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(Circulation Research. 1997;80:95-102.)
© 1997 American Heart Association, Inc.

Articles

Na⁺-H⁺ Exchange Inhibition Protects Against Mechanical, Ultrastructural, and Biochemical Impairment Induced by Low Concentrations of Lysophosphatidylcholine in Isolated Rat Hearts

A.N. Ehsanul Hoque, James V. Haist, Morris Karmazyn

the Department of Pharmacology & Toxicology, Faculty of Medicine, University of Western Ontario, London, Canada.

Correspondence to Dr M. Karmazyn, Department of Pharmacology and Toxicology, University of Western Ontario, Medical Sciences Building, London, Ontario N6A 5C1, Canada. E-mail mkarm@julian.uwo.ca

	Abstract

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Lysophophatidylcholine (LysoPC) accumulates rapidly in the ischemic myocardium and is an important mediator of ischemia-induced cell injury. Na⁺-H⁺ exchange (NHE) inhibition has been demonstrated to protect the ischemic and reperfused myocardium. We determined whether NHE inhibition can also modulate cardiotoxicity produced by LysoPC (3 and 5 µmol/L) in isolated rat hearts. At 3 µmol/L, LysoPC produced a depression in left ventricular developed pressure (LVDP) and elevation in left ventricular end-diastolic pressure (LVEDP), which were 19±7% and 1290±205% of pre-LysoPC values, respectively, after 30 minutes of treatment. In the presence of the NHE inhibitor 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642, 5 µmol/L), LVDP was reduced to only 80.8±8.6%, and LVEDP increased to 270±32% (P<.05 for both parameters). LysoPC significantly depressed tissue ATP, creatine phosphate, and glycogen contents and increased lactate levels, all of which were significantly attenuated by HOE 642. Moreover, marked LysoPC-induced ultrastructural abnormalities, including mitochondrial and myofibrillar disruption, were totally prevented by HOE 642. This protection was mimicked by another NHE inhibitor, methylisobutylamiloride (5 µmol/L). HOE 642 was also effective against injury produced by 5 µmol/L LysoPC although, generally, the protection was less marked than that observed against 3 µmol/L; LVDP depression after 30 minutes was 10.1±4.3% and 41.4±10.4% of pre-LysoPC values in control and HOE 642-treated hearts, respectively (P<.05), whereas corresponding LVEDP elevations were 1629±393% and 990±144% (P>.05). In myocytes superfused with bicarbonate-free buffer subjected to acid loading by NH₄Cl pulsing, pH recovery (as measured by acid flux) was significantly stimulated by 3 µmol/L LysoPC, indicative of NHE activation. Our study shows that cardiac injury produced by low concentrations of LysoPC can be effectively attenuated by NHE inhibition. The results also suggest that the beneficial effects of NHE inhibitors on the ischemic myocardium may be, at least partially, mediated by inhibiting the deleterious effects of LysoPC.

Key Words: lysophosphatidylcholine • isolated rat heart • myocyte • cardiac injury • Na⁺-H⁺ exchange

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocardial NHE represents one of the major mechanisms responsible for restoration of pH_i after ischemia-induced acidosis, although its activation has been proposed to represent an important mediator of tissue injury after the restoration of flow.^{1 2} Accordingly, a large number of studies have now been reported that have demonstrated that pharmacological inhibitors of the antiporter protect the ischemic and reperfused heart (reviewed in References 3 and 4). Although the mechanisms that account for NHE involvement in cardiac injury are not precisely understood, one such mechanism is thought to involve inhibition of Na⁺ influx via the antiporter, because elevation in [Na⁺]_i can then increase [Ca²⁺]_i through Na⁺-Ca²⁺ exchange.^{3 4} As recently reviewed,⁵ this would result in an ionic imbalance contributing to cell injury.

Ischemia- and reperfusion-associated dysfunction represents complex phenomena reflecting a myriad of intracellular alterations. One of the important consequences of ischemia is the production and accumulation of various toxic metabolites that can contribute to tissue injury. Of relevance to the present study, ischemia results in the accumulation of lysophosphoglycerides, particularly LysoPC, in the myocardium.^{6 7} LysoPC is a potent cytotoxic agent that produces cardiac arrhythmias,^{8 9} contractile depression, and cell injury¹⁰ as well as depletion of high-energy phosphate stores and increases in both lactate and free fatty acid levels,¹¹ although the mechanisms for these effects are not well understood. The present study was undertaken to determine whether NHE inhibition protects against LysoPC-induced cardiac injury, because this may explain, at least in part, the salutary effects of antiport inhibition on the ischemic and reperfused myocardium. Moreover, the present study was also based on the fact that LysoPC has been shown to stimulate the activity of PKC,^{12 13} a family of isozymes that is involved in the stimulation of NHE. In fact, PKC-dependent NHE activation has been shown to increase injury of the hypoxic myocardium.¹⁴ To our knowledge, the effects of LysoPC on NHE activity have not been previously studied, nor are there any reports dealing with the potential effect of NHE inhibition on LysoPC-induced cardiac injury. Accordingly, the present study was designed to address these questions by determining the effects of LysoPC on NHE activation in cardiac cells and by assessing the effects of a novel and specific NHE inhibitor, HOE 642,¹⁵ on the direct cardiotoxic properties of this amphiphile on isolated hearts.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Male Sprague-Dawley rats (250 to 300 g) were purchased from Charles-River Canada Ltd (St. Constant, Quebec, Canada). The animals were 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).

Heart Perfusion Studies
Rats were killed by decapitation; and the hearts were immediately removed and placed in cold Krebs-Henseleit buffer (composition below) in order to inhibit any further contractions and then mounted by the aorta on a stainless steel cannula and arranged for retrograde perfusion using the Langendorff method as described previously.¹⁶ The hearts were perfused at a flow rate of 10 mL/min using a peristaltic pump with Krebs-Henseleit buffer of the following composition (mmol/L): NaCl 120, KCl 4.63, KH₂PO₄ 1.17, CaCl₂ 1.25, MgCl₂ 1.2, NaHCO₃ 20, and glucose 8. The buffer was initially equilibrated and then continuously gassed with a 95% O₂/5% CO₂ mixture. The pH of the buffer was 7.4, and temperature was maintained at 37°C by enclosing the entire system in a series of water-jacketed coils. Coronary pressure was measured via a side arm of the perfusion cannula, which was connected to a pressure transducer (Spectramed P23XL). A latex water-filled balloon fixed to a pressure transducer was inserted through the mitral valve into the left ventricle for the determination of LVDP. Positive and negative dP/dt were obtained with a differentiator. LVEDP was adjusted to 5 mm Hg before the start of the experiment by adjusting the volume in the intraventricular balloon with the aid of a micrometer syringe. All determinations of ventricular performance were obtained on-line on a Pentium 586 computer using a Biopac data analysis system (Biolynx Scientific Equipment).

All hearts were initially equilibrated for 30 minutes; after which, LysoPC (3 or 5 µmol/L) was added for a further 30 minutes. To study the effect of NHE inhibition, HOE 642 (5 µmol/L) was added for 15 minutes before LysoPC administration; after which, LysoPC was added as for control hearts. A group of hearts that was perfused for 60 minutes without either LysoPC or HOE 642 was also studied. One set of studies was also performed to determine the effects of another NHE inhibitor, MIA (5 µmol/L), on the cardiodepressant effects of 3 µmol/L LysoPC. We previously reported that this agent is a potent inhibitor of the cardiac NHE and exerts marked protection against the ischemic and reperfused myocardium.¹⁷

Determination of Tissue Metabolites
At the end of the experimental protocol, the hearts were clamped with Wollenberger tongs precooled in liquid nitrogen, removed from the cannula, and stored in liquid nitrogen until the assays were performed. Assays for ATP, creatine phosphate, and lactate were performed on 6% perchloric acid extracts, whereas unextracted pulverized tissue was used to determine glycogen content. Enzymatic assays that were based on changes in extinction at 340 nm (indicative of either an increase or decrease in NADPH or NADH concentrations) were performed after the initiation of specific reactions, as described by Bergmeyer,¹⁸ using a Beckman DU-65 spectrophotometer.

Electron Microscopy
Two hearts from each of the four treatment groups (5 µmol/L LysoPC, 5 µmol/L LysoPC+HOE 642, 3 µmol/L LysoPC, and 3 µmol/L LysoPC+HOE 642) were immediately removed at the end of the perfusion period and perfusion-fixed at a constant perfusion pressure of 80 cm H₂O with 150 mL fixative containing 0.08 mol/L sodium cacodylate, 2% glutaraldehyde, and 1% paraformaldehyde, pH 7.4; after which, sections measuring 1 cmx1 mmx1 mm were cut from the left ventricle. Subsequent processing and sectioning were performed at the Department of Pathology, University Hospital Campus of the London (Canada) Health Sciences Center, with a Lynx automatic tissue processor. Tissues were postfixed with 1% osmium tetroxide and dehydrated with graded ethanol and acetone rinses. The final ethanol/acetone solution was replaced by en bloc stain consisting of a 3:7 ratio of uranyl nitrate/saturated lead acetate solution for 1 hour. The tissues were infiltrated and embedded in Epon-Araldite resin and polymerized overnight at 37°C. Thin sections (60 to 90 nm) were cut with a diamond microtome knife and stained with uranyl acetate and lead citrate. Samples were viewed on an electron microscope (model 109, Carl Zeiss, Inc). Each sample was coded and examined by one of the authors (M.K.) in a "blinded" fashion (without knowledge of the treatment protocol to which it was subjected). Approximately 200 cells were examined from each tissue sample with the assistance of an experienced electron microscopy technician.

Myocyte Isolation, pH_i Determination, and Calculation of J_H
Myocytes were prepared according to a method described previously.¹⁹ After removal, hearts were perfused for 5 minutes with a Ca²⁺-free buffer, pH 7.4, containing (mmol/L) NaCl 120, MgCl₂ 1, KCl 5.4, NaH₂PO₄ 0.33, HEPES 10, and glucose 10. After this initial perfusion, the buffer was changed to one containing 2 mg/mL collagenase (Worthington, type II) and 0.1 mg/mL protease (protease type XIV, Sigma Chemical Co), and the heart was then perfused in a recirculating manner for a further 12 minutes. The collagenase was then washed out for 2 minutes with buffer containing 0.2 mmol/L CaCl₂; after which, the heart was removed, placed in 15 mL of buffer containing 0.2 mmol/L CaCl₂, cut into several pieces, incubated for 15 minutes at 37°C, and then filtered through 210 micron mesh nylon screen, and the filtrate was centrifuged gently for 45 seconds. The buffer was aspirated off, and the cells were resuspended in 50 mL of buffer containing 0.5 mmol/L CaCl₂ for 10 minutes. After this period, the cells were gently centrifuged again, and the buffer was aspirated off. The final cell pellet was suspended in a buffer containing 1 mmol/L CaCl₂. The cells were counted in a hemocytometer and diluted to a concentration of 100 000 cells/mL at a final volume of 5 mL.

To study pH_i, the cells were loaded with 1 µmol/L of the acetoxymethyl ester of the pH-sensitive fluorescent dye BCECF (BCECF-AM) for 30 minutes at 37°C.²⁰ BCECF-loaded cells were then transferred to a chamber (Biophysica Technologies Inc) and mounted on the thermoregulated (37°C) stage of a Zeiss Axiovert 35 inverted microscope. The cells were continuously superfused (bath volume, 1 mL; flow rate, 1 mL/min) with HEPES solution containing 1.0 mmol/L CaCl₂ and field-stimulated with bipolar platinum electrodes at a frequency of 0.5 Hz using a Grass SD6 stimulator. Cells were initially equilibrated for 15 minutes. Fluorescence was detected in individual cells using excitation wavelengths of 440 and 490 nm at an emission wavelength of 530 nm with a Photon Technologies International monochromator-based spectrofluorometer.

ß_i was calculated as described in detail previously^{21 22} and involved the initial 2-minute application of 20 mmol/L NH₄Cl, followed by its stepwise removal; at which time, the values in the calculated change () in [NH₄⁺]_i and the measured pH_i were used to determine ß_i according to the following equation: ß_i=[NH₄⁺]_i/pH_i. These experiments were performed in the presence of 5 µmol/L HOE 642 to block the NHE plus 5 mmol/L BaCl₂ to prevent NH₄⁺ efflux through K⁺ channels.²¹

To produce intracellular acidification, cells were exposed, after the initial 15 minutes of equilibration, for 5 minutes to 20 mmol/L NH₄Cl in the absence or presence of LysoPC, which upon removal produced marked acidification.²³ The recovery from acidosis was monitored for 15 minutes as an indication of NHE activity as cells were superfused with bicarbonate-free buffer, thus precluding participation of other major pH_i regulatory processes. During this period, J_H was determined at 0.1 pH unit intervals according to the following equation: J_H=ß_ixrate of pH_i recovery as described in detail previously.^{21 22} NHE dependence of pH_i recovery was further confirmed by the fact that pH_i recovery was almost completely blunted by HOE 642 as well as MIA, and pH failed to return to normal during the monitoring period.

Chemicals
LysoPC was purchased from Sigma, and MIA was purchased from Research Biochemicals Inc. HOE 642 (cariporide) was a generous gift from Dr Wolfgang Scholz, Hoechst-Marion-Roussel (Frankfurt, Germany). All enzymes and other reagents required for metabolite assays were purchased from Sigma and were of the highest quality available. BCECF-AM was purchased from Molecular Probes. Fixative components used for electron microscopy were purchased from Marivac Limited.

Statistical Analysis
The data were analyzed using ANOVA, followed by a Student-Newman-Keuls test to identify significant treatments. Differences were considered significant at a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of HOE 642 on LysoPC-Induced Changes in Isolated Hearts
The effect of LysoPC on contractile function is summarized in Figs 1 and 2. At 3 µmol/L, LysoPC produced a time-dependent loss in contractility concomitant with a marked elevation in LVEDP; after 30 minutes of perfusion, LVDP decreased to 19±7%, whereas in the presence of HOE 642, LVDP was reduced to only 80.8±8.6% of pre-LysoPC values after 30 minutes of perfusion (Fig 1, top). In addition, LVEDP was elevated to 1290±205% of pre-LysoPC values in hearts treated with LysoPC only. HOE 642 significantly attenuated the ability of 3 µmol/L LysoPC to elevate LVEDP to 271±32% compared with values obtained before the administration of LysoPC (Fig 1, bottom). As shown in Fig 1, the protective effect of HOE 642 against contractile dysfunction produced by 3 µmol/L LysoPC was evident throughout virtually the complete perfusion period except within the first few minutes of LysoPC treatment.

View larger version (17K): [in this window] [in a new window]   Figure 1. Changes in LVDP and LVEDP in isolated hearts exposed to 30 minutes of perfusion with 3 µmol/L LysoPC (LPC) by itself (, n=6) or in the presence of HOE 642 (, n=5). Values are mean±SE and depict percentage of pre-LPC values. Basal values for LVDP before addition of LPC were 74±6 and 73±8 mm Hg for control and HOE 642–pretreated hearts, respectively, whereas LVEDP was 5 mm Hg for all hearts. *P<.05 vs the respective value obtained in the absence of HOE 642.

As is evident from Fig 2, the protective effects of HOE 642 were markedly diminished when the LysoPC concentration was increased to 5 µmol/L. With this concentration of LysoPC, HOE 642 significantly reduced the loss in LVDP only during the last 5 minutes of treatment. Thus, the depression in LVDP was attenuated from 10.1±4.3% of pre-LysoPC values in control hearts to 41.4±10.4% (P<.05) in hearts pretreated with HOE 642, whereas the attenuation of LVEDP was not significantly affected (1629±393% [control] versus 990±144% [HOE 642], P>.05). Representative recordings illustrating responses of developed pressure to 3 µmol/L LysoPC either in hearts treated with LysoPC alone (top) or in hearts pretreated with HOE 642 (bottom) are shown in Fig 3.

View larger version (19K): [in this window] [in a new window]   Figure 2. Changes in LVDP and LVEDP in isolated hearts exposed to 30 minutes of perfusion with 5 µmol/L LysoPC (LPC) by itself (, n=6) or in the presence of HOE 642 (, n=5). Values are mean±SE and depict percentage of pre-LPC values. Basal values for LVDP before addition of LPC were 70±9 and 74±8 mm Hg for control and HOE 642–pretreated hearts, respectively, whereas LVEDP was 5 mm Hg for all hearts. *P<.05 vs the respective value obtained in the absence of HOE 642.

View larger version (11K): [in this window] [in a new window]   Figure 3. Representative recordings demonstrating LVDP in a heart after the addition of 3 µmol/L LysoPC (LPC) alone (control) or in a heart pretreated with 5 µmol/L HOE 642. Arrows indicate time when LPC was administered.

Although the data are not shown, the effects of LysoPC and HOE 642 on both positive and negative dP/dt paralleled the results obtained with respect to LVDP in that HOE 642 significantly attenuated the loss in function particularly in response to the lower LysoPC concentration. There were no changes in coronary perfusion pressure with any treatment (Table 1).

View this table: [in this window] [in a new window]   Table 1. Coronary Perfusion Pressure During LysoPC Treatment in the Presence or Absence of HOE 642

We assessed ultrastructural changes associated with LysoPC treatment in the presence or absence of HOE 642. Fig 4a demonstrates a typical cell of a heart exposed to a 30-minute treatment with 3 µmol/L LysoPC alone. Marked damage is evident with obvious mitochondrial swelling and myofibrillar disruption. The nucleus exhibits chromatin aggregation. This type of injury was observed in 97% of cells examined. In contrast, we were unable to observe any morphological changes in any of the cells examined from hearts exposed to 3 µmol/L LysoPC in presence of HOE 642. As shown in Fig 4b, mitochondria were routinely intact, and sarcomeres appeared normal with evenly distributed chromatin in the nucleus. Fig 5b shows a typical myocyte from a heart perfused with 5 µmol/L LysoPC alone with mitochondrial clearing and myofibrillar disruption being particularly evident. This type of injury was consistent with 100% of the cells examined demonstrating such severity of damage. Gross morphological abnormalities were not observed in the presence of HOE 642, although a moderate degree of mitochondrial swelling was evident (Fig 5c) in 92% of cells examined. Myofibrils appeared normal, with no evidence of contraction bands or sarcomere disruption in any cells.

View larger version (110K): [in this window] [in a new window]   Figure 4. Transmission electron micrographs showing ultrastructural changes in a heart exposed for 30 minutes to 3 µmol/L LysoPC in the absence (a) or presence (b) of HOE 642. Magnification x8100 for both panels.

View larger version (107K): [in this window] [in a new window]   Figure 5. Transmission electron micrographs showing ultrastructural changes in a heart exposed for 30 minutes to 5 µmol/L LysoPC in the absence (b, magnification x8100) or presence (c, magnification x11 880) of HOE 642. A control heart was perfused for 60 minutes without drug treatment (a, magnification x11 880).

We further evaluated whether the effects of LysoPC and the protective influence of NHE inhibition could be associated with improved-energy metabolic status. As shown in Figs 6 and 7, LysoPC treatment was associated with significant depression in ATP, creatine phosphate, and glycogen contents, whereas tissue lactate levels were significantly increased. Metabolic changes were almost completely reversed by HOE 642, such that values were not significantly different from untreated control hearts. This influence of HOE 642 occurred with both LysoPC concentrations despite the reduced ability of this drug to attenuate the cardiodepressant actions of 5 µmol/L LysoPC.

View larger version (31K): [in this window] [in a new window]   Figure 6. Tissue ATP and creatine phosphate contents. Hearts were perfused for 60 minutes without treatment (open bars) or for 30 minutes without treatment followed by 30 minutes of perfusion with 3 µmol/L LysoPC (dotted bars), 3 µmol/L LysoPC+HOE 642 (bars with vertical lines), 5 µmol/L LysoPC (solid bars), or 5 µmol/L LysoPC+HOE 642 (bars with horizontal lines). #P<.05 vs untreated hearts; *P<.05 vs respective value obtained with LysoPC in the absence of HOE 642.

View larger version (24K): [in this window] [in a new window]   Figure 7. Tissue lactate and glycogen contents. Hearts were perfused for 60 minutes without treatment (open bars) or for 30 minutes without treatment followed by 30 minutes of perfusion with 3 µmol/L LysoPC (dotted bars), 3 µmol/L LysoPC+HOE 642 (bars with vertical lines), 5 µmol/L LysoPC (solid bars), or 5 µmol/L LysoPC+HOE 642 (bars with horizontal lines). #P<.05 vs untreated hearts; *P<.05 vs respective value obtained with LysoPC in the absence of HOE 642.

Effect of MIA
We tested the effect of another NHE inhibitor, MIA, on contractile changes produced by 3 µmol/L LysoPC. As summarized in Table 2, MIA also protected against both the cardiodepressant effect of LysoPC as well as the latter's ability to elevate LVEDP. Compared with the effect after HOE 642, a significant effect of MIA was not observed until 20 minutes of LysoPC treatment (Table 2).

View this table: [in this window] [in a new window]   Table 2. Changes in LVDP and LVEDP in Response to LysoPC in the Presence or Absence of MIA

Effect of LysoPC (3 µmol/L) on pH_i Recovery After Acid Loading
Because of the ability of two NHE inhibitors to attenuate the cardiotoxic properties of LysoPC, we considered it of importance to determine whether the latter can activate the antiporter. Our initial plan was to determine the effects of both LysoPC concentrations used in the perfused heart study; however, we report only the effects with 3 µmol/L LysoPC, because higher concentrations produced direct cytotoxic effects, rendering pH_i measurements very difficult and inconsistent. As shown in Fig 8, 3 µmol/L LysoPC significantly accelerated pH_i recovery after the acidosis produced by NH₄Cl pulsing, such that acid efflux was greater under acidic pH_i conditions after NH₄Cl removal. Since these cells were bathed in bicarbonate-free conditions, this rapid acid efflux is likely indicative of greater NHE activation.

View larger version (15K): [in this window] [in a new window]   Figure 8. JH after NH4Cl pulsing–induced acidosis at different pHi values in rat ventricular myocytes in the control cells or in presence of 3 µmol/L LysoPC (LPC). Values are mean±SE with n=14 (except at pHi 6.6, where n=4). *P<.05 vs control. On the right are representative fluorescence recordings showing continuous pHi recovery during the initial 10-minute period following NH4Cl removal and at the end of the 15-minute monitoring period (broken recording segment). Horizontal line indicates 5 minutes of exposure to 20 mmol/L NH4Cl.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NHE likely represents a major contributing factor to cardiac injury associated with myocardial ischemia and reperfusion, as evidenced by extensive studies showing protection against such injury by inhibitors of the antiporter (reviewed in References 3 and 4). These include both amiloride and its N-5 disubstituted derivatives^{24 25 26 27} as well as nonamiloride NHE inhibitors, including HOE 694²⁸ and HOE 642.¹⁵ Despite the well-documented ability of NHE inhibitors to exert salutary effects on the ischemic and reperfused myocardium, the mechanistic bases for the protection remain to be determined. The most widely promulgated hypothesis is that activation of NHE, particularly at reperfusion, produces a large influx in Na⁺, which then results in diminished Ca²⁺ extrusion through the Na⁺-Ca²⁺ exchange pathway, resulting in the intracellular accumulation of toxic levels of Ca²⁺,^{3 4} a process exacerbated by inhibition of the Na,K-ATPase by ischemia, further compromising intracellular Na⁺ regulation. Indeed, studies have shown that the protective effects of amiloride are associated with diminished tissue accumulation of both Na⁺ and Ca²⁺ levels.²⁵ An important role for intracellular events at the time of reperfusion mediating NHE-dependent injury is further highlighted by the fact that administration of NHE inhibitors at the time of reperfusion is associated with marked cardioprotection.^{26 27} Other investigators have proposed that the deleterious effects of NHE and beneficial effects of NHE inhibitors are unrelated to Ca²⁺ but are instead reflective of proton extrusion per se, resulting in a "pH paradox."^{3 29} Taken together, it appears that the beneficial effects of NHE inhibitors against the ischemic and reperfused myocardium are complex and most likely involve multifactorial processes.

The basis for the present study was to further investigate the potential mechanism for the protective effects of NHE inhibition on the ischemic myocardium. We used HOE 642 as the primary drug to probe NHE for this purpose because this agent is a potent inhibitor of the antiporter and is selective against NHE-1, the primary if not sole NHE isoform found in the myocardium.¹⁵ The rationale for determining the effects of NHE inhibition against LysoPC-induced cardiac injury was based on a number of premises. First, ischemia produces a rapid (within 4 to 5 minutes) elevation in LysoPC levels in the myocardium^{6 7} as well as in coronary sinus blood during pacing in patients with coronary artery disease.³⁰ Second, the potent cardiotoxic properties of LysoPC have been well documented and include the development of arrhythmias^{8 9 31} and contractile depression,¹⁰ suggesting that LysoPC accumulation could contribute to ischemia-induced cardiac dysfunction. Third, LysoPC has been shown in a number of studies to activate the Na⁺ channel in the cardiac cell,^{32 33 34} suggesting that it can contribute to the scenario described above for NHE-mediated ischemic and reperfusion injury, particularly since NHE represents a major contributor to Na⁺ entry in the heart.³⁵ It should also be noted that LysoPC also inhibits myocardial Na,K-ATPase activation, which would also contribute to Na⁺ loading under pathological conditions, although it appears that this effect occurs only with very high LysoPC concentrations.³⁶ Last, there is evidence in the literature, albeit indirect, that LysoPC could stimulate NHE activity, which was confirmed in the present study. For example, LysoPC, particularly at low concentrations (<20 µmol/L), stimulates PKC, a major activator of NHE, in different tissues, including vascular endothelium, where a PKC-dependent stimulation of free radical production by LysoPC has been demonstrated.^{12 13 37} To our knowledge, whether LysoPC can activate NHE in the heart has not been previously demonstrated. Accordingly, we considered it of importance to investigate whether NHE inhibition could affect the cardiotoxic properties of low concentrations of LysoPC, because this could further contribute to our understanding to both the mechanisms of LysoPC-induced cardiac injury and the protective effect of NHE inhibition on the ischemic myocardium.

The present results are the first to demonstrate that two structurally dissimilar NHE inhibitors can protect against the deleterious effects of low LysoPC concentrations on the rat heart. The protection was manifested by improved mechanical function, inhibition of ultrastructural damage, and preserved energy metabolite status. Coupled with our observation that LysoPC can activate NHE, our results, when taken together, suggest that NHE could represent an important mediator of cardiac injury produced by LysoPC. It is of paramount importance, however, to stress that NHE inhibition was effective only against low concentrations of LysoPC, with very effective protection against 3 µmol/L of the amphiphiles and reduced benefit against 5 µmol/L, at least with respect to functional and ultrastructural parameters. Two added studies that were performed (n=2, data not shown) support this contention. First, doubling either the HOE 642 or MIA concentrations failed to offer any added benefit to the modest attenuation of the cardiodepressant effect of 5 µmol/L LysoPC afforded by 5 µmol/L HOE 642 (Fig 2), suggesting that the degree of NHE activation is unlikely to be a factor in diminished protection by NHE inhibitors against higher concentrations of LysoPC. Second, when the LysoPC concentration was increased to 10 µmol/L, a total lack of protection by either HOE 642 or MIA was seen with respect to all parameters. Thus, our results could be summarized to indicate that NHE represents an important contributor to cardiac injury associated with low LysoPC amounts, although the antiporter is less important when LysoPC concentrations are increased. The reduced effectiveness of NHE inhibitors to protect against higher concentrations of LysoPC most likely reflects recruitment of other mechanisms, including a direct perturbation of cell membrane integrity via a detergent-like action leading to cell injury.^{38 39}

Because of the complexity of LysoPC-induced cardiac toxicity, we considered it beyond the scope of the present study to carry out a detailed assessment of the potential cellular mechanisms underlying our findings. Extensive research over the past number of years has revealed complex effects of LysoPC on cardiac parameters that appear to be concentration dependent and that may have important bearing on the interpretation of our results. A particularly relevant set of observations is that low micromolar concentrations of LysoPC alter ion channel gating behavior in cardiac myocytes, including increased activity of the Na⁺ channel.^{32 33 34} This would be expected to increase Na⁺ influx and subsequently elevate [Ca²⁺]_i via Na⁺-Ca²⁺ exchange mechanisms. The present study shows that in addition to the LysoPC-induced elevation in [Na⁺]_i via Na⁺ channel activation, it can also potentially increase Na⁺ levels by direct activation of NHE, particularly under acidotic conditions. It is unlikely that NHE activation per se is a causative factor for LysoPC-induced toxicity. However, reduction of Na⁺ influx via NHE exchange would be expected to decrease total [Na⁺]_i after LysoPC exposure, thereby decreasing potential Ca²⁺-overloading conditions. Indeed, it has been shown that although LysoPC, at concentrations similar to those used in the present study, increases myocardial Ca²⁺ levels,⁴⁰ this occurs secondary to increased Na⁺ channel activity and not via a direct effect on either sarcolemmal integrity or activation of Ca²⁺ channels.⁴¹

In conclusion, the present study shows that NHE inhibition effectively attenuates cardiac injury produced by direct administration of low concentrations of LysoPC, although that protection is attenuated with higher LysoPC concentrations. The present study also shows that LysoPC can activate NHE under acidotic conditions. The inhibition of LysoPC-mediated toxicity may further explain the beneficial effects of NHE inhibitors in the ischemic and reperfused myocardium.

	Selected Abbreviations and Acronyms

 

ßi = intracellular intrinsic buffering power HOE 642 = 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate JH = acid flux LVDP = left ventricular developed pressure LVEDP = left ventricular end-diastolic pressure LysoPC = lysophosphatidylcholine MIA = methylisobutylamiloride NHE = Na+-H+ exchange PKC = protein kinase C

	Acknowledgments

 
This study was supported by a grant from the Medical Research Council of Canada. Drs Hoque and Karmazyn were recipients of a Postdoctoral Fellowship and a Career Investigator Award, respectively, from the Heart and Stroke Foundation of Ontario. We thank Dr Wolfgang Scholz of Hoechst-Marion-Roussel (Frankfurt, Germany) for the generous gift of HOE 642. We also thank Elaine Hunter, Department of Pathology, University Hospital Campus of the London Health Science Center, for her advice and assistance with electron microscopy. The authors are also grateful to Drs Jeff Dixon of the Department of Physiology, University of Western Ontario, and Metin Avkiran of St. Thomas' Hospital, London, UK, for their advice concerning the measurement of J_H.

	Footnotes

 
Presented in part at the 18th Annual Meeting of the North American Section of the International Society for Heart Research, Chicago, Ill, June 9-13, 1996.

Received May 22, 1996; accepted October 17, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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