The antioxidant effects of a novel iron chelator salicylaldehyde isonicotinoyl hydrazone in the prevention of H2O2 cardiomyocytes

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1 Cardovascular Research 47 (2000) locate/ cardores locate/ cardores The antoxdant effects of a novel ron chelator salcylaldehyde soncotnoyl hydrazone n the preventon of H2O2 njury n adult cardomyocytes a, b,c a Magda Horackova *, Prem Ponka, Zenoba Byczko a Department of Physology and Bophyscs, Faculty of Medcne, Dalhouse Unversty, Halfax, Nova Scota B3H 4H7 Canada b Lady Davs Insttute for Medcal Research, Jewsh General Hosptal, Montreal H3T 1E2 Canada c Department of Physolgy and Medcne, McGll Unversty, Montreal, Canada Receved 8 February 2000; accepted 31 March 2000 Abstract Objectve: Ths study was desgned to nvestgate the cardoprotectve effect of the novel lpophlc ron chelator salcylaldehyde soncotnoyl hydrazone (SIH) aganst the oxdatve stress exerted by H2O2 through the producton of OH radcal va the Fenton reacton and to compare them wth those of the hydrophlc ron chelator deferoxamne (DFO) and the Na / H exchange nhbtor methylsobutyl amlorde (MIA). Methods: We used long-term cultures of spontaneously beatng adult gunea-pg ventrcular cardomyocytes developed and characterzed prevously n our laboratory. We assessed ther contractle actvty by vdeo-recordng as well as the underlyng Ca transents by Fura 2 fluorescence. In some experments we also recorded these functonal parameters, plus the electrcal actvty (acton potentals) n response to electrcal stmulaton va sucton ppettes, n ndvdual freshly solated myocytes. Results: Exposure of the regularly and synchronously beatng cultured cardomyocytes to 100 mm H2O2 ntally caused a substantal prolongaton of Ca transents accompaned by an rregular contractle actvty, then n contractle arrest and a severalfold ncrease n cytosolc [Ca ] that occurred, wthn 30 mn of H2O2 applcaton. Smlar effects were also observed usng freshly solated cardomyocytes. The latter effects were frst accompaned by sgnfcant prolongaton of the acton potental duraton (APD) wth supermposed early afterdepolarzatons followed by a second phase wth a very fast decrease n APD, contractons, as well as Ca transents and a thrd phase of nexctablty, contractle arrest, ncreased cytoplasmc [Ca ] and a fnal contracture. All these effects were rreversble n both types of preparatons but they could be fully prevented by a 15-mn prencubaton wth 200 mm SIH. Smlar protectve effects were observed wth DFO, but n ths case a much hgher concentraton had to be used (1 mm) and much longer (2 h) prencubaton was needed. By contrast, 5 mm MIA faled to fully protect the cardomyocytes, although a sgnfcant delay (10 mn) of the effects of H2O2 was observed. Conclusons: The data ndcate that SIH provdes a very powerful and very fast protecton aganst the oxdatve stress exerted by H2O2 presumably va the ron-medated Fenton reacton producng hydroxyl radcal ( OH), whereas the protectve effect of DFO s hndred by ts very slow and rather lmted ntracellular entry, and the protecton that MIA exerts va the nhbton of Na / H exchange aganst H2O2 much less effectve Elsever Scence B.V. All rghts reserved. Keywords: Calcum (cellular); Cell culture/ solaton; Contractle functon; e-c couplng; Free radcals 1. Introducton the schemc myocardum [1]. More recently, electron paramagnetc resonance and hstochemcal studes have It has been proposed that oxygen-derved oxdants, such convncngly demonstrated that these oxdants are gener- as the superoxde radcal (O 2), hydrogen peroxde (H2O 2), ated early durng myocardal reperfuson [2 6]. The and the hydroxyl radcal ( OH), could be mportant formaton of these free radcals n bologcal systems s medators of arrhythmas that occur durng reperfuson of catalyzed by ron whch s normally non-reactve because t s bound to protens of ron transport (transferrn) and *Correspondng author. Tel.: ; fax: E-mal address: magda.horackova@dal.ca (M. Horackova). Tme for prmary revew 29 days / 00/ $ see front matter 2000 Elsever Scence B.V. All rghts reserved. PII: S (00)

2 530 M. Horackova et al. / Cardovascular Research 47 (2000) storage (ferrtn). Under certan abnormal condtons, such Natonal Insttutes of Health (NIH Publcaton No , as durng reperfuson njury, actvated oxygen speces revsed 1996). release ron from these protens, and the resultng free ron (Fe ) promotes the formaton of the devastatngly reactve toxc ( OH) Solutons and chemcals Iron-chelatng agents, such as deferoxamne (DFO) have The Ca -free standard HEPES-buffered Tyrode s solubeen shown to nhbt free-radcal formaton and the ton contaned mm NaCl, 3.8 mm KCl, 1.2 mm consequent free radcal tssue damage n some expermen- KH2PO 4, 1.2 mm MgSO 4, 11.1 mm glucose, and 10 mm tal systems [7]. HEPES; ph was adjusted to 7.4 wth NaOH. Ths soluton However, DFO crosses cell membranes neffcently, s referred to hereafter as Ca -free Tyrode s soluton. and ths feature may lmt ts effectveness n vvo [8]. On Salcylaldehyde soncotnoyl hydrazone (SIH,.e. 2- the other hand, a lpophlc ron chelator, salcylaldehyde hydroxybenzal soncotnoyl hydrazone) was syntheszed soncotnoyl hydrazone (SIH), whch enters cells and by Schff base condensaton between 2-hydroxybentssues very effcently [9 11], s a powerful nhbtor of zaldehyde and soncotnc acd hydrazde, as descrbed ron-dependent producton of hydroxyl radcal ( OH) from prevously [10]. The chemcal structure s gven n Fg. 1. H2O 2 [12,13]. Prevously [14] we studed n consderable detal the development of H2O2-nduced changes n membrane po Cardac myocyte preparatons tentals, membrane currents, and correspondng contractle actvty (shortenng) n rat and gunea-pg ventrcular Detals of the solaton of adult gunea-pg carmyocytes, usng the sucton-ppette whole-cell clamp domyocytes have been presented elsewhere [18,19]. method. Brefly, the aorta of anesthetzed gunea pgs ( g) The observed changes n response to mm H2O2 was cannulated and perfused retrogradely for 5 mn at occurred n three phases: a prolongaton of acton potental 358C wth Ca -free Tyrode s soluton. To ths soluton duraton (APD) accompaned by ncreased contractlty, was then added % collagenase (Worthngton followed by a perod of decreased APD and reduced type II, Freehold, NJ), 0.004% trypsn (Sgma Chemcal contractlty, wth a fnal phase of nexctablty and the Co., St. Lous, MO), 0.4% type F (fatty acd free) albumn, myocytes contracture. and 25 mm CaCl 2. The concentraton of collagenase was DFO prevented those changes at much lower concen- chosen to yeld an optmal dssocaton process. Ths tratons and after much shorter prencubaton exposures perfusate was recrculated for 3 5 mn, followng whch when appled ntracellularly (va a sucton electrode) than the ventrcles were removed and slced nto small strps when appled extracellularly. Ths ndcates that the whch were ncubated n a shaker bath for several 15-mn changes were nduced by OH generated ntracellulary n perods. After gentle centrfugaton, cells were pooled and the presence of ron. ths cell suspenson was used for platng after the cells In ths study, we have compared the protectve effects were counted n a specal 1-ml countng chamber aganst oxdatve njury by H2O2 that externally appled (Sedgewck rafter; Gratcules Ltd.; Turnbrdge Wells, UK). SIH exerts wth those of DFO and wth those of the At least 98% of the cells n a preparaton are myocytes Na / H exchange nhbtor methylsobutyl amlorde 6 ( cells/ anmal), of whch.80% are rod (MIA), whch has been ndcated as provdng protecton aganst some effects of H2O 2 [15] and aganst superfuson njury [16,17]. We have used our model of cultured adult gunea-pg cardomyocytes as well as n some experments we also used freshly solated cardomyocytes [18 20] to determne how H2O2 n the presence or absence of the above drugs affects contractle actvty, electrcal actvty, and Ca transents. Our results demonstrate that SIH exerts a very fast and potent cardoprotectve effect aganst oxdatve stress nflcted by H2O 2, whch ndcates the potental therapeutc sgnfgance of ths lpophlc ron chelator aganst cardac schema/ reperfuson. 2. Methods The nvestgaton conforms wth the Gude for the Care and Use of Laboratory Anmals publshed by the US Fg. 1. The chemcal structure of SIH and DFO.

3 M. Horackova et al. / Cardovascular Research 47 (2000) shaped and.90% are vable (as demonstrated by exclu- mm. The membrane restng and acton potentals were son of Trypan Blue). recorded wth patch ppettes where Rel55 10 MV. The ppettes were fabrcated wth a Kopf (two-stage) vertcal 2.3. Culture technques puller. An Ag AgCl brdge connected the ppettes to the nput stage of an amplfer (Axoclamp-Axon Instruments, Cells were cultured n Eagle s mnmum essental Burlngame, CA, USA). The myocytes were ntracellularly medum wth Earle s salts, supplemented as descrbed stmulated through the recordng patch ppette by current prevously [19]. Cytosne 1-b-D arabnofuranosde (10 pulses that were 20 ms n duraton and 1 5 na n mm) was added to the culture medum to mnmze the ampltude at a frequency of 0.2 Hz or 0.5 Hz. The acton growth of fbroblasts, and lamnn (5 mg/cm) was used to potentals and the correspondng shortenngs were recorded coat the 1.2-cm-dameter glass coverslps. We used a by means of pclamp software on a PC-AT 386 computer. 5 2 platng densty (10 myocytes/ cm ) whch we establshed Statstcal comparson of some data has been made usng n a prevous study [18] to be optmal for the culturng of Student s t-test for par data whch are presented as gunea-pg myocytes. means6s.e.m. Plated cells were mantaned n an ncubator at 378C under a 95% ar 5% CO2 atmosphere, and culture meda were replaced twce a week. Cultures used n these experments were 3 4 weeks old, as they are at ths stage 3. Results fully nterconnected and very stable n ther synschronzed beatng rates [20]. In ths study we used regularly spontaneously beatng cultured cardomyocytes for most experments because they are a more stable and more reproducble expermental 2.4. Recordng of contractle actvty n myocytes model for recordng contractle actvty and Ca transents than are freshly solated cardomyocytes. The detals of ths technque have been descrbed However, snce the electrcal actvty could not be prevously [19]. Brefly, 1.2-cm-dameter glass coverslps drectly recorded for prolonged perods of tme n the wth cultured myocytes were placed n a 0.5-ml bath and spontaneously beatng cultures, we also used solated perfused at 3 ml/mn at 35618C. The contractle actvty myocytes n some experments to assess the smlarty of (shortenng) of the myocytes was recorded by a vdeo the waveform of the Ca transents and the confguraton edge-detectng system. The recordng was obtaned by of the acton potental n the presence of H2O2. The recordng the movement of the end of the solated cell or frequency of the contractons and of the Ca transents an ntercellular boundary (edge) between two or more wthn each ndvdual culture were dentcal, the sngle cultured cells whch represented the synchronsed contrac- regular contractons beng accompaned by sngle Ca tle actvty of the whole confluent layer of each culture. transents of approxmately 500 ms duraton (Fg. 2A). Wthn 10 mn of the applcaton of 100 mm H2O2 the Ca 2.5. Measurements of [Ca ] transents became transently largely prolonged (to about 2 s) wth multple peaks, and ther dastolc baselne was Recordng of the Ca transents were performed usng slghtly ncreased, ndcatng elevated restng cytosolc Fura 2-AM methodology []. Cultured (or freshly so- [Ca ] (Fg. 2B). These dramatcally prolonged Ca lated) myocytes were loaded wth 4 mm Fura 2-PE3-AM transents were accompaned by rregular contractle actv- (Teflabs Inc., Austn, TX) for 60 mn at room temperature ty (Fg. 2B). Durng the followng mn, the under an O2 atmosphere. The myocytes were then super- ampltude of the ndvdual contractons decreased whle fused wth control Tyrode s soluton contanng 2.5 mm the ampltude of the Ca transents was not altered much, CaCl for 30 mn at 378C on the mcroscope stage. The 2 despte the fact that, the level of cytoplasmc [Ca ] durng Ca transents were measured usng a PTI-system (South dastole further ncreased and they became shorter and Brunswck, NJ) that measures fluorescence at wavelengths more frequent (Fg. 2C). Wthn the next 10 mn (.e., of 340 and 380 nm. The Ca transents are expressed as mn after H2O2 was appled), the cultures stopped the fluorescent rato 340/ 380. contractng and the Ca transents dsappeared whle the cystolc [Ca ] ncreased further (Fg. 2D), reachng a 2.6. Recordng of the electrcal actvty value of 2 3 tmes the ntal one. Ths state was fully rreversble, and spontaneous actvty dd not return even The freshly solated myocytes were placed nto a small after 120 mn of perfuson wth control Tyrode s soluton (0.5 ml) expermental chamber, superfused (3 ml/ mn) (not shown). wth Tyrode s soluton (ph 7.4; C as recorded H2O2 had smlar effects n the freshly solated contnuously by a thermoprobe n the chamber), and myocytes. The contractle actvty was greatly prolonged gassed wth 100% O. The concentraton of CaCl was 2.5 as a result of an ncrease of the acton potental duraton 2 2

4 532 M. Horackova et al. / Cardovascular Research 47 (2000) Fg. 2. The contractle actvty (upper traces) and Ca transents (lower traces) of 25-day-old spontaneously beatng cultured adult gunea-pg cardomyocytes n Tyrode s soluton (A) and n the presence of 100 mm H2O2 10 mn (B), 20 mn (C), and 30 mn (D). Note that the baselne of Ca (the control dastolc level s represented by the dotted lne) ncreased steadly wth the length of exposure to H2O 2. Smlar results were recorded n another 8 preparatons. (APD), whch reached a maxmum of several seconds wth (e.g. compare Fg. 5A and B) and t fully exerted ts early afterdepolarzatons (EADs) wthn 20 mn of the protectve effect aganst 100 mm H2O2upto50mn applcaton of 100 mm H2O 2 (Fg. 3 B and C). Ths APD the longest perfuson examned (Fg. 5C E). A small prolongaton was accompaned wth consderably pro- ncrease n the ampltude of the contractle actvty was longed Ca transents. Ths effect lasted only a few occasonally observed (Fg. 5E). However, ths occurred mnutes, whle the APD vared from beat to beat between 1 wthout any sgnfcant change n APD whch was: and 3 s (e.g. compare Fg. 3B and C). About 25 mn after msec and ms for control and expermenthe applcaton of H2O2 the APD started to shorten at a tal group, respectvely (n54; P.0.05). very fast rate (Fg. 3D F), and wthn mn after the The hydrophlc ron chelator DFO also had protectve applcaton, the cells became nexctable, and the contrac- effects aganst H2O 2, but only at 1 mm,.e., a fve tmes tons and Ca transents subsded whle the cytosolc hgher concentraton than SIH. In addton, at least 2 h [Ca ] was ncreased two- to threefold (Fg. 3G). Shortly prencubaton wth 1 mm DFO was needed to prevent after that, the cell underwent a contracture. (Fg. 6B) the effects of 100 mm H2O2 observed n ts All the changes n the contractle actvty of the cultured absence. There was usually a slght decrease n the myocytes and the Ca transents observed n the presence frequency of spontaneous beatng n the presence of DFO, of 100 mm H2O2 were completely suppressed when the whch was not analyzed n detal, snce t was fully cultures were prencubated wth 200 mm SIH for 15 mn reversble upon a subsequent 20-mn washout n Tyrode s pror to H2O2 applcaton (Fg. 4B). In order to assure that soluton (Fg. 6C). the effect of SIH dd nor represent merely a delayed Fnally, there was a lttle or no protectve effect of 5 mm process of that observed n ts absence, we washed out MIA aganst H2O2 njury n cultured myocytes. The both drugs after 40-mn exposure to H2O2 and contnued pattern of the effects of 100 mm H2O2 on the contractle perfuson n control Tyrode s soluton for another actvty and the Ca transents was smlar n both the mn. Both the contractle actvty and the Ca transents presence and the absence of MIA, although they developed mantaned a regular frequency and pattern (Fg. 4C). 200 more slowly (Fg. 7B D), wth the fnal stage of rreversmm SIH tself dd not affect the electrcal or contractle ble contractle arrest (Fg. 7D) occurrng mn later actvty or the Ca transents n freshly solated myocytes when MIA was present (n56) than n ts absence.

5 M. Horackova et al. / Cardovascular Research 47 (2000) Fg. 3. The acton potentals (AP) and the correspondng contractle shortenng (c) and Ca transents (nserts) n freshly solated gunea-pg myocyte recorded upon stmulaton at 0.2 Hz n Tyrode s soluton (A), and after100 mm H2O2 had been appled for 15 mn (B), 20 mn (C), 25 mn (D), 27 mn (E) and mn (F and G). Smlar results were obtaned n other 4 myocytes. Note the dfferent tme scales n the ndvdual recordngs and the early afterdepolarzatons (EADs) at the end of the AP (C). 4. Dscusson In the present study we have demonstrated that the applcaton of H2O2 leads to dsturbances n spontaneous contractle actvty n cultured myocytes as a result of prolonged Ca transents, whch n turn seems to be a result of a greatly prolonged APD (as observed n freshly solated myocytes). Furthermore, the ncreased Ca nflux leads to manfold ncrease n dastolc concentraton of cytosolc [Ca ] as demonstrated by the ncrease of the baselne of the Fura-2 fluorescence rato. Ths Ca overload s presumably at least partally responsble for the fnal rreversble nextablty and contractle arrest n the cultured cardomyocytes and the contracture n the freshly solated myocytes. All these effects were fully prevented by the lpophlc ron-chelator SIH n both the cultured and freshly solated myocytes. Recently, SIH was shown to prevent OH-medated (formed from Fe(III) EDTA plus ascorbate) release of TBARS from 2-deoxyrbose as well as the release of Fg. 4. The spontaneous contractle actvty (upper traces) and Ca ethylene from 2-keto-4-methobutyrc acd [13] and to transents (lower traces) of 24-day-old cultured adult gunea-pg car- prevent plasmd pvc-17 DNA strand breaks nduced by domyocytes. The culture has been prencubated for 15 mn n 200 mm SIH (A) before an applcaton of 100 mm H O for 40 mn (B). Ths OH radcals [22]. More recently, when electron para- 2 2 exposure was followed by a 20-mn washout n control Tyrode s soluton magnetc resonance (EPR) wth 5, 5-dmethyl-1-pyrrolne- (C). Smlar results were obtaned n another 7 preparatons. N-oxde (DMPO) was used as a spn trap for OH, SIH

6 534 M. Horackova et al. / Cardovascular Research 47 (2000) Fg. 5. The acton potentals (AP), the correspondng contractle shortenngs (c), and the Ca transents (upper panels) recorded n freshly solated gunea-pg cardomyocytes upon stmulaton at 0.5 Hz n Tyrode s soluton (A), 15 mn after the applcaton of 200 mm SIH (B), and after 100 mm HO 2 2 has been appled n the presence of 200 mm SIH for 20 mn (C), 35 mn (D) and 50 mn (E). Smlar results were obtaned n another 3 myocytes. was shown to nhbt Fe(II)-dependent producton of OH logcal alteratons of cardomyocytes oxdatvely damaged from H2O 2 [12]. Collectvely, these studes ndcated that followng ther exposure to H2O 2. SIH s a powerful nhbtor of ron-medated oxyradcal Although another chelator DFO was reported to formaton n vtro. The present study, ndcates that SIH be also cardoprotectve aganst oxdatve stress durng may also be a strong nhbtor of free-radcal formaton n schema/ reperfuson [23,24], t was sgnfcantly less functonally ntact cells, snce t can prevent electrophyso- potent than SIH n preventng H2O2-nduced electrophy- sologcal changes n cultured cardomyocytes (present study). Ths can be explaned by the fact that SIH crosses cell membranes more effcently than DFO does [10] and thus can effectvely chelate ntracellular ron and prevent ts partcpaton n the Fenton reacton. Indeed, a recent study by Cable and Lloyd [8] provded evdence that DFO s ncapable of effcently crossng membranes, enterng cells only by endocytoss, and that t accumulates manly n the endosome-lysosome complex. Also n concord wth the present results are our earler data [14] demonstratng that the oxdatve njury of freshly solated rat and guneapg cardomyocytes by H2O2 was prevented mmedately followng the applcaton of DFO ntracellulary va a sucton ppette, whle several hour s prencubaton wth a much hgher DFO concentraton was needed when the chelator was appled extracellularly. Snce n the present study we have demonstrated that Ca overload develops as a result of the oxdatve njury caused by H2O 2, we have nvestgated the possble contrbuton to the Ca loadng due to the actvaton of Na 1 / H exchange leadng to ncreased [Na ]. Such an n- 1 crease n [Na ] could possbly subsequently nduce a Fg. 6. The contractle actvty (upper traces) and Ca transents (lower 1 decrease of Na -dependent Ca efflux durng dastole traces) n 26-day-old spontaneously beatng cultured adult gunea-pg 1 and/ or an ncreased Na -dependent Ca nflux durng cardomyocytes prencubated for 2 h n 1 mm DFO n Tyrode s soluton 1 (A), after applcaton of 100 mm H O (n the presence of DFO) for 40 systole va Na / Ca exchange. 2 2 mn (B) and after washout for 20 mn n control Tyrode s soluton (C). Our data ndcate that although blockng the Na / H Smlar results were obtaned n another 3 preparatons. exchange by the hghly potent and selectve nhbtor 5 mm

7 M. Horackova et al. / Cardovascular Research 47 (2000) Fg. 7. The contractle actvty (upper traces) and Ca transents (lower traces) n 25-day-old spontaneously beatng cultured adult gunea-pg cardomyocytes after 30 mn of prencubaton wth 5 mm MIA (A) and upon a subsequent addtonal applcaton of 100 mm H2O2 for 15 mn (B), 30 mn (C), and 45 mn (D). Note the ncreasng dastolc level of [Ca] n C and D; the dotted lne represents the basal level of [Ca] n the absence of H2O 2. Smlar results were obtaned n another 5 experments. MIA [16,17] dd sgnfcantly slow down the process of the References oxdatve njury by H2O 2, t dd not prevent t. These data ndcate that some of the Ca load may be related to the [1] Mannng AS, Hearse DJ. Reperfuson-nduced arrhythmas: mecha- 1 Na nflux va Na / H exchange, pontng out the nsm and preventon. J Mol Cell Cardol 1984;16: subsequent role of the Na / Ca exchanger. [2] Arroyo CM, Kramer JH, Dckens BF, Weglck WB. Identfcaton of free radcals n myocardal schema/ reperfuson by spn trappng However, t s lkely that most of the Ca overload s a 1 wth ntrone. DMPO FEBS Lett 1987;2: drect consequence of largely prolonged Na nflux va the [3] Garlck PB, Davs MJ, Hearse DJ, Slater TF. Drect detecton of free 1 2 Na channels that results n Ca accumulaton va Na - radcals n the reperfused rat heart usng electron spn resonance Ca exchange, as ndcated by our earler study [14]. We spectroscopy. Crc Res 1987;51: have demonstrated that both the ncreased APD and the [4] Shlafer M, Brosamer K, Forder JR, Smon RH, Ward PA, Grum CM. contractle force nduced by H O could be fully blocked Cesum chlorde as a hstochemcal marker of hydrogen peroxde n 2 2 reperfused schemc hearts. J Mol Cell Cardol 1990;22: by tetrodotoxn. The secondary effect of the H2O2 produc- [5] Vandeplassche G, Hermans C, Thone F, Borgers M. Mtochondral ng a fast shortenng of APD may be due to actvaton of hydrogen peroxde generaton by NADH-oxdase actvty followng KATP channels as suggested by Tokube et al. [25]. regonal myocardal schema n the dog. J Mol Cell Cardol Thus, our results demonstrate a hghly protectve capac- 1989;: ty of SIH aganst free-radcal-medated tssue damage and [6] Zweer JL, Flaherty JT, Wesfeldt ML. Drect measurement of free strongly suggest that ths chelator may be useful n the radcal generaton followng reperfuson of schemc myocardum. Proc Natl Acad Sc USA 1987;84: treatment of ron-medated oxdatve stress under varous [7] Hershko C. Control of dsease by selectve ron depleton: a novel pathologcal stuatons n the heart such as schema/ therapeutc strategy utlzng ron chelators. Balleere s ` Cln reperfuson. Hence, SIH deserves further nvestgaton n Haematol 1994;7: more complex expermental models n vvo. [8] Cable H, Lloyd JB. Cellular uptake and release of two contrastng ron chelators. J Pharm Pharmacol 1999;51: [9] Epsztejn S, Kakhlon O, Glcksten H, Breuer W, Cabantchk I. Acknowledgements Fluorescence analyss of the lable ron pool of mammalan cells. Anal Bochem 1997;248: [10] Ponka P, Borova J, Neuwrt J, Fuchs O, Necas E. A study of We would lke to thank Mr. P. Kng for hs edtoral ntracellular ron metabolsm usng pyrdoxal soncotnoyl hyassstance n preparng the manuscrpt. Ths work was drazone and other synthetc chelatng agents. Bochm Bophys Acta 1979;586: supported by grants from the New Brunswck Heart and [11] Zannnell G, Glcksten H, Breuer W et al. Chelaton and moblza- Stroke Foundaton and the Medcal Research Councl of ton of cellular ron by dfferent classes of chelators. Mol Pharmacol Canada (MT 4128) to Dr. M. Horackova. 1997;51:

8 536 M. Horackova et al. / Cardovascular Research 47 (2000) [12] Hermes-Lma M, Santos NCF, Yan J, Andrews M, Schulman HM, [19] Horackova M, Huang MH, Armour JA, Hopkns DA, Mapplebeck Ponka P. EPR spn trappng and 2-deoxyrbose degradaton studes C. Cocultures of adult ventrcular myocytes wth stellate gangla or of the effect of pyrdoxal soncotnoyl hydrazone (PIH) on OH ntrnsc cardac neurons from gunea-pgs: spontaneous actvty and formaton by the Fenton reacton. Bochm Bophys Acta pharmacologcal propertes. Cardovasc Res 1993;27: ;1426: [20] Horackova M, Byczko Z. Dfferences n the structural characterstcs [13] Schulman HM, Hermes-Lma M, Wang EM, Ponka P. In vtro of adult gunea-pg and rat cardomyocytes durng ther adaptaton antoxdant propertes of the ron chelator pyrdoxal soncotnoyl and mantenance n long-term cultures: confocal mcroscopy study. hydrazone (PIH) and some of ts analogs. Redox Rep 1995;1:373 Exp Cell Res 1997;237: [] Horackova M, Morash B, Byczko Z. Altered transarcolemmal Ca [14] Beresewcz A, Horackova M. Alteratons n electrcal and contrac- transport modfes the myofbrllar ultrastructure and proten metabotle behavor of solated cardomyocytes by hydrogen peroxde: lsm n cultured adult ventrcular cardomyocytes. Mol Cell Bopossble onc mechansms. J Mol Cell Cardol 1991;23: chem 2000;204: 33. [15] Duan J, Moffat MP. Potental cellular mechansms of hydrogen [22] Hermes-Lma M, Nagy E Ponka P, Schulman HM. The ron chelator peroxde-nduced cardac arrhythmas. J Cardovasc Pharmacol pyrdoxal soncotnoyl hydrazone (PIH) protects plasmd puc ;19: DNA aganst OH-medated strand breaks. Free Rad Bol Med [16] Karmazyn M, Moffat MP. Role of Na / H exchange n cardac 1998;25: physology and pathophysology: medaton of myocardal reperfu- [23] Karwatowska-Prokopczuk E, Czarnowska E, Besęwcz A. Iron son njury by the ph paradox. Cardovasc Res 1993;27: avalablty and free radcal nduced njury n the solated schaemc/ [17] Moffat MP, Karmazyn M. Protectve effects of the potent Na/ H reperfused rat heart. Cardovasc Res 1992;26: exchange nhbtor methylsobutyl amlorde aganst post-schemc [24] Khald MA, Ashraf M. Drect detecton of endogenous hydroxyl contractle dysfuncton n rat and gunea-pg hearts. J Mol Cell radcal producton n cultured adult cardomyocytes durng anoxa Cardol 1993;25: and reoxygenaton: s the hydroxyl radcal really the most damagng [18] Horackova M, Byczko Z, Mallet-Frotten L. Immunohstochemcal radcal speces Crc Res 1993;72: analyss of the adaptaton of adult gunea-pg cardomyocytes n [25] Tokube K, Kyosue T, Arta M. Effects of hydroxyl radcals on K ATP long-term cultures and n cocultures wth cardac neurons: a novel channels n gunea-pg ventrcular myocytes. Pflug Arch-Eur J model for studes of myocardal functon. Mol Cell Bochem Physol 1998;437: ;172: