Orally administered NHE1 inhibitor cariporide reducesacute responses to coronary occlusion and reperfusion

RACHAEL A. HUMPHREYS,1 JAMES V. HAIST,1 SUBRATA CHAKRABARTI,2QINGPING FENG,3 J. MALCOLM O. ARNOLD,3 AND MORRIS KARMAZYN1

Departments of 1Pharmacology and Toxicology, 2Pathology, and 3Medicine,University of Western Ontario, London, Ontario, Canada N6A 5C1

Humphreys, Rachael A., James V. Haist, SubrataChakrabarti, Qingping Feng, J. Malcolm O. Arnold,and Morris Karmazyn. Orally administered NHE1 inhibi-tor cariporide reduces acute responses to coronary occlusionand reperfusion. Am. J. Physiol. 276 (Heart Circ. Physiol. 45):H749–H757, 1999.—Na1/H1 exchange (NHE) mediates myo-cardial ischemic and reperfusion injury. We examined theeffects of dietary administration of the potent and selectiveNHE1 inhibitor cariporide on acute responses to coronaryartery ligation and reperfusion in the anesthetized rat. MaleSprague-Dawley rats received control rat chow or an identicaldiet containing 3 parts per million of cariporide for 1 wkbefore 225 min of occlusion of the left main coronary artery or45 min of occlusion followed by 180 min of reperfusion. Heartswere excised and divided into left ventricle, right ventricle,and interventricular septum for analysis of NHE1 mRNAexpression and apoptosis by staining with terminal deoxy-nucleotidyl transferase-mediated nick end labeling. Ischemiaand reperfusion were associated with a threefold elevation inNHE1 mRNA expression in control animals that was signifi-cantly reduced in cariporide-fed rats. Cariporide reducedmortality from 26% of animals to 0%. The incidence of allarrhythmias was significantly reduced, including ventricularfibrillation (from 42 to 0%) and ventricular tachycardia (from81 to 15%), as well as the number of ventricular prematurebeats (from 70 6 12 to 17 6 6). Cariporide moderately reducedapoptosis only in the reperfused left ventricle to values notsignificantly greater than those in sham-operated animals,and this was associated with a significantly higher ratio ofBcl-2 to Bax. This study suggests that NHE inhibition withdietary cariporide represents an effective management ofacute postinfarction responses.

sodium-hydrogen exchange; HOE-642; infarction; arrhyth-mias; sodium-hydrogen exchanger subtype 1 messenger ribo-nucleic acid expression; apoptosis

THE MYOCARDIAL Na1/H1 exchanger (NHE) representsone of the major mechanisms for intracellular pHregulation in response to ischemia-induced acidosis(15). Extensive studies over the past number of yearshave provided evidence that, in addition to its role inintracellular pH regulation, the exchanger likely con-tributes to cardiac injury associated with myocardialischemia and reperfusion (reviewed in Refs. 6 and 11).This concept is based on extensive evidence that phar-macological inhibition of NHE results in cardiac protec-tion that is generally independent of experimentalmodel, animal species, or the type of NHE inhibitor

used (6, 11). Moreover, protection appears to be associ-ated with beneficial effects with respect to a wide arrayof parameters ranging from inhibition of necrosis andantiarrhythmic effects to an antiapoptotic influence (1,6, 11).

One NHE inhibitor that is of particular interest is4-isopropyl-3-methylsulfonylbenzoylguanidine meth-anesulfonate (HOE-642), which has been assigned thenonproprietary name cariporide. In contrast to amilo-ride analogs, which are nonspecific NHE inhibitors(12), cariporide appears to be a selective inhibitor of theNHE1 isoform, which is the primary, if not sole, cardiacNHE subtype, thereby rendering it particularly attrac-tive for therapeutic interventions for cardiac disorderswhile minimizing the potential for side effects (19). It isof substantial interest that cariporide is currently inclinical trials to assess its potential influence in high-risk cardiac patients with acute coronary syndromes(GUARDIAN study). Recently, another NHE1-selectiveinhibitor, EMD-85131, has been demonstrated to po-tently inhibit infarct size in dogs even when adminis-tered after coronary artery occlusion (7). The vastmajority of studies demonstrating beneficial effects ofNHE inhibitors including cariporide have utilized ei-ther in vitro approaches or, when whole animal modelswere used, direct infusion of the active compound.Because of the potential clinical value of cariporide inthe treatment of heart disease, the present study wasundertaken to assess whether a 1-wk dietary adminis-tration of cariporide alters immediate postligation re-sponses in anesthetized rats subjected to coronaryartery occlusion with or without reperfusion. We as-sessed the effect of this approach in terms of expressionof the NHE1 gene and the incidence of apoptosis. Ofparticular importance was the detailed assessment ofmortality and ventricular arrhythmias, because bothare critical events occurring in patients with acutemyocardial infarction.

METHODS

Experimental protocol. Male Sprague-Dawley rats (250–350 g) were initially randomized to receive either the controlrat chow or an identical diet containing 3 parts per million ofcariporide ad libitum. After 1 wk of feeding, the animals wereweighed and then anesthetized with pentobarbital sodium(50 mg/kg ip) and buprenorphine (0.03 mg/kg sc). Animalswere then intubated and ventilated (10 ml/kg, 70 breaths/min) with air using a rodent respirator (model 683, HarvardApparatus). A standard lead I electrocardiogram (ECG) wasrecorded (model 79E, Grass recorder), and a thermistor probewas inserted into the rectum to measure core temperature,which was maintained at 37–38°C with the aid of a heatingpad. An incision was made in the skin on the left side of the

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0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society H749

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chest, and the pectoral muscles were retracted to expose theribs. An incision was then made through the fourth intercos-tal space, the ribs were retracted, and the pericardium wasincised to allow access to the heart. A 6-0 braided silk sutureattached to a 10-mm micropoint reverse cutting needle wasthen placed under the left main coronary artery ,2 mm fromits origin. The ends of the suture were threaded through alength of polyethylene tubing, forming a snare, which couldthen be used to ligate and reperfuse the coronary artery asrequired. In sham-operated animals the ligature was put inplace but was not tied. Animals were subjected to either aprolonged 225-min ischemic period or a 45-min period ofischemia followed by 180 min of reperfusion. In all groupsnegative pressure was applied 45 min after initial placementof the ligature (immediately after ligature removal for thereperfusion group), and the chest wall was sutured closed inthree layers (ribs, muscle, and skin). The animals weremaintained for a further 180 min, after which they werekilled by decapitation and the hearts excised for subsequentdetermination of NHE1 expression and apoptosis. In addi-tion, blood was collected for determination of plasma caripo-ride levels. For the latter, plasma was sent on dry ice toHoechst-Marion-Roussel laboratories (Frankfurt, Germany)for analysis by HPLC.

Arrhythmia measurement and assessment. The ECG wasobtained with platinum needle electrodes inserted subcutane-ously for a 10-min stabilization period before coronary liga-tion, after which the ECG was determined for a further55-min period. This represented 55 min of ischemia innonreperfused hearts or 45 min of ischemia plus 10 min ofreperfusion in hearts subjected to reflow. Initial experimentsrevealed that virtually all ischemia-induced arrhythmiasoccurred during a 10- to 30-min postligation period. More-over, arrhythmias were almost completely absent duringreperfusion (see DISCUSSION). Ischemia-induced ventriculararrhythmias were analyzed according to the Lambeth Conven-tion guidelines for the analysis of experimental arrhythmias(22). All ventricular premature beats were recorded. Thepercentage of incidence and duration of ventricular tachycar-dia and ventricular fibrillation, when present, were alsonoted. Ventricular tachycardia was defined as a run of four ormore beats at a rate faster than resting sinus rhythm.Ventricular fibrillation was defined as a signal for whichindividual QRS complexes could no longer be distinguishedfrom one another and for which a rate could no longer bemeasured. Ventricular fibrillation was considered irrevers-ible if it did not revert within 3 min of onset. Mortality wasalso recorded.

RNA isolation. Hearts for NHE expression studies weredivided into left ventricle, right ventricle, and interventricu-lar septum and stored in liquid nitrogen. RNA was subse-quently isolated using TRIzol reagent (Canadian Life Tech-nologies, Burlington, ON, Canada). Tissues were homogenized,and RNA was extracted with chloroform followed by centrifu-gation to separate the solution into aqueous and organicphases. RNA was recovered from the aqueous phase byprecipitation with isopropyl alcohol and suspended in diethylpyrocarbonate-treated water. Quantitation of RNA was per-formed by determining the absorbency at 260 and 280 nm.

Reverse transcription-polymerase chain reaction. First-strand cDNA synthesis was performed using the SuperScriptII system (Canadian Life Technologies). RNA (10 µg) wasadded to oligo(dT) primers (Canadian Life Technologies),denatured at 65°C for 10 min, and quenched on ice for 10 min.RT was carried out by the addition of 200 units of Moloneymurine leukemia virus-reverse transcriptase and 1 mmol/ldNTP and 10 mmol/l dithiothreitol at 42°C for 50 min in a

total reaction volume of 20 µl. The reaction was terminated bya 15-min incubation at 70°C. The resulting RT products werestored at 220°C.

The amplification was carried out using the followingcDNA sequences. For NHE1, primer 1 (58-TACGGTACCCT-GCTCTTCTGCCTC-38) and primer 2 (58-GATGCATGCG-GATCTCCTCCTCCTT-38), with a predicted product size of649 bp, were used (4). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified simulta-neously for each sample in a separate set of tubes using thesame quantity of RT product. For GAPDH, primer 1 (58-TATTGGCCGCCTGGTCACCA-38) and primer 2 (58-CCACCT-TCTTGATGTCATCA-38), with a predicted product size of 746bp, were used. PCR reactions were performed in a totalvolume of 50 µl containing 13 PCR buffer, 2.5 mmol/l MgCl2,250 µmol/l dNTP mix, 0.5 µmol/l of each amplification primer,2.5 units Taq polymerase (Canadian Life Technologies), and 4µl of the RT product. The amplification was carried out asfollows: 45 s at 94°C (denaturation), 45 s at 60°C (annealing),and 1 min at 72°C (extension) for 30 cycles. In initial studieswe observed that this thermal cycling reaction was optimalfor both NHE and GAPDH, with both reactions remainingwithin the linear (logarithmic) phase of amplification over arange of template concentrations.

Electrophoresis. The amplification products were analyzedon 3% agarose gels stained with ethidium bromide in 13Tris-acetate-EDTA buffer. Ten microliters of each PCR prod-uct were loaded in each lane and electrophoresed at 110 V for90 min. At the end of the electrophoresis the gel wasvisualized under ultraviolet light. Positive amplifications forNHE1 were demonstrated as single bands at 649 bp. GAPDHamplification was demonstrated as a single band at 746 bp.

Southern blotting. The specificity of the amplification wasconfirmed by Southern blotting following the transfer of thePCR products from gel onto nylon membranes after denatur-ation and neutralization. Hybridization with biotinylatedamplification product-specific oligoprobe was carried out us-ing the NHE1 oligoprobe 58-TAGACCTGTTGGCTGT-GAAGA-38 (4). A similar amplification product-specific oli-goprobe (58-GGATGCATTGCTGACAATCTT-38) was used forGAPDH. The detection was carried out by incubation of nylonmembranes in the dark at 37°C for 2 h with the use of a nitroblue tetrazolium-5-bromo-4-chloro-3 indolylphosphate sys-tem (Oncor, Gaithersburg, MD) for visualizing the specificblue bands. Semiquantification of the RT-PCR products wascarried out by densitometry using a Hewlett-Packard 4Cscanner and Mocha software (Jandel Scientific). The densito-metric values were expressed as arbitrary units per micro-gram of total RNA added to the RT reaction.

Detection of apoptosis. A 3-mm transverse section, 2 mmfrom the site of ligation through the center of the myocar-dium, was obtained and separated into left ventricular, rightventricular, and interventricular septal regions. The tissueswere fixed in 10% buffered formaldehyde and then embeddedin paraffin. Paraffin sections (4–6 µm) were prepared andplaced on positively charged slides for in situ apoptosisdetection using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyribonucleotide triphosphate-digoxigenin nickend labeling (TUNEL) technique. Slides were incubated with20 µg/ml proteinase K for 15 min at room temperature andthen washed in deionized water. Endogenous peroxidase wasinactivated with 3% H2O2 and methanol for 5 min at roomtemperature. The subsequent staining procedure was carriedout according to the manufacturer’s instructions (ApopTag,Oncor). Briefly, sections were rinsed and preincubated withequilibration buffer for 10 min, and digoxigenin-labeled dUTPand dATP in TdT buffer were added to cover the section,

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which was then incubated in a humidified chamber at 37°Cfor 60 min. The reaction was stopped by immersing the slidesinto a stop/wash buffer. After sections were washed in PBS(0.01 mol/l, pH 7.4), they were incubated in anti-digoxigeninperoxidase for 30 min at room temperature. The slides werestained for 6 min with 1 mg/ml 3,38-diaminobenzidine (DAB)and 0.3% H2O2 in PBS and counterstained with Harrishematoxylin. Negative control slides were processed at thesame time at which the TdT enzyme was excluded. The slideswere examined by light microscopy at 3400 magnification,and the number of apoptotic cells per microscopic field wascounted and expressed as the number of cells per 100high-power fields. Adjacent hematoxylin-eosin stained sec-tions were examined to confirm that apoptotic cells repre-sented cardiac myocytes.

Immunostaining for Bax and Bcl-2. To assess the presenceof Bax and Bcl-2, we utilized immunohistochemical tech-niques as described in detail previously (13). Formalin-fixed,paraffin-embedded tissue sections (5 µm) were mounted onpoly-L-lysine-coated slides. Samples were deparaffinized bythree changes of 2 min each with xylene followed by dehydra-tion with 100, 95, and 70% ethyl alcohol (two changes, 2 minfor each alcohol concentration). After dehydration, sampleswere incubated for 5 min in PBS followed by antigen retrieval(boiling by microwaving for 10 min) in 10 mM citrate buffer,pH 6.0. Samples were cooled for 20 min in citrate buffer,washed twice in PBS for 5 min, and then blocked for 45 min inTris-Na-K (TNK) solution (100 mmol/l Tris, 550 mmol/l NaCl,and 10 mmol/l KCl, pH 7.6–7.8) containing 2% BSA, 0.1%Triton X-100, and 3% normal goat serum. Rabbit polyclonalanti-mouse/rat Bcl-2 or anti-mouse/rat Bax primary antibody(1:700 dilution, Pharmingen, Mississauga, ON, Canada) wasadded to the blocking solution, and the samples were incu-bated overnight at room temperature in a humidified cham-ber. Slides were then washed three times for 5 min each withPBS, endogenous peroxidases were blocked with 3% H2O2 inmethyl alcohol for 5 min, and samples were then washedthree times with PBS. A Vectastain elite ABC kit (VectorLaboratories, Burlington, ON, Canada) was used for stainingthat was carried out according to kit instructions. Color wasdeveloped by incubating slides for 5 min in TNK solutioncontaining 1 mg/ml DAB and 0.3% H2O2. Counterstainingwas done with Harris hematoxylin.

Slides were evaluated at a 3400 magnification. The inten-sity of DAB staining was graded by one of us (J. V. Haist)using similar criteria described previously (13) and using thefollowing scale: 0, no staining; 1, light staining; 2, moderatestaining; and 3, intense staining. In each field of view, theapproximate percentage of the field at each intensity wasrecorded, and the average for 50 fields at each intensity wascalculated for each experiment. A histochemical score wasdetermined for each slide by calculating the sum of the stainintensity multiplied by the percentage of the field for all fourintensity grades and then dividing by 100 (17). For negativecontrol slides the primary antibody was left out. No stainingwas observed in these samples. Normal rat pancreas wasused for positive control staining. Bax staining was observedin acinar but not islet cells, in agreement with Krajewski andco-workers (13). Bcl-2 staining was evident in both acinar andislet cells but not in the ducts, in agreement with Hockenberyet al. (8).

Statistical analysis. Data for mortality and percent inci-dence of arrhythmic events were analyzed using chi-squareanalysis. Duration of arrhythmias and incidence of ventricu-lar premature beats were analyzed using Mann-Whitney’srank sum test. Analysis of NHE1 expression and the inci-dence of apoptosis was done using Kruskal-Wallis one-way

ANOVA. A value of P ,0.05 was considered to indicate asignificant difference.

RESULTS

Animal presurgical data. The pooled mean bodyweights for control and cariporide-fed animals were295 6 6 (n 5 58) and 286 6 5 g (n 5 44), respectively(P . 0.05). The mean plasma concentration of caripo-ride after 1 wk of feeding was 41 6 8 ng/ml.

NHE1 mRNA expression. We assessed NHE1 expres-sion at the end of the ischemic or reperfusion periods inleft ventricle as well as the interventricular septumand right ventricle. These data are summarized in Fig.1, which shows the ratios of NHE1 to GAPDH expres-sion, with the latter being completely unaffected by anytreatment (data not shown). Compared with shamanimals, both ischemia and ischemia-reperfusion wereassociated with an approximately threefold elevationin NHE1 mRNA expression in animals fed a controldiet. Cariporide feeding alone had no effect on NHE1expression in animals not subjected to coronary liga-tion (data not shown). However, in cariporide-fed ratssubjected to ligation, the elevation in left ventricular

Fig. 1. Na1/H1 exchanger (NHE1) mRNA expression in left ventricle(A), right ventricle (B), and interventricular septum (C) of heartssubjected to various treatments. Values represent densitometricmeasurement of RT-PCR products after Southern hybridization.GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Values aremeans 6 SE; n 5 10 for each treatment group. *P , 0.05 vs. sham;#P , 0.05 vs. respective values obtained in absence of cariporide.

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NHE1 expression was significantly less than that ob-served in animals fed the control diet. As also summa-rized in Fig. 1, B and C, no effects were observed ineither the interventricular septum or right ventricles inthese animals. Examples of individual Southern blotsof RT-PCR reaction products for various treatments areprovided in Fig. 2. As noted earlier, there was no effectof any treatment on the mRNA expression of theGAPDH housekeeping gene (Fig. 2).

Mortality. The total incidence of mortality in allanimals is summarized in Fig. 3A. In control animals amortality rate of 26% (11 of 43 animals) was observed,whereas no deaths occurred in animals fed the caripo-ride diet. Ventricular fibrillation was readily identifiedas the cause of death in five animals subjected to ECGmeasurement at the time, whereas six animals diedduring the 3-h recovery period.

Incidence of arrhythmias. Virtually no arrhythmiasoccurred during the reperfusion, most likely reflectingthe prolonged ischemic duration (see DISCUSSION). More-over, arrhythmias were generally restricted to theperiod of 10–25 min after coronary artery ligation. Theincidence of arrhythmias during ligation was pooled forall animals irrespective of subsequent reperfusion. Thedata for ventricular fibrillation are summarized in Fig.3B, which shows that 42% demonstrated this type ofarrhythmia. The average duration of fibrillation was23 6 9 s. Although the data are not shown, shamanimals did not demonstrate any arrhythmias irrespec-tive of dietary regimen. In contrast, fibrillation wascompletely absent in animals receiving the cariporidediet (Fig. 3). Figure 4 demonstrates the incidence ofectopic activity (Fig. 4A) as well as an analysis ofventricular tachycardia in terms of incidence (Fig. 4B)and duration (Fig. 4C). The number of ventricularpremature beats averaged 70 6 12 for control diet-fedanimals, which was significantly decreased to 17 6 6 bycariporide. Moreover, 35 of 43 control animals (81%)demonstrated ventricular tachycardia, which was sig-nificantly reduced to 15% (5 of 38) by dietary cariporide,as was the duration of ventricular tachycardia, from40 6 9 to 4 6 1 s (P , 0.05). Examples of ECG recordingsfrom two control rats showing the types of arrhythmiasobserved in this study are provided in Fig. 5.

Incidence of apoptosis. We next quantified the num-ber of apoptotic nuclei in three regions of the heart afterocclusion with or without subsequent reperfusion. Thesedata are summarized in Fig. 6. In the left ventricle theincidence of apoptosis was significantly elevated bycoronary artery ligation as well as by both ligation andreperfusion. Cariporide failed to attenuate apoptosis inthe left ventricle after sustained ischemia, such thatvalues were identical to those seen in animals fed thecontrol diet. However, in the reperfused left ventricle,an ,50% decrease in the number of apoptotic nucleiwas evident such that the values obtained in this groupwere not significantly higher than those seen in the shamgroup. Very few apoptotic nuclei (note differences in scalein Fig. 6) were found in either the right ventricle orinterventricular septum, with no significant differencesamong any of the treatment groups. Examples of positivelyTUNEL-stained nuclei in sham hearts and heartssubjected to occlusion and reperfusion from control orcariporide-fed animals are provided in Fig. 7.

Fig. 2. Representative examples of Southern blotsshowing NHE1 or GAPDH mRNA expression in shamhearts (lane A) or hearts subjected to coronary occlu-sion without (lane B) or with (lane C) reperfusion.

Fig. 3. Summary of incidence of mortality (A) and ventricularfibrillation (VF; B) in animals subjected to coronary artery occlusionon either a control diet or a diet containing cariporide. *P , 0.05 vs.control.

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Bcl-2 and Bax expression. To further characterizeapoptosis, we next used a histochemical technique tosemiquantify the expression of Bcl-2 and Bax in heartssubjected to various treatments. As summarized inTable 1, ligation with or without reperfusion produced amodest elevation in Bcl-2 expression, although none ofthese effects were significant compared with those insham animals. A trend toward elevated Bax expressionwas similarly observed in response to occlusion, al-though a significant increase was seen only in reper-fused hearts. The elevation in Bax expression in thesehearts was completely prevented by dietary cariporide,resulting in a significantly increased ratio of Bcl-2 toBax that was not observed with any other treatmentgroup. Examples of Bcl-2 and Bax immunohistochemi-cal staining are provided in Fig. 8.

DISCUSSION

The present study demonstrates that in vivo coro-nary artery occlusion and reperfusion increases NHE1expression in the infarcted left ventricle of the rat,which is associated with increased apoptosis and a high

incidence of ventricular arrhythmias. Moreover, wedemonstrated that dietary administration of the NHEinhibitor cariporide attenuates these early postinfarc-tion responses. The mean plasma levels of cariporide(41 6 8 ng/ml or ,0.1 µM) is a concentration that hasbeen shown to inhibit NHE1 by ,68% in rat cardiacmyocytes in which intracellular acidosis was producedby ammonium chloride pulsing (20). There is now goodevidence that inhibition of NHE produces cardioprotec-tive effects in the ischemic and reperfused myocardiumin a wide range of experimental models and animalspecies (reviewed in Refs. 6 and 11). The protectiveeffect of NHE inhibitors likely reflects diminishedintracellular Ca21 loading, because blocking the NHEwill result in reduced Na1 influx via the antiporter. As aresult, more effective Ca21 removal through the Na1/Ca21 exchanger would occur (18). An important ad-vance in the development of potentially useful cardiopro-tective agents that target NHE has been thedevelopment of compounds less related to the amiloridestructure with generally greater potency and specificityagainst NHE, namely, HOE-694 and HOE-642 (19). Ofthese two compounds, HOE-642 is of particular interestbecause it is a specific inhibitor of the primary, if notsole, isoform NHE1, which is found in the mammalianmyocardium (19). Moreover, HOE-642, which has beenassigned the nonproprietary name cariporide, has beenfound to exert substantial protection against variousforms of myocardial injury (9, 10, 16, 19, 20). Inaddition, this drug is currently under clinical evalua-tion in a multicentered international study to evaluateits potential effects in patients with acute coronarysyndromes (GUARDIAN study).

The present study was carried out to assess whetherdietary preadministration of cariporide alters the earlyresponses to occlusion and may be of importance withrespect to potential development of this drug, or otherNHE inhibitors, for oral use in cardiac therapeutics. Inaddition, we examined in detail the effects of thistreatment on the development of arrhythmias, includ-ing ventricular fibrillation, as well as mortality aftercoronary ligation. Moreover, early changes in NHE1mRNA expression were determined in view of evidencethat in vitro ischemia increases expression of theantiporter in isolated rat hearts (2). Finally, we deter-mined whether apoptosis is affected by dietary adminis-tration of the NHE inhibitor. A 1-wk feeding regimenwas determined based on initial plasma sampling forcariporide concentrations that showed that plasmalevels retained steady-state values during this period.

Potential role of NHE in early postinfarction re-sponses. The present report shows that coronary arteryocclusion in this model is associated with a stimulationof NHE1 expression in the left ventricular myocardiumwith no significant effect on either right ventricle orinterventricular septum. It is well known that NHEactivity is stimulated by myocardial ischemic and reper-fusion conditions, and to our knowledge this is the firstdemonstration that coronary artery occlusion can alsostimulate NHE1 expression. As noted above, this find-ing is in agreement with an earlier report that global

Fig. 4. Quantification of ventricular premature beats (VPB; A) andincidence (B) and duration of ventricular tachycardia (VT; C) inanimals subjected to coronary artery occlusion on either a control dietor a diet containing cariporide. *P , 0.05 vs. control.

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ischemia in the isolated rat heart increases NHE1mRNA (2). The relevance of increased NHE1 expres-sion to early postocclusion responses is unclear, al-though it is logical to suggest that increased NHEexpression will result in subsequent increased proteinsynthesis of the exchanger, suggesting that the myocar-dial chronic response to infarction may involve alteredNHE1 protein expression. Accordingly, because caripo-ride blunted the increased mRNA expression in theinfarcted mycardium, the long-term consequence ofthis effect would be important to assess. The reason forthe somewhat unexpected effect of cariporide in attenu-ating the elevation in NHE1 mRNA expression isunclear at this time, although it may be a consequenceof diminished injury due to inhibition of NHE activity,resulting in modulated production of factor(s), yet to bedetermined, that are important in the regulation ofNHE1 gene expression. Alternatively, it could be sug-gested that NHE1 expression is regulated by NHEactivity.

A major thrust of the present study was to assessarrhythmias in detail, because most sudden cardiacdeaths, which represent ,50% of all deaths afterinfarction, can be attributed to ventricular fibrillation.In our model, the occurrence of arrhythmias was re-stricted only to the ligation period, with no arrhyth-mias observed during reperfusion. The lack of reperfu-sion-associated arrhythmias is in agreement with theconcept that the incidence of these arrhythmias isinversely related to the preceding duration of ischemia,which may in turn reflect the degree of cell injury. Ourresults show that dietary cariporide is effective in

preventing arrhythmias produced by coronary arteryocclusion. These findings are in agreement with aprevious report (19) demonstrating these effects in ratsadministered either intravenous or oral cariporide,particularly in terms of abolition of ventricular fibrilla-tion. The mechanism underlying the ability of dietarycariporide to prevent ischemia-induced arrhythmiasmay reflect the fact that NHE inhibitors can attenuateelectrolyte disturbances in the ischemic myocardium,including attenuation of both Ca21 and Na1 elevation(18). Moreover, NHE inhibition can block the directdetrimental effects of lysophosphatidylcholine as wellas H2O2, two products whose levels are increased in theischemic myocardium and that can contribute to devel-opment of arrhythmias (9, 10).

Our study further demonstrates that dietary caripo-ride modestly inhibits the development of apoptosis;however, the effect of cariporide occurred only withrespect to apoptosis associated with 180 min of reperfu-sion following 45 min of ischemia, with no effect againstapoptosis produced by prolonged (225 min) ischemiaalone. We utilized durations of occlusion and reperfu-sion identical to those recently reported by Fliss andGattinger (5) using a similar model. Our results agreewith that study in terms of the similar level of apoptosisobserved during either ligation or reperfusion. Theselective salutary effect of cariporide seen in our studyagrees with the concept proposed by these investigatorsof potentially different mechanisms of inducing apopto-sis in the sustained-ligated versus occluded-reperfusedmyocardium, under which conditions neutrophil activa-tion may play an important role. Moreover, it appears

Fig. 5. Electrocardiogram recordings from 2 control rats subjected to coronary artery occlusion showing types ofarrhythmias observed in this study. In rat no. 82 (top), a run of ventricular fibrillation followed by tachycardia isevident 3 min postligation (post-lig) that then reverses to normal rhythm such that, 4–5 min postligation,tachycardia followed by irreversible ventricular fibrillation is observed. Rat no. 84 (bottom) demonstrates primarilyventricular premature beats, which were first apparent at 6 min postligation.

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that various properties of apoptotic nuclei appear to bedifferent between those found in the infarcted versusreperfused myocardium (5). It is interesting that neutro-phil activation is a pH-dependent phenomenon, and,indeed, neutrophil-induced cardiac injury can be attenu-ated by NHE inhibition (3). Although it is intriguing tospeculate that inhibition of neutrophil activation con-tributed to the reduction in apoptosis in the reperfusedmyocardium, it should be added that cariporide alsoinhibits apoptosis in the ischemic and reperfused iso-lated rat heart devoid of neutrophils, suggesting thatthe antiapoptotic influence of the drug likely reflectsmultifaceted mechanisms (1). The inability of caripo-ride to affect apoptosis associated with ligation indi-

cates that apoptosis and ischemia-induced arrhyth-mias are unlikely to be related in this model. To furtherexamine the effect of cariporide on apoptosis, we car-ried out histochemical assessment of the protoonco-genes Bcl-2 and Bax, which are important modulatorsof apoptosis by virtue of their abilities to inhibit orstimulate the process, respectively (14, 23). BecauseBcl-2 promotes cell survival, whereas Bax acceleratescell death, the ability of cariporide to increase the ratio

Fig. 6. Quantitative assessment of apoptosis in left ventricle (A),right ventricle (B), and interventricular septum (C) after varioustreatments. Values are means 6 SE and indicate terminal deoxy-nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive nuclei as described in METHODS. *P , 0.05 vs. sham treat-ment group. Note differences in scale in A vs. B and C.

Fig. 7. Micrographs demonstrating positively TUNEL-stained nuclei(arrows) in left ventricles from a sham rat heart (A) or heartssubjected to occlusion and reperfusion without (B) or with (C)cariporide.

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of Bcl-2 to Bax, primarily by preventing Bax upregula-tion, may explain, at least in part, the beneficial effectsof this drug on the occluded-reperfused myocardium.However, on the basis of the data, cause-and-effect

relationships between these proteins and the incidenceof apoptosis cannot be firmly established.

Potential clinical relevance. In conclusion, our studydemonstrates that dietary administration of the NHEinhibitor cariporide attenuates various early eventsassociated with coronary artery occlusion and reperfu-sion. Studies in the rat myocardium must be inter-preted cautiously; however, the potent ability of dietarycariporide to prevent arrhythmias, particularly in termsof abolishing ventricular fibrillation and, indeed, mor-tality postocclusion, is encouraging for the develop-ment of potentially effective agents for patients at riskof myocardial infarction. The relevance of increasedNHE expression or the reduction in apoptosis in theacutely infarcted myocardium requires further studies.However, apoptosis has recently been shown to be theearliest and most predominant form of cell death inpatients with myocardial infarction (21). Of particularrelevance to the present study, that report (21) also demon-strated that apoptosis is further accelerated by reperfu-sion. As such, the selective attenuation of the latter bycariporide may be of further clinical importance.

Table 1. Histochemical semiquantification of Bcl-2and Bax expression in rat heart

Treatment n Bcl-2 Bax Bcl-2/Bax

Sham 5 1.1860.12 0.7260.19 1.4960.21Sham1cariporide 5 1.2460.17 0.7760.11 1.4260.18

Ligation 7 1.6560.19 1.1760.09 1.4160.13Ligation1cariporide 7 1.4060.18 0.9860.12 1.4260.17

Ligation-reperfusion 8 1.5860.12 1.3760.16* 1.1560.14Ligation-reperfusion1

cariporide 9 1.7160.19 0.6160.05† 2.8160.03‡

Values for Bcl-2 and Bax depict histochemical scores obtained asdescribed in METHODS; n 5 no. of hearts examined in group. Bcl-2/Bax,ratio of Bcl-2 to Bax. *P , 0.05 vs. both sham groups; †P , 0.05 vs.ligation-reperfusion group not treated with cariporide; ‡P , 0.05 vs.both sham groups and ligation-reperfusion group not treated withcariporide (ANOVA).

Fig. 8. Micrographs demonstrating Bcl-2 and Baximmunohistochemical staining in left ventriclesfrom a sham rat heart (A) or hearts subjected toocclusion and reperfusion without (B) or with (C)cariporide.

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We thank Hoechst-Marion-Roussel for providing the diets used inthis study.

This study was supported by a grant from the Medical ResearchCouncil of Canada. M. Karmazyn is a Career Investigator of theHeart and Stroke Foundation of Ontario.

Address for reprint requests: M. Karmazyn, Dept. of Pharmacol-ogy and Toxicology, Univ. of Western Ontario, Medical Sciences Bldg.,London, ON, Canada N6A 5C1.

Received 17 July 1998; accepted in final form 22 October 1998.

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