C Pharmacology & Toxicology 2000, 86, 222–227. Copyright CPrinted in Denmark . All rights reserved

ISSN 0901-9928

Ultrastructural Evidence of the Protective Effect of Naπ/Hπ

Exchange Inhibition on the in vitro Damage Induced byIschaemia Reperfusion in the Interventricular Septum of

the Rabbit HeartPedro Salinas1, Pablo Gil-Loyzaga2 and Santos Barrigon1

1Department of Pharmacology, School of Medicine, Complutense University and 2Cell Culture Center (CAI-UCM),Complutense Univiversity, Avda Complutense s/n, 28040 Madrid, Spain

(Received October 26, 1999; Accepted January 6, 2000)

Abstract: We investigated the effects of the Naπ/Hπ antiporter inhibitor, dimethylamiloride, on myocardial injury after 1h global ischaemia and 30 min. reperfusion in the isolated arterially perfused interventricular septum of the rabbit heart.After ischaemia and reperfusion challenge, dimethylamiloride significantly increased the recovery of developed tension ina dose-dependent manner, and significantly decreased the maximal increase in resting tension. Ultrastructural analysis ofmyocytes submitted to the experimental in vitro model supported functional maintenance of physiologically-like con-ditions. Where myocardial portions were submitted to ischaemic conditions and reperfusion, myocyte cell damage reachedusual characteristics of infarct-like induced lesions. Intracellular oedema, severe disruption of myofibrils with loss ofmuscle striation and both swelling and fragmentation of mitochondria were the main characteristics observed. Di-methylamiloride treatment clearly modifies ultrastructural findings towards the normalization of cell shape and structure,only a slight-middle intracellular oedema and contraction bands were found. On the basis of the present results, we suggestthat the protective effects exhibited by dimethylamiloride on the ischaemic myocardium are compatible with its Naπ/Hπ antiporter inhibition properties, they diminish Naπ accumulation and then either Ca2π overload or non-exocytoticnoradrenaline release during the ischaemia and reperfusion challenge.

The Naπ/Hπ exchanger was initially suggested by Deitm-er & Ellis (1980) in sheep Purkinje fibers in experimentsusing Naπ- and pH-sensitive microelectrodes, and 22Naπ

flux studies, and further characterized by Pierce & Philipson(1985) in dog cardiac sarcolemmal vesicles. The Naπ/Hπ

exchanger is an integral plasma membrane protein thatcatalyzes the electroneutral exchange of extracellular Naπ

for intracellular Hπ with a 1:1 stoichiometry. Virtually alltissues investigated possess this type of antiporter, which isparticularly important in the regulation of pHi especiallyafter acid loading (Piwnica-Worms et al. 1985). In the lastfew years, four Naπ/Hπ exchanger isoforms have beencloned (Naπ/Hπ exchanger 1–4), the Naπ/Hπ exchanger 1being ubiquitously expressed, growth-factor-activated, andamiloride-derivative-sensitive. Moreover, this isoform seemsto be predominant in myocardial tissue (Yun et al. 1995;Noel & Pouyssegur 1995). Dimethylamiloride is one of the5-amino-substituted derivatives of amiloride which has highpotency for inhibiting the Naπ/Hπ exchanger of cell mem-branes (Vigne et al. 1984).

Myocardial ischaemia induces progressive acidification ofthe myocardial cells. By activation of the Naπ/Hπ exchangemechanism [Naπ]o is increased at the end of the ischaemia,

Author for correspondence: Santos Barrigon, Department of Phar-macology, School of Medicine, Complutense University, Avda.Complutense s/n, 28040 Madrid, Spain (fax (34) 91 3941463, e-mailBarrigon/EUCMAX.SIM.UCM.ES).

thereby increasing the [Ca2π]i through the Naπ/Ca2π ex-changer (Tani & Neely 1989). Over the past years increasingamounts of data have been published indicating that inhi-bition of Naπ/Hπ exchange during myocardial ischaemiadecreases the calcium overload and protects the myocardialcells during ischaemia and reperfusion (Levitsky et al.1998).

Myocardial cell damage by ischaemia follows a progress-ive increase in the degree of lesion from the early and stillreversible state (Bloom & Cancilla 1969) to the irreversiblestate and necrosis by long-term ischaemic exposure (Fer-rans & Butany 1983). Morphometrical and ultrastructuralstudies have clearly demonstrated that pharmacological in-terventions can significantly reduce the extent of myocardialnecrosis, but also the characteristics of cell lesions (Perez-Cao et al. 1989 & 1994). Therefore, ultrastructural changesin cardiac myocytes submitted to ischaemic models couldclearly help to define the functional situation of the cardiacmuscle (Perez-Cao et al. 1994). Thus, the use of a revisedversion of the major ultrastructural criteria of myocardialalterations might help classify and compare differentmodels of ischaemia, both in vivo and in vitro, but also tocompare different treatments apparently acting on differentcell level or step of lesions.

In spite of growing evidence about improved functionalrecovery, attenuated 45Ca2π-uptake and reduced release ofintracellular enzymes by ischaemia reperfusion challengeunder inhibition of the Naπ/Hπ exchanger (Levitsky et al.

223MYOCARDIAL ISCHAEMIA PROTECTION BY Naπ/Hπ EXCHANGE INHIBITION

Table 1.

Major criteria of myocardial alteration (from: Fukuhara 1985;Ganote & Humphrey 1985; Perez-Cao et al. 1994).

Grade 0: Normal myocyte.Grade 1: Reduction of glycogen granules, and mitochondrial ma-

trix density.Evidence of vacuoles.Some mitochondrias slightly swollen.

Grade 2: High reduction of glycogen granules and mitochondrialmatrix density.

Slight swelling of mitochondrias, including cristaebreakdown and presence of dense bodies.Margination and clearing of nuclear chromatin.Mild intermyofibrillar oedema, and contractionbands.

Grade 3: Lack of glycogen granules.High swelling of mitochondrias, including absence ofcristae and dense bodies.Severe intermyofibrillar oedema with sarcomere dis-ruption.Intracellular oedema.Contraction bands.

Grade 4: Severe intracellular oedema and cell membrane break-down.

Severe swelling and cristae breakdown of mitocond-rias.Some mitochondrias appeared contracted with densematrix.Severe intermyofibrillar oedema.Contraction bands.Chromatin severely clumped.

1998), the conclusive morphometrical and ultrastructuralstudies have not been carried out. Therefore, the aim of thepresent study was to examine if the protection exerted bythe inhibition of the Naπ/Hπ exchanger on ischaemia-re-perfusion challenge at the functional level could be corre-lated with ultrastructural changes in cardiac myocytes sub-mitted to an in vitro model of myocardial ischaemia.

Materials and Methods

Experimental preparation. The experimental preparation was theisolated arterially perfused interventricular septum of the rabbit ac-cording to Shine et al. (1976), Bourdillon & Poole-Wilson (1982),and Weiss et al. (1984). The investigation conforms with the Guidefor the Care and Use of Laboratory Animals (1985). Male New Zea-land White rabbits (2- 2,5 kg body weight) were anticoagulated in-travenously with 2000 UI sodium heparin, anaesthetized with anoverdose of pentobarbitone (120 mg intravenously) and the heartsrapidly removed. Within 4 min. the septal artery, a branch of theleft coronary artery, was cannulated and the interventricular septumimmediately perfused (2.1 ml/min. per g wet weight) by means of avariable speed peristaltic pump (Gilson, Minipuls HP4). The tri-angular perfused area of the septum was dissected, mounted in aPlastic chamber, and its apex attached to a Grass FT03 transducer.The developed tension and the rate of tension development (∫dT/dt) were recorded continuously in a model 79D Grass polygraph.Septa were electrically stimulated with one pulse of 15 V and 5msec. duration at a basal rate of 1.5 Hz (Grass, SD9) by means ofa platinum electrode inserted in the muscle and a surface silver elec-trode in close apposition (less than 2 mm apart), and perfused witha standard solution containing (mmol/l): NaCl, 130; KCl, 6; MgCl2,

1; CaCl2, 1.5; NaHCO3, 9–11; NaH2PO4, 0.43; D-glucose, 5.5 (pH7.4). Solutions were bubbled with carbogen (95% O2, 5% CO2) dur-ing the whole experimental duration. Septal temperature (32æ) wasmonitored throughout by means of a thermistor needle insertedinto the muscle, and maintained by warming the perfusate with athermostatically controlled heating coil and by heating the chamberwith a water-filled radiator through which N2 was bubbled. Themuscle surface temperature was continuously monitored by a ther-mistor placed near to the septal surface. Under such conditionsmuscle temperature was stable to ∫0.25æ throughout all the experi-ment duration. In all experiments, an equilibration period of 40min. was allowed before any intervention.

Effects of the Naπ/Hπ exchange inhibition on the rabbit septa afterischaemia-reperfusion challenge. Global ischaemia was induced bystopping the flow through the perfusion cannula during 1 h. Afterthis period of ischaemia, reperfusion was done by starting the per-fusion for another additional 30 min. In these experiments, themechanical parameter analyzed to evaluate the protective effect ofNaπ/Hπ exchange inhibition against the ischaemia-reperfusion in-jury was the recovery of developed tension, expressed as percentageof its preischaemic value and the maximal increase in resting ten-sion. In an ischaemic control group of seven experiments, this pro-tocol was followed without pharmacological intervention. In an-other group of experiments dimethylamiloride, 0.1 mM (nΩ3), 1 mM(nΩ6), and 10 mM (nΩ6) was added to the perfusate during 30 min.previous to global ischaemia and during reperfusion. No additionaleffects were shown when the dimethylamiloride concentration washigher than 10 mM.

Transmission electron microscopy. Following the 30 min. reperfusionperiod, samples of 1¿1¿1.5 mm of heart tissue obtained from thebase near to the septal artery, were fixed for 2 hr with 0.5 M sodiumcacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2%paraformaldehyde. After 15 min. washout in Sorensen buffer (0.1M KH2PO4 plus 0.1 M Na2HPO4; pH 7.4), samples were fixed againfor 2 hr in a 2% osmium tetroxide solution in Sorensen buffer, andcounterstained for 30 min. in a 2% uranyl acetate solution inMichaelis buffer (0.025 M; pH 7; 440 mOsmol/l; 2 nM calcium).Samples were then dehydrated by a graded series of acetone rinses

Fig. 1. Mechanical effects of global ischaemia (60 min.) and reper-fusion (30 min.) on the isolated arterially perfused interventricularsepta of the rabbit in (A) control conditions and (B) after 30 min.of perfusion with dimethylamiloride, 10 mM, in preischaemia andduring reperfusion. Lower record: developed tension. Upper record:the first differential of developed tension with respect to time. Hori-zontal calibration: time, 10 min. Vertical calibrations: dT/dt, g/s(upper) and tension, g (lower).

PEDRO SALINAS ET AL.224

and, finally in propylene oxide. Tissues were infiltrated and embed-ded in Araldite resin, and polymerized overnight in a 70æ oven. Thinsections (20–40 nm) were cut using an ultramicrotome (LKB IIIUltratome) and collected on 400 mesh copper grids. Preparationswere stained with uranyl acetate and lead citrate, and analyzed byusing a HU 12A Hitachi electron microscope. In order to classifylesions a previous score (Fukuhara 1985; Ganote & Humphrey1985) was used with some modifications according to table 1 (Perez-Cao et al. 1994).

Drugs. Dimethyl amiloride was obtained from Research Biochem-icals Incorporated, Natick, USA. The drug was dissolved directlyin the perfusate.

Statistical analysis. Data are presented as the mean∫SD for eachgroup and statistical analysis was carried out by using the Student’sunpaired t-test. Differences between groups were considered as sig-nificant when P,0.05.

Results

The effects of Naπ/Hπ exchange inhibition on the ischaemiareperfusion challenge in the contractile parameters of the rab-bit septa.

After the onset of the ischaemia, developed tension fell in aprogressive manner, being close to zero at 10 min. (fig. 1A).During this period, a gradual rise in resting tension wasseen. After 3–4 min. reperfusion, tension further raised tomaximum. After 30 min. reperfusion, developed tension re-covered 19.7∫1.7% of its preischaemic value. The maximumpositive and negative rates of change of developed tensionwere apparently modified in a similar way.

Perfusion with increasing dimethylamiloride concen-trations, 0.1 to 10 mM, 30 min. before the ischaemic inter-vention and during reperfusion significantly increased therecovery of developed tension after the ischaemia and reper-fusion intervention in a dose-dependent manner (6.6, 66.5and 132%, respectively) (fig 1B; table 2). The maximal in-crease in resting tension also was diminished in a dose-de-pendent manner by dimethylamiloride, up to 56.9% of theischaemic control value of 13.1∫0.93 g.

Table 2.

Effects of Naπ/Hπ exchanger inhibition on the recovery of de-veloped tension, and on the maximal increase in resting tension(MRT) in isolated rabbit septa after a 1 hour global ischaemia and30 minutes reperfusion challenge. DMAΩdimethyl-amiloride.

n MRT (g) Recovery (%)

CONTROL 7 13.10∫0.93 19.7∫1.7

DMA, 0.1 mM 4 11.95∫2.35 21.0∫4.2DMA, 1.0 mM 6 7.41∫1.24** 32.8∫7.4*DMA, 10.0 mM 6 5.65∫0.87** 45.7∫5.0**

Recovery of developed tension expressed as the mean of individualpercentages (calculated by division of the developed tension at theend of 30 min. reperfusion by that immediately before the onset ofthe ischaemia) for each experiment.Values expressed as mean∫S.D. * P,0.05 ** P,0.01.

Fig. 2. Control myocardial slices in in vitro maintenance exhibiteda normal structure. a) Myocytes from control slices, exhibiting theirusual irregular interdigited cell nuclei (asterisk), a regular myofila-ment arrangement (empty stars) and glycogen granules in the cyto-plasm. Some rare vacuoles and electrodense bodies were also found(arrows). b) Most of mitochondria (asterisk) contained normal ma-trix density and a tightly packed crest. c) An intercalated disc (ar-rowheads) showing filaments anchored on their transversal por-tions, some vacuoles or electrondense bodies were also found intheir vicinity. Magnification a: ¿15,000; b: ¿20,000; c: ¿22,000.

Ultrastructural analysis.Control myocardial slices after in vitro perfusion.

Control myocytes submitted to in vitro perfusion for 1 h,showed a healthy shape even when some vacuolar cytoplas-mic system reorganization was noticed (fig. 2). Myocyte cellnuclei exhibited their normal interdigited periphery, closeto which some vacuoles or dense bodies were observed (fig.2A). Myofibrilar organization showed a usual sarcommerpattern without disorganization or lysis of the contractilemyofilaments (fig. 2A). Mitochondrias were spread outthroughout the cytoplasm with a rounded normal shapewith normal crest organization (fig. 2B), although somerare vacuoles were found among them. Glycogen granuleswere observed (fig. 2B and C). Intercalated discs, where ac-tin filaments are anchored on transversal portions (fig. 2C),were completely conserved. Some vacuoles or electrondensebodies were found in the vicinity of intercalated discs (fig.2C). In conclusion, the myocytes of heart portions submit-ted to in vitro conditions could be considered as lesion freecells showing a score of grade 0–1 (see table 1).

225MYOCARDIAL ISCHAEMIA PROTECTION BY Naπ/Hπ EXCHANGE INHIBITION

Fig. 3. Effects of in vitro ischaemia and reperfusion on cardiac myo-cytes. The structural damage resulted from 1 hr of in vitro ischaemiaand 30 min of reperfusion could be similar to myocardial infarct-like lesions. a) Semipanoramic magnification showed an importantintracellular oedema with sarcomere disruption and disorganization(stars). b) and c) An important number of mitochondria, in particu-lar those located close to the cell periphery, appeared swollen (aster-isk). Some myelin-like membrane distortions were also observed (ar-rowhead). d) Intracellular oedema (stars) was particularly evidentaround the cell nucleus. Magnification a: ¿12,000; b: ¿40,000; c:¿40,000; d: ¿20,000.

Effects of in vitro ischaemia and reperfusion on cardiac myo-cyte ultrastructure.

After one hr of in vitro ischaemia and 30 min of reperfusion,large myocardial infarct-like lesions were present in allsamples. The structural damage to cardiac myocytes causedby in vitro ischaemia and reperfusion was characterized byan important intracellular oedema (fig. 3A), which induceda lack of the myofibril arrangement and striation pattern.Severe intermyofibrilar oedema, sarcomere disruption andcomplete disorganization or even lysis of the contractile my-ofilaments were observed (fig. 3A). A significant numberof mitochondrias, in particular at the myocyte periphery,appeared swollen with membrane disruptions and myelinforms (fig. 3B and C). Intracellular oedema was particularlyevident around the cell nucleus (fig. 3D). In conclusion, themajority of myocytes of heart portions submitted to isch-aemia and reperfusion during in vitro maintenance reachedan alteration score of grade 4 (see table 1).

Protection derived from the use of dimethylamiloride duringin vitro ischaemia and reperfusion of myocardium.

After one hr of in vitro ischaemia and 30 min of reperfusionin the presence of 30 min dimethylamiloride (10 mM) pre-treatment, most of the myocytes examined seemed quite un-affected, or with only minor and reversible modifications(fig. 4A). Myofibrils were preserved exhibiting the usual stri-ation pattern of the sarcomeres, even when most of themyocytes showed a slight-mild intracellular oedema (fig.4A). Glycogen granules were present in the sarcoplasm butwere sparse. The majority of mitochondria appeared intact,with normal shape and crest organization (fig. 4A, B andC). Some vacuoles, probably related to rare mitochondriadamaged after swelling, were found in large groups of nor-mal ones (fig. 4B). Additionally, some myocytes containedcontraction bands (fig. 4B and C) where the myofibril hadcollapsed and was distorted. Some of the contraction bandswere found close to the intercalated discs without alter-ations of the intercellular contacts (fig. 4C). In conclusion,the majority of myocytes of the heart portions submitted toischaemia and reperfusion in dimethylamiloride treatmentshowed a maximal alteration score of grade 1–2 (table 1).

Fig. 4. Protection due to the use of dimethylamiloride in the in vitroischaemia and reperfusion of myocardium. a) A small number ofmyocytes exhibited slight myofilament disruption (star) and slight-mild intracellular oedema (asterisk). b) Some mitochondria ap-peared swollen (asterisks) in large populations of normal ones.Some contraction bands (white arrowheads) were observed. c)Eventually some contraction bands (arrows) were found in thevicinity of normally-shaped intercalated discs. Magnification a:¿22,000; b: ¿17,000; c: ¿18,000.

PEDRO SALINAS ET AL.226

Discussion

The results of the present study demonstrate that infusionof dimethylamiloride protected, in a dose-dependent man-ner, the isolated rabbit heart from the deleterious effects ofan ischaemia and reperfusion challenge when it was presentduring either ischaemia or the reperfusion period. More-over, this protection was evident not only in functional butalso in morphometrical and ultrastructural studies. Thesedata support and extend previous observations that di-methylamiloride protects rabbit myocytes from 45 min.ischaemia after cardioplegic arrest of the whole heart(Koike et al. 1996), and decreases the magnitude of celldeath in the isolated arterially perfused rabbit papillarymuscles when evaluated by the trypan blue uptake method(Kaplan et al. 1995). There are several publications that thisdrug also exhibits myocardial protective effects on modelsof ischaemia and reperfusion in other animal species such asrat (Meng & Pierce 1990), guinea-pig (Moffat & Karmazyn1993) and pig (Klein et al. 1995).

We found the maximal cardioprotective effects at a con-centration of 10 mM, which has been proven as effective inthe blockade of the Naπ/Hπ exchanger in the rabbit heartwithout affecting cellular energy (Koike et al. 1996). More-over, concentrations as high as 20 mM have been describedas nearly specific for the Naπ/Hπ exchanger, with no sig-nificant effect on the protein kinase, Naπ/Ca2π exchangeror the sodium channel (Kaplan et al. 1995). Recently it hasbeen shown that as short as 1 min. exposure to dimethylam-iloride immediately at the start of reperfusion protected themyocardium against the deleterious effect of 1 hr ischaemiaand reperfusion challenge (Maddaford & Pierce 1997). Thefast dimethylamiloride protective effect rules out other di-methylamiloride non-specific effects that require severalminutes to develop (Kaplan et al. 1995), and points to theinhibition of the Naπ/Hπ exchanger as the main mechanisminvolved in the cardioprotective effect of dimethylamiloride.In fact, during myocardial ischaemia intracellular acidosisdevelops quickly, and activates the Naπ/Hπ exchanger toextrude Hπ out of the cell. The activation of the exchangerincreases the influx of extracellular sodium at the end ofthe ischaemia, which could secondarily increase the [Ca2π]ithrough the Naπ/Ca2π exchanger inducing calcium over-load (Tani & Neely 1989).

Apart from this main mechanism, other mechanisms notdirectly related to the calcium exchange could account forthe dimethylamiloride protective effects. In this respect, itis well documented that during ischaemia, a progressive re-lease of endogenous norepinephrine from adrenergic nerveterminals within the myocardium occurs, and that this re-lease could have a wide range of possible deleterious conse-quences (Jennings & Reimer 1981; Todd et al. 1985a & b).The ischaemia-induced noradrenaline release has been pro-posed as non-exocytotic (i.e. calcium-independent), throughthe neuronal catecholamine uptake (uptake 1), and relatedto the intraneuronal sodium accumulation. Moreover theNaπ/Hπ exchanger has been identified as the predominant

pathway of sodium entry into the sympathetic nerve endingin ischaemia, and the Naπ/Hπ exchanger inhibition mark-edly suppresses the ischaemia-induced noradrenaline release(Schoming et al. 1988; Gerber et al. 1999). In our experi-mental conditions, a modification of catecholamine releaseby dimethylamiloride could account for its effects on isch-aemia-induced damage. Nevertheless, the partial contri-bution of the suppression of the catecholamine release tothe overall dimethylamiloride cardioprotective effect re-mains to be evaluated.

The ultrastructural findings reported here seem to indi-cate that in vitro maintenance of myocardial portions innormoxic perfusion did not affect the structure and fine cellorganization of myocytes. Also, experimental ischaemia andreperfusion exhibited alterations similar to those observedin other in vivo models (Jennings & Reimer 1981; Perez-Caoet al. 1989 & 1994). The main cell alterations involved inischaemic injury to the myocardium were present. Myocar-dial ischaemia usually result in intracellular oedema, ac-cumulation of lipid droplets, appearance of contractedbands, severe disruption of myofibrils, both swelling andfragmentation of mitochondria, glycogen granules loss,condensation of nuclear chromatin and karyolysis (Perez-Cao et al. 1994). All these results led us to propose a modi-fied table of progressive score for the alterations dependingon the severity of lesions (see table 1). Hypoxia has beenlargely involved in the pathogenesis of myocardial infarct-like pathologies either in clinical or in experimental iso-proterenol-induced myocardial necrosis. Some similar find-ings have been observed in clinical infarcts, in experimentalin vivo models and in the present results (Rona et al. 1959;Ferrans et al. 1969; Fleckenstein 1971; Maroko & Braun-wald 1976; Jennings & Reimer 1981; Todd et al. 1985a & b;Rona 1985; Bhimji et al 1986; Nayler 1988). These similar-ities validate our present model from an anatomical pointof view.

The present study also demonstrates that dimethylami-loride significantly reduces the myocardial necrosis in-duced by ischaemia and reperfusion in an in vitro myocar-dial model suggesting that it may also delay ischaemic in-jury. The significant reduction in myocardial lesions withdimethylamiloride, from a score of grade 4 to score ofgrade 1–2, and similar to that obtained with calcium-channel blockers (Perez-Cao et al. 1989 & 1994), indicatesthat dimethylamiloride had important mitochondrial pro-tection abilities and may induce reduction in intracellularoedema.

AcknowledgementsThis study was supported in part by 083/30/98 research

grant from the CAM and PR 269/98–8172 from the multi-disciplinary research fund of the University Complutense,Madrid, Spain. The authors are grateful to Dr. EstherO’Shea for her comments and English language revision ofthe manuscript, and to Ms. T. Rodriguez-Benito for hertechnical assistance in electron microscopy.

227MYOCARDIAL ISCHAEMIA PROTECTION BY Naπ/Hπ EXCHANGE INHIBITION

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