of colored microspheres
In 8 animals colored microspheres (15.5 µm in diameter) were used to analyze myocardial blood flow as previously described [23]. In these animals, the left atrium was catheterized with a 20G cannula, through which 1.8·10⁶ yellow microspheres (Triton Technology) were injected at baseline, 3.6·10⁶ blue microspheres 40 min after coronary occlusion, and 3.6·10⁶ red microspheres 5 min after reflow. Arterial blood was withdrawn from the descending aorta through a Medicut catheter (Sherwood Medical) at a rate of 12 ml·min^–1 (2400-003 infusion pump, Harvard Apparatus) for the 10 s before and the 90 s following each injection of microspheres. This blood was frozen for ulterior analysis.

2.6 Segment length measurements
The digitized signals stored on the hard disk were analyzed by means of commercially available software (Enhanced Graphics Acquisition and Analysis, RC Electronics). Segment length measurements were performed as previously described [20]. The system automatically performed measurements in the ischemic and control segments at the times identified as end-diastole and end-systole in the control segment. Systolic shortening was calculated as the ratio of systolic shortening/end-diastolic length (EDL) according to the formula:

Amplitude of segment length change was defined as the difference between the maximal and the minimal segment length during the cardiac cycle. Segment length measurements were performed only during sinus rhythm, before, and after 1, 5, 15, 30 and 55 min of coronary occlusion, and after 1, 5, 15, 30 min and 1, 2, 3, 4 and 5 h of reperfusion. Segment length measurements were expressed as percent of the values immediately before coronary occlusion. The development of rigor contracture was estimated from the reduction in the amplitude of segment length change (difference between maximal and minimal length through the cardiac cycle) that occurred during the coronary occlusion period. This reduction was referred to the amplitude of segment length change observed after 5 min of coronary occlusion. At this time contractile failure is fully established, and changes in segment length through the cardiac cycle can be considered passive. The development of rigor contracture offers resistance to these passive changes and reduces their amplitude.

2.7 Postmortem studies
After 5 h of reperfusion, the LAD was reoccluded and 5 ml of 10% fluorescein was injected into the left atrium. The heart was excised immediately and immersed in Ringer solution at 4°C, and cut in 5–7 mm slices perpendicular to its long axis. All the slices from each heart were illuminated from the basal side with ultraviolet light to outline the area at risk as previously described [22].

2.7.1 Analysis of subendocardial blood flow
Transmural myocardial samples from the area at risk and control myocardium in the fourth slice were obtained. The samples were divided into subepicardial and subendocardial halves, and the subendocardial half (0.31±0.6 g) was immediately frozen until analysis. These myocardial samples and the reference blood samples previously obtained were digested by incubation at 72°C with 4 mol·l^–1 KOH. The microspheres were extracted by filtration, and each sample was processed with 100 µl dimethylformamide (Sigma Chemical). To quantify the dye content of the samples, their photometric absorption was determined (Shimadzu UV 160A). Regional myocardial blood flow (ml·min^–1·g^–1 of wet tissue) was calculated as the specific absorbance of each dye per sample multiplied by the withdrawal rate of reference arterial blood and divided by the specific absorbance in reference blood [24].

2.7.2 Measurement of infarct size
All the slices from all hearts were imaged under ultraviolet light by means of a Sony TR 705E Hi8 video camera. Instead of the fourth slice, used for analysis of regional blood flow, the apical surface of the fifth slice was illuminated. The images were digitized on line into 768x576 pixel images by connecting the videocamera output to a Matrox IP8 digitization card (Matrox Electronic Systems, Dorval, Que., Canada). The slices were then incubated at 37°C in 1% triphenyltetrazolium chloride, buffered at pH=7.4 for 5–10 min, and imaged again under white light using the same camera and digitization card. A reference scale was also digitized in each experiment. The zone at risk and the area of necrosis from all the slices were measured in the digital images by using a commercially available software (Image Pro-Plus, Media Cybernetics, Silver Springs, MA, USA). The mass of myocardium at risk and infarct size were calculated from these measurements and the weight of slices as previously described [20].

2.8 Histological analysis
The third slice, starting from the apex, was processed for histology using the double embedding method with a combination of celloidin and paraffin. Six-micron-thick sections including the whole right and left ventricles were obtained using a Leitz 1400 microtome (Heidelberg, Germany) and mounted on 10x14 cm pieces of glass. The sections were stained with Masson's trichrome. The area of necrosis was quantified on the sections as previously described [21]by using a modification of the method of Miyazaki et al. [25]. A transparent overlay 0.1 mm thick was placed over the coverglass of the tissue section and examined with a Nikon Labphot microscope and a binocular lens (Olympus stereo microscope, SZ 111) at a magnification of 100x and 200x. The area of necrosis was carefully marked on the transparent overlay with a sharp stainless-steel needle with a 10 µm tip (Microlance 25G, 5/8, 0.5x16, Becton and Dickinson, Fraga, Spain). Color photographs of the whole section with the photographic overlay were obtained. These slides were digitized and the areas of necrosis measured by digital planimetry. The measurements of the area of necrosis on both sides of the third slice, beginning from the apex as measured by triphenyltetrazolium staining, were averaged and compared to the area of contraction band necrosis in one histological section from that slice. All experimental procedures were approved by the Research Commission of the Hospital General Vall d'Hebron and conformed to the Helsinki Declaration.

2.9 Statistical analysis
Statistical analysis was performed by using commercially available software (SPSS PC+4.0). Statistical comparisons between groups were performed by analysis of variance (ANOVA) after having assessed the data for normal distribution. Individual comparisons were performed by means of the Less Significant Difference test. Changes in physiological parameters throughout the experiment were assessed by Multiple ANOVA analysis for repeated measurements. A critical P-value of 0.05 was used for all tests. All values are expressed as means±s.e.m.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
3.1 Serum HOE642 concentrations
The i.v. administration of 3 mg/kg of HOE642 resulted in reproducible plasma drug levels, peaking at 6.7±2.7 µM, 5 min after injection. Serum concentrations showed an exponential decay, and were 2.7±1.3, 1.7±0.5 and 0.7±0.3 µM, respectively, at 30, 90 and 240 min after injection.

3.2 Hematologic data and blood biochemistry
No significant change was observed in platelet count, partial thromboplastin time, or plasma concentrations of glucose, creatinine, Na⁺ or K⁺ between the beginning and the end of the experiment, except for a small but significant increase in hematocrit (from 23.4±0.7 to 26.4±0.8%, P = 0.01), and no between group differences were observed in any of these determinations. At the end of the experiment, plasmatic creatinine kinase activity in the control group (6166±1381 IU/100 ml) was greater than in the HOE preCO group (2614±557 IU/100 ml) but not significantly different from the HOE preR and HOE preR+IC and control groups (4598±673 and 5815±1555 IU/100 ml respectively). The i.v. injection of HOE642 or placebo did not induce any significant change in blood gases or pH.

3.3 Hemodynamic data and coronary blood flow
Basal heart rate and aortic blood pressure showed no between-group differences (Table 1). Coronary blood flow at the LAD was 26.1±2.0 ml/min before coronary occlusion, without between group differences. The i.v. injection of HOE642 was not accompanied of any detectable change in heart rate, aortic pressure or coronary blood flow. During the first minutes of reperfusion, blood flow in the LAD increased well above the basal values in all groups. This increase in LAD flow was significantly more pronounced in the HOE preCO group (Fig. 2).

View this table: [in this window] [in a new window]   Table 1 Hemodynamic data

 

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Changes in LAD blood flow throughout the experiment. Reactive hyperemia was significantly more marked in animals receiving HOE642 before coronary occlusion (closed rhombuses) than in those receiving the drug before reperfusion with (open rhombuses) or without (closed circles) intracoronary infusion, or in controls (open circles). *P<0.05 respect to the other 3 groups.

 
3.4 Arrhythmias
Three (8.3%) of the 36 animals presented ventricular fibrillation during coronary occlusion, 22±2 min after the onset of ischemia; 1 animal was from the HOE preR+IC, and the remaining 2 were controls. During reperfusion 4 animals presented ventricular fibrillation 49±27 s after reflow; 1 of these animals was from the HOE preR group and 3 from the Control group.

Twenty-seven animals (75%) presented runs of accelerated idioventricular rhythm or ventricular tachycardia during the first 60 min of reperfusion (Fig. 3), with marked between-group differences (P = 0.002): these reperfusion arrhythmias occurred less frequently in the HOE preCO and the HOE preR+IC (3 and 2 animals, respectively), than in the HOE preR and Control groups (6 and 16 animals, respectively). However, their time of onset was not significantly different in the HOE preCO group (18.1±17.4 min after reflow) as compared to the HOE preR, HOE preR+IC, and Control groups (2.6±2.6, 0.5±0.5, and 2.5±1.6 min after reflow respectively, P = 0.14). The total duration of these arrhythmias during the first 60 min of reperfusion tended to be shorter in the HOE preCO group (1.19±0.90 min) than in the remaining three groups (3.76±1.31, 4.1±2.1 and 5.43±2.6 min, respectively, in the HOE preR, HOE preR+IC and Placebo groups.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Representative traces of the first 25 min of reperfusion from the HOE preCO group, receiving HOE642 before coronary occlusion (top) and the Control group, receiving vehicle only (bottom). The discontinuities correspond to the points where the recording speed was increased to 25 mm/s for approximately 5 s, and that have been removed. No reperfusion arrhythmias are observed in the animal that had received HOE642, while the control animal presented multiple episodes of accelerated idioventricular rhythm (*). In the trace from the HOE preCO group the aortic pressure curve (AP) shows a transient episode of hypotension caused by an additional dose of thiopental (arrow), and is progressively damped during the last minutes of recording. The calibrations of the electrocardiogram (ECG), ischemic and control segment length (IS and CS, respectively), and coronary blood flow (F) are different in the two experiments. R=reperfusion.

 
3.5 Regional wall function
Before coronary occlusion end-diastolic segment length in the LAD territory was 11.8±0.7 mm and systolic shortening 28.0±1.3%, without between-group differences. Coronary occlusion produced a rapid increase in end-diastolic segment length, and abolition of systolic shortening. After 5 min of coronary occlusion end-diastolic length and systolic shortening were respectively 109.7±1.1 and 14.1±9.3% of basal value, without between group differences (P = 0.99 and P = 0.71, respectively). At this time, the amplitude of segment length change was 2.42±0.18 mm, and similar in animals that had received HOE642 before coronary occlusion to those who did not (2.12±0.37 and 2.53±0.2 mm, respectively, P = 0.31). During the remaining 50 min of coronary occlusion end-diastolic length and systolic shortening remained stable, and at the end of coronary occlusion were respectively 108.5±1.6 and 10.6±4.3% of basal values without differences between groups (P = 0.76 and P = 0.98, respectively). However, the amplitude of segment length change underwent a marked reduction during the occlusion period. This reduction was less marked in the HOE preCO than in the remaining groups (Fig. 4), and at the end of the occlusion period the amplitude of segment length change averaged in the HOE preCO group 79.5±9.5% of the value after 5 min of occlusion while in the remaining 3 groups it averaged only 61.1±10.26% of the value observed after 5 min of occlusion. MANOVA analysis detected a significant time–treatment interaction (P = 0.045), indicating a different progression of ischemic rigor in the presence and in the absence of HOE642. Reperfusion induced an marked and abrupt reduction in end-diastolic segment length (shrinkage) in animals from the Control and HOE preR groups. This shrinkage was not observed in the HOE preCO group, and was less pronounced in the HOE preR+IC group (Fig. 5). Systolic shortening remained markedly reduced in all animals throughout the reperfusion period, without between-group differences. No inter-group differences in end-diastolic length or systolic shortening of the control segment were observed throughout the experiment.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Reduction in the amplitude of passive segment length change throughout the ischemic period. Values are expressed as percent of the amplitude observed 5 min after coronary occlusion. A progressive reduction in the amplitude of segment length change, indicating the development of rigor contracture, was observed in all animals. This reduction was less pronounced in animals that had received HOE642 before coronary occlusion (closed circles) than in those that had not received the drug (open circles). After 30 min of coronary occlusion the reduction was already evident in the absence of HOE642 but not in its presence. *P = 0.04 respect to animals not having received HOE642 before coronary occlusion.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Changes in end-diastolic length during the reperfusion period. A rapid reduction of end-diastolic length (segment shrinkage) was observed in animals receiving i.v. HOE642 before reperfusion (HOE preR, closed circles) and in control animals (open circles), but not in those receiving i.v. HOE642 before coronary occlusion (closed rhombuses) or receiving i.v. HOE642 before reperfusion plus intracoronary infusion into the area at risk (open rhombuses). *HOE preCO and HOE preR+IC, P<0.05 vs HOE preR and Control; +HOE preCO, P<0.05 with respect to the remaining groups.

 
3.6 Regional myocardial blood flow
Before coronary occlusion, subendocardial blood flow was 1.3±0.4 ml/min/g in the LAD territory and 1.7±0.2 ml/min/g in control myocardium (P = 0.20). Coronary occlusion did not significantly modify blood flow in control myocardium (2.2±0.4) but abolished blood flow in the LAD territory (0.0±0.1 ml/min/g), and this reduction was not different in the HOE preCO (–0.08±0.1 ml/min/g) or the Control group (0.08±0.2, P = 0.55). Five minutes after reflow, subendocardial blood flow was slightly below basal values in reperfused myocardium (0.5±0.2 and 0.9±0.1 mi/min/g, respectively, in HOE preCO and Control groups, P = 0.13), and remained unchanged in control myocardium (2.3±0.4 ml/min/g).

3.7 Infarct size and histology
The area at risk averaged 15.6±1.8 g, without differences between the HOE preCO, HOE preR, HOE preR+IC, and Control groups (18.3±2.8, 12.9±1.2, 14.7±1.7 and 15.3±1.06 g, respectively (P = 0.28), or 12.54±0.93, 9.77±0.58, 11.23±0.94 and 11.99±0.82%, respectively, of ventricular mass, P = 0.29). Infarct size involved 7.22±0.8 g, with significant between-group differences (P = 0.026): 3.75±1.29, 5.65±0.47, 9.59±1.14 and 9.03±1.39 g, respectively, in the HOE preCO, HOE preR, HOE preR+IC and Control groups. These differences were even more evident when infarct size was expressed as a percent of ventricular mass (Fig. 6): infarct size averaged 45.52% of ventricular mass and was much smaller (16.34±4.95%, P<0.0001) in the HOE preCO group than in the remaining 3 groups (55.27±3.92).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Infarct size, as a percent of the area at risk in the 4 study groups. HOE PreCO = i.v. injection of HOE642 10 min before coronary occlusion; HOE preR = i.v. injection of 10 min HOE642 before reperfusion; HOE preR+IC = the same as that for HOE preR plus intracoronary injection of HOE642 into the area at risk during the last 5 min of coronary occlusion. *P<0.0001 with respect to the remaining groups.

 
Histological analysis demonstrated that infarcts were composed almost exclusively of contraction band necrosis in all groups. The area of contraction band necrosis involved 19.3±5.4% of the heart slice in animals receiving HOE642 before coronary occlusion, which was significantly less (P = 0.05) than in animals receiving HOE642 before reperfusion, HOE642 by intracoronary infusion, or placebo (26.2±5.3, 39.6±5.3 and 39.2±8.1%, respectively).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
This study demonstrates that pretreatment with the new, highly selective Na⁺–H⁺ exchange inhibitor, HOE642, before a transient coronary occlusion has a marked protective effect against the development of ischemic rigor in the in situ pig heart. The reduced extent of rigor results in less hypercontracture and necrosis during subsequent reperfusion. Inhibition of Na⁺–H⁺ exchange only during reperfusion by means of direct intracoronary infusion of HOE642 into the area at risk prevents reperfusion arrhythmias, but has no effect on final infarct size. Treatment with intravenous HOE642 immediately before reperfusion has no detectable effect. These results indicate that the anti-infarct effect of Na⁺–H⁺ exchange inhibition is mediated by a reduction in the extent of rigor contracture during myocardial ischemia.

4.1 Previous studies
Previous studies have described a reduction in infarct size in animals pretreated with amiloride or amiloride derivatives [14, 16, 18]as well as with more selective inhibitors of NHE-1 Na⁺–H⁺ exchanger [17, 26]when administered before ischemia. Conflicting results have been reported, however, on the efficacy of Na⁺–H⁺ exchange inhibitors administered immediately before or during reperfusion [27]. Some authors have described protection against post-ischemic disfunction [17]or myocardial necrosis [26], while others have found no beneficial effect [15–17]. Two recent studies have used the in situ pig heart model to analyze the effect of HOE694 on infarct size when given before or during coronary occlusion. Klein et al. [17]observed that HOE694 was effective in reducing myocardial necrosis in the pig heart submitted to 45 min of coronary occlusion and 24 h of reperfusion when given before coronary occlusion, but not when given i.v. before reperfusion. In contrast, Rohmann et al. [26]found a 51% reduction of infarct size with HOE694 in pigs submitted to 60 min of coronary occlusion and 2 h of reperfusion when given i.v. before reperfusion. The results of the present study may help to explain these discrepancies since they suggest that inhibition of Na⁺–H⁺ exchange during the initial seconds of reperfusion may have some beneficial but transient effect. This transient protective effect could explain the reduction in infarct size observed by Rohmman et al. after only 2 h of reperfusion in pigs receiving HOE694 at the end of the occlusion period.

4.2 Ischemic rigor and hypercontracture
Rigor contracture can be easily observed in isolated, unrestrained cardiomyocytes under anoxia, simulated ischemia or metabolic inhibition, as a reduction of about 50% in cell length. In in-situ cardiomyocytes rigor does not result in such a reduction in cell length, but can be detected as a decrease in compliance [1]. Once rigor appears, accelerated Ca²⁺ rise and ATP depletion follow, and reoxygenation usually results in hypercontracture [2]. Reoxygenation of cells not presenting rigor virtually never induces their hypercontracture.

The effect of Na⁺–H⁺ exchange inhibition on the development of rigor contracture has been analyzed in previous studies in the isolated rat heart [10, 28]. Moffat et al. observed a reduction in the elevation of resting tension during zero-flow ischemia in the isolated rat hearts pretreated with 1 µM methylisobutil amiloride [10], while Murphy described a slight delay of approximately 2 min in the rise of resting tension in the ischemic hearts pretreated with 1 mM amiloride [28]. However, Meng et al. [29]found no effect of 5-(N,N-dimethyl)amiloride at 20 µM in the ischemic isolated rat right ventricle. The reasons for the discrepancies in the isolated rat studies are not clear. The results of the present study are in agreement with recent observations showing that inhibition of Na⁺–H⁺ exchange with HOE642 prevents or delays rigor contracture in isolated rat cardiomyocytes and perfused rat hearts submitted to metabolic inhibition or hypoxia [20]. The reduction in the number of cells developing rigor contracture during energy deprivation resulted in a parallel reduction of the number of cells developing hypercontracture during subsequent reoxygenation, even when reoxygenation was performed in the absence of HOE642 [29].

The mechanism of the protective effect of HOE642 against the development of rigor contracture is not clear. It has been suggested that inhibition of Na⁺–H⁺ exchange could preserve cell energy by different mechanisms, including enhanced intracellular acidosis. However, several studies have failed to detect any significant effect of Na⁺–H⁺ exchange inhibition on the time course of intracellular acidification [7, 28, 30]or ATP depletion [7, 12, 28]in ischemic myocardium. Since studies in isolated cells have shown that cytosolic Ca²⁺ only begins to rise after the development of rigor contracture [2], the protective effect of Na⁺–H⁺ exchange inhibition against Ca²⁺ overload [28]cannot explain its effect against rigor. The best-documented effect of Na⁺–H⁺ exchange inhibition on ischemic myocardial cells is a limitation of Na⁺ gain [28, 30, 31], which cannot be easily connected with its protective effect against rigor contracture.

In the in situ heart, HOE642 could still have a beneficial effect on intracellular ATP by improving collateral blood flow, a mechanism that cannot be investigated in the isolated heart model where most of the studies analyzing the metabolic effects of Na⁺–H⁺ exchange inhibition have been performed. However, this possibility seems unlikely in the present study. No difference or trend towards a better collateral flow in animals receiving HOE642 before coronary occlusion was observed in this study, in agreement with previous studies showing that during acute coronary occlusion in the pig heart collateral flow was virtually nil, below the resolution of the microspheres technique [32, 33]. Moreover, the intravenous injection of the drug did not induce any change in LAD flow during the 10 min elapsed after the i.v. injection and coronary occlusion. The significantly more pronounced hyperemic response observed in the HOE preCO during the reperfusion period raises the question of its possible contribution to the observed beneficial effects of Na⁺–H⁺ exchange. However, this possibility seems very unlikely. The increased hyperemia cannot be explained by a direct vasodilatory effect of HOE642, since it was not observed in the HOE preR or HOE preR+IC groups, in which the plasma HOE642 concentration during the early reperfusion period are predicted to be higher. In addition, analysis of regional blood flow demonstrated that subendocardial flow during early reperfusion was not higher in the HOE preCO group than in controls. The increased hyperemic response can reflect the reduced infarct size in this group.

Reperfusion-induced hypercontracture resulting in contraction band necrosis produces a rapid, pronounced, and sustained reduction of end-diastolic fiber length (myocardial segment shrinkage) which can be monitored with the aid of intramyocardial piezoelectric crystals [34]. In the present study segment shrinkage of the reperfused myocardium occurred in all groups of treatment except in the group receiving i.v. HOE642 before coronary occlusion and showing smaller infarcts. These results are in agreement with those from Klein et al., who observed that myocardial shrinkage after 24 h of reperfusion was prevented by i.v. HOE694 administered before, but not during, coronary occlusion [17]. In the present study, however, animals receiving intracoronary HOE642 infusion into the area at risk before reperfusion differed from those receiving placebo or i.v. HOE642 before reperfusion in that they did not present shrinkage upon restoration of coronary blood flow. These results suggest that the lack of any effect of i.v. HOE642 given before reperfusion is partly due to inadequate Na⁺–H⁺ exchange blockade during the initial seconds of reperfusion with this administration schedule. Due to the virtually absent collateral flow in the pig heart [32, 33], the drug administered by i.v. injection during coronary occlusion does not reach the ischemic myocardium. The large area of contraction band necrosis observed in hearts from the HOE preR+IC group is in contrast to the absence of shrinkage in this group during early reperfusion. Although the present results are consistent with the occurrence of delayed shrinkage in the HOE preR+IC, they do not provide definitive statistical evidence of such delayed hypercontracture.

4.3 Arrhythmias
The small number of animals presenting ventricular fibrillation during either coronary occlusion or reperfusion prevents analysis of the possible effect of Na⁺–H⁺ exchange inhibition of this arrhythmia. This analysis was however possible in the case of reperfusion arrhythmias in the form of idioventricular accelerated rhythm or runs of slow ventricular tachycardia with atrio-ventricular dissociation that occur almost invariably in the pig heart model during reperfusion after moderately prolonged coronary occlusion [35]. These reperfusion arrhythmias have been related to increased automatism secondary to increased cytosolic Ca²⁺ occurring during reperfusion [36], and in the present study were significantly attenuated in the HOE preCO and HOE preR+IC groups. These results are consistent with a protective effect of Na⁺–H⁺ exchange inhibition against reperfusion arrhythmias, and with inadequate inhibition in the group that received the drug intravenously during coronary occlusion which was less than in the remaining two groups.

4.4 Study limitations
The dose of HOE642 used in this study was selected to obtain a serum concentration above 0.5 µM at the time of reperfusion in all groups, a concentration which has been shown to reduce by more than 90% amiloride-sensitive Na⁺ influx in Na⁺–H⁺ exchange-deficient fibroblasts transfected with the NHE-1 subtype of exchanger [18]. Although serum concentration was only measured in a subset of animals, the highly consistent concentrations observed in animals made it extremely unlikely that the target serum concentration was not attained in animals in which concentrations were not measured. Due to the exponential decay of HOE642 concentrations after i.v. administration, the design of the study necessarily resulted in higher serum concentrations throughout the reperfusion period in the groups receiving the i.v. injection of the drug before reperfusion as compared to the HOE preCO group. The additional amount of HOE642 administered by intracoronary infusion in the HOE preR+IC group could have resulted in still higher concentrations in this group. However, this additional amount (0.47±0.04 mg) was negligible compared to the much larger i.v. dose (98.7±6.1 mg). It can therefore be assumed that serum concentrations of HOE642 were the same in the HOE preR and HOE preR+IC groups, and that the only difference between both groups was the local HOE642 concentration in the area at risk at the time of reflow.

Intracoronary catheterization and associated coronary endothelial injury could have adversely influenced infarct size in the HOE preR+IC group [23]. In order to rule out this possibility, an additional group of 7 experiments was performed according to exactly the same protocol as in the HOE preR+IC group except that no intracoronary infusion was performed. Infarct size in this group (34.6±7.4% of the area at risk) was not larger than in the control group, indicating that the lack of beneficial effect of HOE642 in the HOE preR+IC group was not due to an adverse effect of intracoronary catheterization.

	5 Conclusion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Inhibition of Na⁺–H⁺ exchange during coronary occlusion by i.v. HOE642 delays the onset of ischemic rigor and reduces reperfusion-induced arrhythmias, hypercontracture, and infarct size. The magnitude of the protection afforded by pretreatment with HOE642 against myocardial injury secondary to transient coronary occlusion, larger than that observed with any previously described intervention, makes selective Na⁺–H⁺ exchange blockade a promising therapeutic strategy in patients with acute ischemic syndromes.

Time for primary review 23 days.

	Acknowledgements

 
HOE642 was kindly provided by Hoechst AG (Frankfurt/Main, Germany). The authors acknowledge the excellent technical work of Carmen González Bravo. This work was partly supported by Grants 95/0465 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, and by Concerted Action PL95/1254, BIOMED II Program from the European Union.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 

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