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1 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; 2 Division of Cardiology, Department of Medicine, University of Louisville, Louisville, Kentucky 40202; and 3 Boehringer Ingelheim Pharma KG, D-88397 Biberach an der Riss, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Numerous studies have examined the effect of Na+/H+ exchanger (NHE) inhibition on the myocardium; however, the effect of NHE-1 inhibition on neutrophil function has not been adequately examined. An in vivo canine model of myocardial ischemia-reperfusion injury in which 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion was used to examine the effect of NHE-1 inhibition on infarct size (IS) and neutrophil function. BIIB-513, a selective inhibitor of NHE-1, was infused before ischemia. IS was expressed as a percentage of area at risk (IS/AAR). NHE-1 inhibition significantly reduced IS/AAR and reduced neutrophil accumulation in the ischemic myocardium. NHE-1 inhibition attenuated both phorbol 12-myristate 13-acetate- and platelet-activating factor-induced neutrophil respiratory burst but not CD18 upregulation. Furthermore, NHE-1 inhibition directly protected cardiomyocytes against metabolic inhibition-induced lactate dehydrogenase release and hypercontracture. This study provides evidence that the cardioprotection induced by NHE-1 inhibition is likely due to specific protection of cardiomyocytes and attenuation of neutrophil activity.
myocardial infarction; sodium-hydrogen exchange; sodium-hydrogen exchanger-1
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ALTHOUGH A NUMBER OF STUDIES have demonstrated that inhibition of the Na+/H+ exchanger (NHE) protects against cardiac arrhythmias, myocardial stunning, and myocardial infarction, the vast majority of these experiments have been conducted in isolated hearts or in isolated cardiomyocytes (17). A number of reports have suggested that inhibition of NHE ultimately leads to a decreased accumulation of cytosolic Ca2+ and its subsequent detrimental consequences on cardiomyocytes (10, 29, 30). Interestingly, not only is the NHE isoform-1 (NHE-1) the predominant isoform in cardiomyocytes (23), but it is also the predominant isoform in neutrophils (11), which also contribute to arrhythmias and myocardial infarction (3, 6, 7). Several reports using less selective inhibitors of NHE-1 have demonstrated that inhibition of NHE attenuates several neutrophil functions (9, 11, 26-28). However, to date, the in vivo and in vitro effects of NHE-1 inhibition on neutrophil activity have not been thoroughly examined.
We have demonstrated previously that in vivo, BIIB-513, a specific and selective inhibitor of NHE-1, significantly reduces myocardial infarct size (13). To further delineate the cells through which this cardioprotection is achieved, we examined the effects of NHE-1 inhibition in vivo and in vitro on neutrophils and cardiomyocytes. The present study demonstrates for the first time in vivo that NHE-1 inhibition attenuates neutrophil accumulation in the ischemic myocardium. Furthermore, in vitro, NHE-1 inhibition attenuates neutrophil activation and protects isolated rabbit cardiomyocytes against metabolic inhibition-induced damage. These data suggest that the cardioprotection observed from NHE-1 inhibition in vivo is due not only to specific cardiomyocyte protection but may also be due to an attenuation of neutrophil activity.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials. To perform the present studies, BIIB-513 was used. All reagents were obtained from Sigma Chemicals (St. Louis, MO) or GIBCO-BRL (Gaithersburg, MD) unless otherwise indicated.
Ischemia-reperfusion protocol.
A standard myocardial ischemia-reperfusion protocol was employed,
as described previously (14). Dogs were assigned to one of
two groups in a randomized fashion. Either saline (control group) or
BIIB-513 (3.0 mg/kg) was infused intravenously for 15 min immediately
before left anterior descending (LAD) artery occlusion (Fig.
1). All dogs were subjected to 60 min of
LAD artery occlusion and 3 h of reperfusion. In both groups,
hemodynamic measurements and arterial blood gas analysis were obtained
before LAD artery occlusion, at 30 min during the 60-min occlusion, and
every hour after reperfusion. Regional myocardial blood flows were
determined at 30 min during the 60-min occlusion period and at the end
of the experiment.
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1 · g1,
3) heart rate was >180 beats/min at the beginning of the
experiment, or 4) more than three consecutive attempts were
needed to convert ventricular fibrillation with low-energy direct
current pulses.
Determination of myeloperoxidase activity. Neutrophil accumulation in the myocardium was measured by determining myeloperoxidase (MPO) activity according to previously published methods (22). The change in absorbance at 460 nm was measured spectrophotometrically. Each sample was tested in duplicate. MPO activity was determined by comparison with a standard curve generated using purified MPO (Sigma). The data are expressed as MPO activity (units) per gram weight of tissue.
Histopathological analysis. At the end of 180 min of reperfusion, tissue samples were obtained from the AAR. The tissues samples were snap-frozen in isopentane (maintained in liquid nitrogen) and embedded in Tissue-Tek optimum-cutting temperature compound (Miles, Elkart, IN) as previously described (2). Frozen sections of the myocardial samples were stained by hematoxylin and eosin and then were evaluated using a Zeiss Axioplan II microscope. Samples were visualized at X40 magnification.
Neutrophil activation studies. Canine whole blood was collected in 1% anticoagulant citrate dextrose (Miles, Ontario, Canada). Neutrophils were isolated using the Polymorphprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's protocol for the isolation of human neutrophils. Residual red blood cells were removed via hypotonic lysis, and the remaining neutrophils were resuspended in Dulbecco's PBS (DPBS) with 10 mM glucose and adjusted to 106/ml. Cell preparations were >95% neutrophils as determined by Wright's stain and forward scatter/side scatter profiles on the FACScan (Becton-Dickinson, Palo Alto, CA).
BIIB-513, diluted in DMSO, was added to 1 ml of isolated cells to give final concentrations from 0.1 to 1000 µM. After incubation at room temperature for 10 min the cell suspensions were placed at 37°C for 15 min before the addition of 1,000 nM platelet-activating factor (PAF). Cells were incubated at 37°C for 30 min and then were placed on ice for 10 min. One microgram of FITC-conjugated anti-human CD18 monoclonal antibody clone MHM23 (Dako, Glostrup, Denmark), previously shown to cross-react with canine CD18 (31) or FITC-conjugated mouse IgG1 (isotype control), was added to the cell suspension and incubated at 4°C for 60 min. After incubation, the cells were diluted 1:2 in DPBS with 10 mM glucose and analyzed on a FACScan. For analysis of the respiratory burst, a flow cytometric assay was employed to quantitate the oxidation of 2',7'-dichlorodihydrofluorescein during the respiratory burst of neutrophils (1). Briefly, neutrophils (106/ml) were incubated at 37°C for 15 min with 5 µM of 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA; Molecular Probes, Eugene, OR). After being loaded with H2DCF-DA, cells were washed one time in DPBS with 10 mM glucose to remove the residual H2DCF-DA. The cells were resuspended in PBS with 10 mM glucose and adjusted to 106/ml. BIIB-513, diluted in DMSO, was added to 1 ml of isolated cells to give final concentrations from 0.1 to 100 µM. The final DMSO concentration was <0.005%. Cells were incubated at room temperature for 10 min and then were placed at 37°C for 15 min before the addition of equal volumes of either PAF at a final concentration of 1,000 nM, phorbol 12-myristate 13-acetate (PMA) at a final concentration of 400 ng/ml, or vehicle (100% DMSO or 95% ethanol). Cells were incubated at 37°C for 30 min and then were analyzed for fluorescence using a FACScan instrument.Metabolic inhibition of rabbit cardiomyocytes. Ca2+-tolerant cardiac myocytes were isolated from adult New Zealand White rabbits according to previously published methods (24). Myocytes were subjected to metabolic inhibition by incubating the cells in glucose-free Krebs buffer (pH 6.5) containing (in mM) 137 NaCl, 3.8 KCl, 0.5 MgCl2, 0.9 CaCl2, 20 sodium lactate, 10 2-deoxyglucose, 0.75 sodium hydroxysulfite, and 10 HEPES (8). After incubation for 2 h at 37°C, the metabolic inhibition buffer was collected and replaced with culture media. The myocytes were "reoxygenated" for 3 h after which the cells were examined microscopically. The percentage of hypercontracted myocytes was calculated by counting the number of rod-shaped cardiomyocytes within the same three randomly chosen grids immediately before and after metabolic inhibition. Hypercontracted myocytes were identified as those with a length-to-width ratio less than 3:1 (32). Next, culture medium was removed and pooled with the metabolic inhibition buffer. After removal of the "reoxygenation" buffer, myocytes remaining on the plate were scraped into hypotonic lysis buffer (10 mM Na-HEPES and 2 mM EDTA, pH 7.4). As an additional index of cell injury, lactate dehydrogenase (LDH) activity was measured in the supernatants and lysates using a standard assay kit (Sigma), and the percentage of the total amount of LDH released into the supernatants in response to metabolic inhibition was calculated. Various concentrations of the NHE-1 inhibitor BIIB-513 or the equivalent vehicle (DMSO <0.1%) were present in both the metabolic inhibition buffer and the reoxygenation buffer. All experiments were performed in triplicate.
Statistical analysis. All values are expressed as means ± SE unless otherwise noted. Differences between groups in hemodynamics and blood gases were compared by use of a two-way (for time and treatment) ANOVA with repeated measures. Differences between groups in MPO activity, LDH release, hypercontracture, tissue blood flows, AAR, and IS were compared with a two-tailed t-test. Analysis of covariance was used to determine whether the relation between transmural collateral blood flow and IS differed between the control and drug-treated groups. For all experiments, differences between groups were considered significant if the P value was <0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Exclusions and hemodynamic and blood gas data. A total of eight dogs were initially used in this study. None were excluded; thus, eight dogs successfully completed the protocol and were included in data analysis.
Table 1 summarizes the hemodynamic and blood gas data. There were no significant differences within or between the groups throughout the experiment with regard to heart rate, mean arterial pressure, rate-pressure product, and left ventricular (LV) derivative of pressure versus time. There also were no significant differences in pH, PO2, and PCO2 within and between groups at the times studied (data not shown).
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Infarct size measurements.
Figure 2 summarizes the effect of
administration of the NHE-1 inhibitor BIIB-513 before ischemia on IS
expressed as a percentage of the AAR (IS/AAR). Administration of
BIIB-513 resulted in significant (P < 0.05) reductions
in IS, IS/LV (Table 2), and IS/AAR (Fig. 2). There were no significant differences in LV weight, AAR, or AAR/LV
between groups (Table 2). There also were no differences in transmural
collateral blood flow between groups, indicating that both groups were
subjected to equivalent degrees of ischemia (Table 2). However, a plot
of transmural collateral blood flows versus IS/AAR demonstrated that
the regression line describing this relationship in BIIB-513-treated
animals was shifted down compared with the control group (data not
shown), indicating that for any level of collateral blood flow, the
IS/AAR would be predicted to be smaller in NHE-1-inhibited animals.
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Neutrophil accumulation and activity.
As shown in Fig. 3, a significant
reduction in MPO activity within the infarcted myocardium was observed
in dogs treated with 3.0 mg/kg of BIIB-513 when compared with control
animals (P < 0.001). To ensure that the decrease in
MPO activity observed was not due to direct effects of BIIB-513 on the
assay, BIIB-513 (10 µM final concentration) was added directly to
control and tissue samples. No attenuation of MPO activity was observed
(data not shown). To visually confirm the decrease in neutrophil
accumulation in BIIB-513-treated animals, hematoxylin- and
eosin-stained frozen sections of AAR myocardium were examined. As shown
in Fig. 4, although a marked neutrophil
accumulation was observed in AAR from control animals, few neutrophils
were found within the AAR in BIIB-513-treated animals.
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Effect of NHE-1 inhibition on isolated cardiomyocytes.
To determine whether the cardioprotective effects observed in vivo were
due to specific cardiomyocyte protection, isolated rabbit
cardiomyocytes were subjected to metabolic inhibition-induced injury.
Inhibition of NHE-1 with BIIB-513 resulted in significant cardiomyocyte protection. Both hypercontracture (Table
3 and Fig.
7) and LDH release were significantly
attenuated compared with control cells (P < 0.001 and
P < 0.05, respectively) at 0.1-3 µM BIIB-513.
Thus NHE-1 inhibition directly protects cardiomyocytes.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The results of the present study demonstrate in an in vivo canine model of myocardial ischemia-reperfusion injury that administration of the specific and selective new NHE-1 inhibitor BIIB-513 results in a reduction of myocardial infarct size. Although a causal relationship between the attenuation of neutrophil activation and accumulation and the reduction in infarct size has not been definitely proven, the observations that BIIB-513 protects cardiomyocytes and reduces activation of neutrophils at a similar range of concentrations in vitro and that BIIB-513 reduces neutrophil accumulation in vivo as assessed histologically and by MPO activity suggest that its effect on neutrophils may be at least partially responsible for the protection observed.
NHE in the myocardium during ischemia-reperfusion. With myocardial ischemia, an increase in intracellular H+ ensues that activates the NHE, resulting in the extrusion of H+ and the influx of Na+ (10). Increases in intracellular Na+ correlate with increases in intracellular Ca2+, presumably through effects on the Na+/Ca2+ exchanger (10, 30). The net effect of increasing intracellular Na+ is an accumulation of Ca2+ in the ischemic myocardium that contributes to cellular damage, resulting in arrhythmias and contraction band necrosis (29). With reperfusion, extracellular H+ rapidly decreases again, establishing a large intracellular-to-extracellular H+ gradient that activates the NHE again, leading to an abnormally large accumulation of Ca2+ during reperfusion (30) that contributes to reperfusion arrhythmias, myocardial contracture, and necrosis (29). Interestingly, although early reperfusion of ischemic myocardium leads to salvage of the jeopardized tissue, reperfusion also exacerbates the injury sustained during the ischemic period via the mechanisms outlined above and via a subsequent inflammatory response. With reperfusion, leukocytes adhere to the coronary microvasculature (25) and subsequently transmigrate into the myocardium, followed by the release of various inflammatory mediators such as oxygen radicals, which induce further damage of the myocardium (4-7, 15). NHE inhibition with less specific inhibitors of NHE-1 also has been shown to inhibit neutrophil function in vitro (9, 11, 26-28). Thus the current study examined the effects of specific inhibition of NHE-1 on cardiomyocyte protection and neutrophil activity.
Cardiomyocyte protection from metabolic inhibition.
To confirm that the cardioprotection observed in vivo was due to direct
cardiomyocyte protection by NHE-1 inhibition, metabolic inhibition/reoxygenation of isolated rabbit cardiomyocytes was employed. Inhibition of NHE-1 via treatment with increasing
concentrations of BIIB-513 resulted in a concentration-dependent
reduction of cardiomyocyte damage that was significant at
concentrations 
100 nM. As shown in Table 3 and Fig. 7, inhibition of
NHE-1 with BIIB-513 resulted in a significant reduction of LDH release
and a profound attenuation of hypercontracture, respectively. Previous studies have demonstrated that administration of NHE-1 inhibitors prevented postischemic myocardial contracture and Ca2+
clumping (12, 16, 18). Cardiomyocyte hypercontracture
results from an abnormal accumulation of Ca2+ during
ischemia (20, 30). Although several studies have
demonstrated that NHE inhibition reduces Ca2+ accumulation,
more recent data suggest that NHE blockade does not result in
differences in the recovery of Ca2+ or Na+
levels but rather that the persistent acidosis acts to prevent hypercontracture due to reduced Ca2+ sensitivity of the
myofibrils (20, 33). Furthermore, NHE inhibition
attenuates phase-2 cytosolic Ca2+ oscillations
(20), which are shifts in Ca2+ between the
cytosol and sarcoplasmic reticulum that occur when Ca2+
load exceeds the capacity of the sarcoplasmic reticulum. Cytosolic Ca2+ oscillations contribute to reperfusion arrhythmias,
and Ca2+ overload leads to eventual cardiomyocyte death
(10, 29, 30). Thus prevention of Ca2+
overload/oscillation may result in the prevention of contraction band
necrosis, which translates in vivo to the decreased infarct size
observed in the current study.
NHE-1 inhibition differentially affects neutrophil activation. This report demonstrates for the first time that NHE-1 inhibition attenuates the accumulation of neutrophils within the ischemic-reperfused myocardium (Figs. 3 and 4) and suggests that the reduction in myocardial infarct size observed in vivo was due in part to the effects of NHE-1 inhibition of neutrophil function. However, the interpretation of this observation is complex. Because BIIB-513 treatment markedly reduced myocardial infarct size, the ensuing inflammatory response might also be diminished, resulting in decreased neutrophil accumulation. Alternatively, in vitro, NHE inhibition has been reported to block neutrophil chemotaxis (28) and respiratory burst (27); therefore, the decreased accumulation may be due to direct effects on neutrophils. To determine whether specific inhibition of NHE-1 affects neutrophil activation, isolated canine neutrophils were stimulated with PAF, which is increased in coronary sinus blood after myocardial ischemia-reperfusion (19), or PMA, an activator of protein kinase C. Both PAF and PMA activate neutrophils, causing upregulation of the CD11b/CD18 integrin complex that facilitates neutrophil binding to endothelial cells and cardiac myocytes (6), and both stimulate the neutrophil respiratory burst (7, 19). NHE-1 inhibition via pretreatment of neutrophils with up to 1,000 µM of BIIB-513 did not attenuate the PAF-mediated induction of CD18 expression (Fig. 5). Similar data were obtained with PMA-stimulated neutrophils (data not shown). However, a concentration-dependent attenuation of both PAF- and PMA-induced respiratory burst was observed (Fig. 6, A and B, respectively). Interestingly, the dose of PAF employed is approximately 10 times the concentrations found in ischemia-reperfused coronary sinus samples (19); thus, the inhibition of respiratory burst in vivo may in fact be greater. Thus neutrophils may be able to bind but not exert detrimental effects on the cells with which they interact. However, because CD18 upregulation does not translate to adherence, NHE-1 inhibition may decrease neutrophil adhesion. Furthermore, chemotaxis of human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) has been shown to be inhibited by NHE-1 inhibition. Thus NHE-1 blockade in vivo may inhibit chemotaxis, a postulate supported by the marked attenuation in neutrophil accumulation within the ischemic-reperfused myocardium that occurred with BIIB-513 treatment. Recently, in a model of canine myocardial ischemia-reperfusion injury employing 90 min of ischemia, it has been demonstrated that a significant proportion of myocytes within the infarct region are viable at the time of reperfusion and lose viability during reperfusion, suggesting specific cell death due to reperfusion injury (21). Consistent with this study is the fact that, after myocardial ischemia-reperfusion injury, neutrophils accumulate in the ischemic myocardium where they contribute to myocardial damage (3, 6, 7). The current in vivo data, together with the data on the effects of NHE-1 inhibition on isolated cardiomyocytes and neutrophils, suggest that NHE-1 inhibition may protect myocardial cells within the AAR that otherwise may have been irreversibly injured upon reperfusion and that NHE-1 inhibition also may reduce neutrophil-mediated myocardial damage that occurs after reperfusion.
Numerous studies examining Na+/H+ exchange during myocardial ischemia and reperfusion suggest that inhibition of NHE-1 ultimately prevents Ca2+ overload/oscillations and its deleterious consequences in cardiomyocytes. Although the current data support this hypothesis, the data also demonstrate for the first time that the cardioprotection observed in vivo is more complex. Inhibition of NHE-1 also attenuates neutrophil accumulation and activation, suggesting that the reduction in myocardial infarct size observed in vivo also may be due, in part, to effects on neutrophil activity.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Rongang Wang and Dr. Peter J. Newman (Blood Research Institute, Blood Center of Southeastern Wisconsin) for assistance with the FACScan instrument. We also thank Anna Hsu (Medical College of Wisconsin) and Jim Tarara (Mayo Clinic and Foundation) for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-08311 and by a grant from Boehringer Ingelheim Pharma KG.
Current address for R. J. Gumina: Dept. of Internal Medicine, Mayo Clinic and Foundation, Rochester, MN 55905.
Address for reprint requests and other correspondence: G. J. Gross, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: ggross{at}post.its.mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 June 1999; accepted in final form 21 February 2000.
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) or stimulated (+) with 1,000 nM platelet-activating
factor (PAF) in the absence or presence of 10 µM BIIB-513. Each assay
was performed in duplicate. Data shown are derived from 1 of 4 separate
comparable experiments. * P < 0.05 vs. the control
group.
