Cardiovascular Research

Cardiovascular Research 1998 40(2):290-296; doi:10.1016/S0008-6363(98)00183-7
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Hydrogen peroxide induced impairment of post-ischemic ventricular function is prevented by the sodium–hydrogen exchange inhibitor HOE 642 (cariporide)

Mary Lee Myers^a,*, Parviz Farhangkhoee^a and Morris Karmazyn^b

^aDivision of Cardiovascular Surgery, London Health Sciences Centre—Victoria, London, Ontario, Canada
^bDepartment of Pharmacology and Toxicology, The University of Western Ontario, London, Ontario, Canada

^* Corresponding author. 370 South Street-C101, London, Ontario, N6B 1B8, Canada. Tel.: +1-519-667-6646; Fax: +1-519-667-6834.

Received 12 October 1997; accepted 23 March 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Sodium–hydrogen exchange (NHE) activation is a major mechanism of cardiac injury produced by ischemia and reperfusion. In addition, NHE may mediate the direct effects of hydrogen peroxide (H₂O₂) in normally perfused hearts. The present study was done to determine whether H₂O₂ at low concentrations producing mild myocardial depression affects post-ischemic recovery of function and to determine the ability of the NHE inhibitor HOE 642 to modulate this effect. Methods: Isolated Langendorff-perfused rat hearts with a left ventricular balloon inflated to an initial end-diastolic pressure of 5 mmHg were subjected to 90 min of global zero-flow ischemia followed by 60 min reperfusion. In Study 1, hearts were randomized for perfusion with or without H₂O₂ (20 µM) for 15 min before ischemia and throughout reperfusion. In Study 2, identical experiments were done except that the hearts were pretreated with the NHE inhibitor HOE 642 (5 µM). Function was assessed by determining intraventricular pressures. Results: Recovery of developed pressure in Study 1 after 10 min reperfusion was 60.3±8% of pre-ischemic values in control hearts whereas this was reduced to 29.9±10% in hearts treated with H₂O₂ (P<0.05). After 60 min of reperfusion recovery of developed pressure was 80.3±5.2% and 60.7±7% in control and H₂O₂-treated hearts, respectively (P<0.05). Recovery of rates of pressure development (+dP/dt) and relaxation (–dP/dt) paralleled the effects seen with developed pressure. Moreover, these effects were associated with significantly elevated end-diastolic pressure during the last 20 min of reperfusion. In Study 2, HOE 642 completely prevented the deleterious effect of H₂O₂, both with respect to ventricular recovery and to the elevation in end-diastolic pressure during reperfusion. Conclusions: Our results show that very low concentrations of H₂O₂ significantly impair recovery of function in this rat model of myocardial ischemia–reperfusion. Moreover, our results suggest that this effect is likely dependent on NHE activity and can be prevented by treatment with the NHE inhibitor HOE 642.

KEYWORDS HOE 642 (cariporide); Sodium–hydrogen exchange; Hydrogen peroxide; Myocardial oxidative injury; Rat heart

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
One of the major mechanisms whereby myocardial pH is restored following acidosis is via the sodium–hydrogen exchanger (NHE) which functions by extruding protons in exchange for sodium ions in a 1:1 stoichiometric relationship. Although there are at least five NHE isoforms, the NHE-1 isoform predominates in myocardial tissue and can be pharmacologically inhibited by amiloride and its N-5 disubstituted derivatives [1], as well as by more specific and selective compounds such as HOE 642 (cariporide: 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methane sulphate) [2]. Inhibition of NHE has been shown to provide significant protection in a variety of models of myocardial ischemia–reperfusion with consistent improvement in functional recovery, metabolic status, attenuation of arrhythmias, preservation of cellular ultrastructure and inhibition of apoptosis [1–6]. On the other hand, it has been shown that agents that activate NHE such as endothelin-1 [7]or alpha₁ adrenergic agonists [8]can induce cardiac injury and arrhythmias through NHE dependent mechanisms.

Although reversal of intracellular acidosis is essential for cell viability, marked activation of NHE at the time of reperfusion may paradoxically exacerbate tissue damage and dysfunction. There is evidence that the sodium influx in exchange for hydrogen results in an increase in intracellular calcium levels [9], likely secondary to reduced calcium efflux because of the reduction in the sodium gradient driving the sodium–calcium exchanger. The resulting intracellular calcium overload has multiple potential deleterious effects but would theoretically be reduced or prevented by NHE inhibition. NHE activation during ischemia per se likely also contributes to tissue injury as evidenced by NMR studies demonstrating an inhibition of the increases in both sodium and calcium levels in ischemic myocardium by NHE inhibitors [10]. Moreover, NHE inhibitors are generally more effective when administered prior to ischemia rather than at the time of reperfusion only [1, 4]. There is also evidence that NHE inhibitors may protect against other forms of injury such as the direct deleterious effects of lysophosphatidylcholine, a toxic metabolite produced by ischemic myocardium [11]. Furthermore, NHE inhibitors have been shown to provide protection to the myocardium from the deleterious effects of hydrogen peroxide (H₂O₂) but not against free radical generation [12]. This is of particular interest in that there is evidence that H₂O₂ contributes significantly to myocardial ischemia–reperfusion injury [13]. The present study was accordingly carried out to assess the effect of a very low level of H₂O₂ on the myocardial response to ischemia–reperfusion and to further assess the ability of the NHE inhibitor HOE 642 to modulate this effect.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animals
Studies were carried out using male Sprague–Dawley rats (250–300 g) purchased from Charles-River Canada (St. Constant, Quebec, Canada) or Harlan Sprague–Dawley Inc (Indianapolis, IN, USA) and maintained in the health sciences Animal Care facility of the University of Western Ontario in accordance with the guidelines of the Canadian Council on Animal care (Ottawa, Ontario, Canada).

2.2 Isolated heart preparation
The animals were decapitated and the hearts rapidly removed and placed in cold Krebs–Henseleit buffer (composition below). The aortas were cannulated for perfusion on a non-recirculating Langendorff apparatus at a flow-rate of 10 ml/min with Krebs–Henseleit buffer (composition in mM: NaCl 120, KCl 4.63, KH₂PO₄ 1.17, CaCl₂ 1.25, NaHCO₃ 20, glucose 8). The buffer was continuously gassed with a 95% 0₂–5% CO₂ mixture and maintained at 37°C. Left ventricular function was assessed by means of a latex balloon inserted through the mitral valve and secured in the left ventricle. Distilled water was injected into the balloon using a micrometer syringe to obtain a left ventricular end-diastolic pressure of 5 mmHg and this was subsequently left unadjusted for the duration of the protocol. The rates of left ventricular pressure development (+dP/dt) and relaxation (–dP/dt) were determined electronically with a differentiator amplifier. Perfusion pressure, which reflects coronary vascular resistance in this constant flow model, was monitored through a side-arm of the aortic cannula by means of a fluid-filled catheter connected to a pressure transducer. Right ventricular pacing wires were inserted to maintain the heart rate at 300 beats/min. Pacing was maintained throughout both ischemia and reperfusion.

2.3 Experimental protocol
The intent of the study was to assess the potential protective effect of NHE inhibition on H₂O₂ induced inhibition of post-ischemic recovery. Thus it was decided to expose non-control hearts to a mild insult before and after ischemia to mimic a physiologic source of injury. Preliminary studies were carried out using an infusion of 10 µM H₂O₂. With this concentration, however, there was no detrimental effect on either baseline function or recovery of developed pressure after reperfusion. The concentration of H₂O₂ was accordingly increased to 20 µM in order to produce significant inhibition of post-ischemic ventricular recovery.

Hearts were perfused for 45 min prior to a 90 min period of global normothermic ischemia. Initial reperfusion was at 5 ml/min for 2 min, then 8 ml/min for the next 3 min before returning to the pre-ischemic flow-rate of 10 ml/min for a total reperfusion period of 1 hour. Such staged reperfusion has been shown to improve recovery of function, potentially because of less pronounced NHE activation secondary to more gradual restoration of extracellular pH [14]. Two separately randomized studies were carried out in sequence. In Study 1, hearts were randomized for perfusion with or without H₂O₂ (20 µM) for 15 min before ischemia and throughout reperfusion. Study 2 consisted of the same control and H₂O₂ groups but both groups also received the NHE inhibitor HOE 642 (5 µM) for 30 min prior to ischemia as well as throughout reperfusion.

2.4 Chemicals
H₂O₂ was purchased from BDH (Toronto, Ontario, Canada). HOE 642 (cariporide) was a generous gift from Dr. Wolfgang Scholz, Hoechst–Marion–Roussel (Frankfurt, Germany).

2.5 Statistical analysis
Experimental data were analysed using analysis of variance (ANOVA) followed by a Student–Newman–Keuls test to identify significant treatments with a two-way ANOVA for repeated measures being used to assess cardiac function data. Baseline data in Table 1 were analyzed using Student's t-test for paired and unpaired data where appropriate. Differences were considered significant when P<0.05.

View this table: [in this window] [in a new window]   Table 1 Baseline hemodynamic function

 

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
As shown in Table 1, there were no significant differences between the control and H₂O₂ groups with regard to baseline function after the initial 30 min equilibration period. Administration of H₂O₂ resulted in a modest but significant attenuation in developed pressure although there was still no significant difference in function between the control and H₂O₂ groups in each study just prior to ischemia (Baseline 2).

Representative recordings demonstrating the effects of H₂O₂ in terms of developed pressure in the presence or absence of HOE 642 are shown in Fig. 1. The results for post-ischemic recovery of developed pressure in Studies 1 and 2 are summarized in Fig. 2. In Study 1, H₂O₂ significantly depressed recovery of developed pressure after reperfusion compared to control hearts. Thus, after 10 min of reperfusion, developed pressure in control hearts had recovered to 60.3±8% of the pre-ischemic level compared to only 29.9±10% in H₂O₂-treated hearts. This significant difference in recovery was sustained throughout the reperfusion period. In Study 2, administration of HOE 642 had no effect on the magnitude of recovery after reperfusion in control hearts although there was a tendency towards a more rapid recovery with peak recovery occurring after 10 min of reperfusion (Fig. 2, bottom) compared to control hearts not treated with HOE 642 (Fig. 2, top). The most striking observation, however, was the complete inability of H₂O₂ to affect recovery of function in the presence of the NHE inhibitor. For example, developed pressure in H₂O₂ hearts after 10 min of reperfusion was 74.4±9% which was not significantly different from the 81.3±12% recovery in the control group. As shown in Fig. 3, identical effects of H₂O₂ were seen with respect to +dP/dt as well as the ability of HOE 642 to prevent these effects.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative recordings showing changes in left ventricular developed and diastolic pressure in a control heart subjected to ischemia and reperfusion (top panel) and the effect of 20 µM H2O2 alone (middle panel) or in the presence of 5 µM HOE 642 (bottom panel). Tracings obtained in individual heart preparations depict pressures at various time points during ischemia and reperfusion. BL1=30 min of pre-ischemic perfusion; BL2=45 min of pre-ischemic perfusion. Vertical bar indicates 60 mmHg pressure.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Left ventricular developed pressure (as a percentage of baseline) during 60 min of reperfusion following 90 min of ischemia. * P<0.05. Upper panel—Study 1; without HOE 642. Lower panel—Study 2; with HOE 642

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Rate of left ventricular pressure development (as a percentage of baseline) during 60 min of reperfusion following 90 min of ischemia. * P<0.05. Upper panel—Study 1; without HOE 642. Lower panel—Study 2; with HOE 642

 
To assess diastolic function both diastolic pressure (Fig. 4) and rates of relaxation (–dP/dt, Fig. 5) were monitored. The elevation in diastolic pressure for Studies 1 and 2 is presented in Fig. 4. The initial rapid increase in diastolic pressure with reperfusion was unaffected by any treatment. However, in Study 1 diastolic pressure was significantly elevated by H₂O₂ during the last 20 min of reperfusion (Fig. 4, top). In contrast, HOE 642 completely attenuated this effect (Fig. 4, bottom). Moreover, as shown in Fig. 5, the rate of left ventricular relaxation was significantly depressed in the H₂O₂ group in Study 1. Following 10 min of reperfusion, –dP/dt was only 25.9±9.2% in the H₂O₂ group versus 55.5±9.5% in the control group. This magnitude of difference was sustained throughout reperfusion. In contrast, HOE 642 completely prevented the ability of H₂O₂ to depress –dP/dt.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Diastolic pressure (in mmHg) during 90 min of ischemia followed by 60 min of reperfusion. * P<0.05. Upper panel—Study 1; without HOE 642. Lower panel—Study 2; with HOE 642

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Rate of left ventricular relaxation (as a percentage of baseline) during 60 min of reperfusion following 90 min of ischemia. * P<0.05. Upper panel—Study 1; without HOE 642. Lower panel—Study 2; with HOE 642

 
Coronary perfusion pressure showed a tendency to increase modestly with time during reperfusion. However, as summarized in Table 2, levels remained virtually identical among the four groups.

View this table: [in this window] [in a new window]   Table 2 Coronary perfusion pressure (mmHg)

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
NHE activation appears to play a significant role in the pathophysiology of myocardial ischemia–reperfusion injury. Pharmacologic inhibition of the exchanger has been shown to provide significant protection in a variety of models of myocardial ischemia–reperfusion [1–5]. By virtue of its ability to extrude hydrogen from the cell in exchange for sodium, the exchanger provides one of the major mechanisms for maintaining intracellular pH [15]. Paradoxically, however, it is well-established that marked activation of NHE at the time of reperfusion contributes to tissue damage and dysfunction. Although intracellular pH is restored, there is a large influx of sodium in exchange for hydrogen, which is further aggravated by the ischemia-induced depression of the sodium–potassium pump [16]. The reduction in the sodium gradient driving the sodium–calcium exchanger results in decreased calcium efflux and relative calcium overload with its multiple potential deleterious effects. In support of this concept, the improved functional recovery with NHE inhibition has been shown to be associated with a relative decrease in both sodium and calcium levels in the reperfused myocardium [9]. In addition, there is evidence that NHE activation during ischemia per se also contributes to cardiac injury which may explain the superior myocardial protection seen when NHE inhibitors are administered prior to ischemia [1, 4].

Myocardial ischemia–reperfusion is a complex phenomenon, however, and there is evidence that there are other potential mechanisms underlying the protective effects seen so consistently with NHE inhibition such as attenuation of the direct toxic effects of lysophosphatidylcholine and H₂O₂ [11, 12]. The present study further addressed the potential importance of NHE in mediating the deleterious effects of H₂O₂ by determining whether the depression in post-ischemic recovery of function seen with a low concentration of H₂O₂ could be modified by the NHE inhibitor HOE 642. H₂O₂ was added prior to ischemia as well as during reperfusion as levels have been previously demonstrated to be elevated during both ischemia and reperfusion [17, 18]. In addition, exogenous production of H₂O₂ would occur in vivo as a result of neutrophil accumulation in the ischemic area [19]. The model of staged reperfusion utilized has been shown to enhance post-ischemic recovery [14]. The mechanism for this protection is not known with certainty although we postulate, as proposed by other investigators [14], that it may be related to delayed restoration of extracellular pH at the onset of reperfusion thereby attenuating NHE activation [20]. Our findings support this concept as the ultimate magnitude of functional recovery utilizing this reperfusion strategy was not altered by HOE 642, although early recovery was somewhat more rapid. Our results show, however, that the NHE inhibitor prevents the deleterious effects of H₂O₂ on recovery of myocardial function following ischemia–reperfusion. Hearts in Study 1 exposed to H₂O₂ showed a sustained and significant depression in recovery of contractile function following ischemia when compared to the control group. This was not observed in the presence of HOE 642. These results further reinforce the concept that some of the beneficial effect so consistently demonstrated with NHE inhibitors in myocardial ischemia–reperfusion may be due to an ability of these agents to limit the deleterious effects of H₂O₂. It is unlikely that the salutary effects of NHE inhibitors with respect to H₂O₂ demonstrated here as well as previously [12]are related to antioxidant properties as these drugs were ineffective against a free radical generating system consisting of purine and xanthine oxidase [12].

There is a large body of experimental evidence indicating that cytotoxic oxygen metabolites contribute to myocardial ischemia–reperfusion damage and dysfunction [21]. The relative importance of the various oxygen metabolites remains the subject of some debate but evidence from a number of sources indicates that H₂O₂ is a significant mediator of myocardial injury. Studies carried out in crystalloid perfused isolated rat [13]and rabbit [22]hearts suggest that H₂O₂ is the most important oxygen metabolite in reperfusion injury even in the absence of neutrophils. Slezak et al. [17]demonstrated a 250% increase in myocardial H₂O₂ content after 30 min of ischemia in the rat heart, followed by a 600% increase in H₂O₂ content after 2 min of reperfusion. Conversely, levels of catalase and glutathione peroxidase, the enzymes that degrade H₂O₂, are known to be decreased by myocardial ischemia [23]. An increase in pre-ischemic tissue catalase levels through pre-treatment with endotoxin [24]or heat-shock [25]has been shown to improve post-ischemic recovery of ventricular function, while the beneficial effect of perfusing ischemic rat hearts with low concentrations (0.5%) of erythrocytes appeared due to scavenging of myocardial H₂O₂ by catalase and/or glutathione within the red cell [26]. In addition, H₂O₂ appears to be the primary mediator of the contractile dysfunction produced by activated neutrophils [27].

Although it produced a direct negative inotropic effect on its own, the 20 µM concentration of H₂O₂ used in this study is relatively low. Using isolated cardiomyocytes, Janero et al. [28]found that an initial bolus of 50 µM was required to elicit significant cellular injury. A concentration of 150 µM was used in the study of Hoque and Karmazyn [12], but the isolated hearts in that study were not exposed to the additional oxidative stress of ischemia and reperfusion. Based on the present observations it appears that a relatively low concentration of H₂O₂ possessing modest negative inotropic effects may result in significant injury when combined with additional pathological factors such as myocardial ischemia. In this regard, it is interesting that plasma H₂O₂ levels in patients undergoing coronary bypass surgery were found to increase from 80±8 µM/ml before to 155±13 µM/ml immediately after cardiopulmonary bypass [29], an increased production presumed largely secondary to complement-activated neutrophils. Our study suggests that this could be a sufficiently high concentration to potentially exert deleterious effects on the ischemic myocardium.

The precise mechanisms by which H₂O₂ compromises recovery is not known. H₂O₂ has been shown to have a number of deleterious effects on cardiomyocytes including membrane lipid peroxidation [28], alteration in ion channel function [30], nucleotide depletion [12, 28], increases in intracellular calcium [30, 31]and contractile dysfunction [13, 27]. A unifying mechanism of cell injury secondary to H₂O₂ has not been identified [28, 32]but there are potential links between H₂O₂ induced cardiotoxicity and NHE. H₂O₂ has been shown to exert effects via protein kinase C [33], a group of isoenzymes that also plays an important role in NHE activation [4, 5]. In addition, Kaminishi et al. [30]have provided evidence that H₂O₂ inhibits the sodium–potassium ATPase pump resulting in an increase in cardiomyocyte calcium levels secondary to sodium–calcium exchange. There is also evidence that H₂O₂ inhibits myocardial sodium–calcium exchange [34, 35], an effect which would further exacerbate elevated intracellular calcium levels due to reduced calcium removal via this process. In summary, this study demonstrates a significant beneficial effect of the NHE inhibitor HOE 642 on the deleterious effect of H₂O₂ in the heart subjected to ischemia and reperfusion. Further study will be required to better characterize this link and to identify the mechanisms involved.

Time for primary review 30 days.

	Acknowledgements

 
This study was supported by the Department of Surgery, University of Western Ontario, and by the Medical Research Council of Canada. Dr. Karmazyn is a Career Investigator of the Heart and Stroke Foundation of Ontario. We thank Dr. Wolfgang Scholz of Hoechst–Marion–Roussel, Frankfurt, Germany for gift of HOE 642.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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