Cardiopascular Research ELSEVIER CanEovpeular B3山6l204》1l5-l22 Wh球cbevicrcuenlocalac3 Preconditioning delays Ca2-induced mitochondrial permeability transition Laurent Argaud",Odile Gateau-Roesch",Lara Chalabreysse,Ludovic Gomez" Joseph Loufouat",Francoise Thivolet-Bejui",Dominique Robert Michel Ovize ASEE2 LgbaraloP径sl6 LyNurd Linire忙Cnde Brrar以Lae【&om Buckoiller93乃Ljoe.Fran "Lsdaroire a'Amie Psnologive./Misiaf Loais Podel.(on Frnce Digutawn d tiner f fminnon didlrake,Hipi Edoard Hemrol Ljoe.Franr Received23ugd20间3：ecerad is revisod frrm 150e起ter26 acrepled 5 Noember203 Te好rimary review2ds Abstract Objective:We imestigated whether scheic peecondponing (PC)may modity mnochonddal pemeability tronsitiou (MPD)pore opening.Methods:In pmel 1,New Zealand White rbils nderwen eierno imerventie (sham group)or 10 min ofischemia folloed by 5 min or repertusion,peecoded (PC)or not (C;coutrol)by one episode of's min of ischemin rad 5 min of repertasion.Rabburs were preeated by cither saline or the MPT poee inhihie cyelosperin A (CsA)ar iss noe-immmppressive derivative C29 (10 mpkg.IV bols)Hearts wee harvested and mitochondra isoled for further esminuced MPTsngCseve micro-electrode. In protocul 2.C and PC hearts uderwenl 30 min of ichemis ind 4 h of reperfasion They were preeated eiher by saline,CA or Ca29,as n pemocol 1.Infirct se w essessed by trphemytrerrazolium ond apoptoss by TUNEL stoining Resalts:In protooo 1,the Ca overlead reqeired induce MPT pore opening was significaly higher is PC than in C heiets.CA and C29 signifieantly sd the Ca overoad required tor MPT pore opening.In peoccol 2,mean utarct size averzged 25%of'the risk region Cs.Cs29 treated hears versus 15%in PC and 55%in cummk (Pe0.05 vs.C.P=rs vs.PC)Cardomyocyte apopksis was significandy reduced by PC and cyeloporn e2 ment wh a围6 a apopto候k如dex ot less than2 %a eit世er arcup versus田ore than11%acs.Cc通elusio:Ths2g声hut delayed opening of MPT pore my play a majee role in ischemie PC. 2003 European So:iety of Cardiolgy.Pablished by Elsevier B.V.All rghts reserved. Ky4 srdr Precendn3wne:《女tm4a山rc Minchondriv.Apoglacs:hecmis L.lnt女oduction (AP)collapse,uncoupling of the respiratory chain,efflux of Ca and release of'small proteins such as cytochrome c Ischemie preconditioning (PC)has been shown to limit 17].MPT is mastly induced hy Ca overload,especially both necrotie and apoptotic cardiomyocyte oll death 11-4]. when it is associated with adenine nucleotide deplction. Yet,the mechanim of this cardiprotoctaon remains unclear. increased inorganic phosphate cnneentration,oxidative Ischemia-reperfisian affects various key mitochondrial fi stress,all features of ischemia-reperfusion [8).MPT pore tions.including ATP production,Cahomeostasis,oxygen opening is inhibited by the immunasuppeessive drug cyclo derived fice radical production,and permeability transition sporin A (CA)that can protect the heart from ischemia- 5].Mitochoedral permealslity transition (MPT)is due to repertisioe inin vitro models [9-13]MPT appears to be a opening of a large porre in the iner mitnchondrial memhrane, critical event in the transition from reversible to irreversible mhcee structure remains incompleely known [6)MPT pore myocardial injury following an ischemic isult [14]. opening causes matrix swelling.inner membrane polential Whether ischemic PC alters MPT pore opening.and ahether this may explain its antinecrotic and antiapoptotic (omipotre atoe al:11-4-T71-70174:be477 effects remains urclear.Therefore.our objective was to 717A ktermine:(1)whcther milochondria.direetly isolaled from E-nnfavfnpe:luoL出aw丘L.u preconditioned hearts,display any alertion of Ca"in- niozroccefelcrua-wl垂M.0y匹l doced MPT pore opening.and,(2)whether in vivo phamma. 0008-63635.soe Buet tratkr C 2003 Errupeaa Socity of Cardialgy.P-liherl by Eleevir B.V.All rights nerved doi-10 101 candianes 3011.11.c0t
Preconditioning delays Ca2+-induced mitochondrial permeability transition Laurent Argauda , Odile Gateau-Roescha , Lara Chalabreysseb , Ludovic Gomeza , Joseph Loufouat a , Francoise Thivolet-Be´juib , Dominique Robert c , Michel Ovizea,* a INSERM E0226, Laboratoire de Physiologie Lyon-Nord, Universite´ Claude Bernard Lyon I, 8, Avenue Rockefeller, 69373 Lyon, France bLaboratoire d’Anatomie Pathologique, Hoˆpital Louis Pradel, Lyon, France c De´partement d’Urgence et de Re´animation Me´dicale, Hoˆpital Edouard Herriot, Lyon, France Received 25 August 2003; received in revised form 15 October 2003; accepted 5 November 2003 Time for primary review 22 days Abstract Objective: We investigated whether ischemic preconditioning (PC) may modify mitochondrial permeability transition (MPT) pore opening. Methods: In protocol 1, New Zealand White rabbits underwent either no intervention (sham group) or 10 min of ischemia followed by 5 min of reperfusion, preceded (PC) or not (C; control) by one episode of 5 min of ischemia and 5 min of reperfusion. Rabbits were pretreated by either saline or the MPT pore inhibitor cyclosporin A (CsA), or its non-immunosuppressive derivative Cs29 (10 mg/kg, IV bolus). Hearts were harvested and mitochondria isolated for further assessment of Ca2+-induced MPT using a Ca2+-sensitive micro-electrode. In protocol 2, C and PC hearts underwent 30 min of ischemia and 4 h of reperfusion. They were pretreated either by saline, CsA or Cs29, as in protocol 1. Infarct size was assessed by triphenyltetrazolium, and apoptosis by TUNEL staining. Results: In protocol 1, the Ca2+ overload required to induce MPT pore opening was significantly higher in PC than in C hearts. CsA and Cs29 significantly increased the Ca2+ overload required for MPT pore opening. In protocol 2, mean infarct size averaged 25% of the risk region in CsA/Cs29 treated hearts versus 15% in PC and 55% in controls ( P < 0.05 vs. C, P= ns vs. PC). Cardiomyocyte apoptosis was significantly reduced by PC and cyclosporin treatment with a mean apoptotic index of less than 2% in either group versus more than 11% in controls. Conclusion: This suggests that delayed opening of MPT pore may play a major role in ischemic PC. D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Preconditioning; Calcium (cellular); Mitochondria; Apoptosis; Necrosis 1. Introduction Ischemic preconditioning (PC) has been shown to limit both necrotic and apoptotic cardiomyocyte cell death [1– 4]. Yet, the mechanism of this cardioprotection remains unclear. Ischemia-reperfusion affects various key mitochondrial functions, including ATP production, Ca2+ homeostasis, oxygenderived free radical production, and permeability transition [5]. Mitochondrial permeability transition (MPT) is due to opening of a large pore in the inner mitochondrial membrane, whose structure remains incompletely known [6]. MPT pore opening causes matrix swelling, inner membrane potential (DWm) collapse, uncoupling of the respiratory chain, efflux of Ca2+ and release of small proteins such as cytochrome c [7]. MPT is mostly induced by Ca2+ overload, especially when it is associated with adenine nucleotide depletion, increased inorganic phosphate concentration, oxidative stress, all features of ischemia-reperfusion [8]. MPT pore opening is inhibited by the immunosuppressive drug cyclosporin A (CsA) that can protect the heart from ischemiareperfusion in in vitro models [9– 13]. MPT appears to be a critical event in the transition from reversible to irreversible myocardial injury following an ischemic insult [14]. Whether ischemic PC alters MPT pore opening, and whether this may explain its antinecrotic and antiapoptotic effects remains unclear. Therefore, our objective was to determine: (1) whether mitochondria, directly isolated from preconditioned hearts, display any alteration of Ca2+-induced MPT pore opening, and, (2) whether in vivo pharma- 0008-6363/$ - see front matter D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cardiores.2003.11.003 * Corresponding author. Tel.: +33-4-7877-7074; fax: +33-4-7877- 7175. E-mail addresses: laurent.argaud@chu-lyon.fr (L. Argaud), ovize@rockefeller.univ-lyon1.fr (M. Ovize). www.elsevier.com/locate/cardiores Cardiovascular Research 61 (2004) 115 – 122
116 L.Argond r ol/ovenouosrakir是ah6Jw0W长2 cological reproduction of this in vitro alteration might protect Protocol 1 Protocol 2 the ischemie-reperfused beart from necrosis and apoplosis. Sham L'trm CsA 2.Methods Cs29 2.1.Surgical preparation C-C8A C C:C29 The investigation con forms to the Guide for e Care and Lise af Lahortory Auimaly puhlished by the US Nationnal C-CsA ■ Institule of Health (NIH Publicalion No.85-23.revisod C-Cs29 1991. PCA■ Male New Zealand White rabbits,weighing 22-2.5 kg PC ■ were anesthetized by intrarmuscular injections of xylazine (5 P℃-Cs29■■ mg/kg)and ketamine (50 mgkg).as previously deseribed [15]An intmvens infison ofa mixture of xybzine (21- 50 Hgikg per min)and ketamine (40-100 ugkg per min) PC-Cs29■■H was then maintained throughout the experiment After a Fle 1 Experinel design.Potooel 1:ettect of ischemo PC and midline cervical incision,a tracheotomy was pertormed and cyceosorin us MPT pore opetiag All xus uderwesl a 30-mn amimals were ventilated with room air.A cannula was eapriiran priod Shan undewnE n icheni Paoounltiuned and inserted into the right irteral jugulr vein for administra- ererml groaps usderweat 10 mn of iochano fallewrad hy 5 min nf tion of'drugs and fluids and inlo the lefl carolid arkry for reperdon PC conssed of one eprsede ne$mir af ichemia and 5 mn of measurement of blood pressure.After an intravenous holus repernion.Cc6ogu单P,6 cond tiened gup,C,sfe自 A:C29.mu-inmatuppooiv:detivalive of C lie.pocif MPT administration of fentanyl (10 mkg),a left thoracotomy pone rpening irhhtrel:C.CA ard CC2,CA and Cevpretreated was performed in the fourth left intercostal space.Despite cnrmmk:mopecmvly.P'1kA and M-(x29.(xA and (2u-prrtrened the fact that opioids can protect the ischemic heart,admin- om们a,spectmely.Cs4,C290fe5n0B时saW istration of fentanyL likely did not alter the results since it buls al lhe begirtite uf experinb (amL Puloal 2:cfleel of was performed in a simalr way in all experimental groups. ichaic化dido红puia闭ind证ard apuploe.Al roapo underwesl 30 mis of bchertia and 4 h of ueperfasion.CsA.Cs29 oe The paicardium was openal and the heart expoed.A 3.0 iw@xD国V®he bmni球风cT由u3k size silk suture nttached to a small curved neede wis passed aund a marginal branch of the left circumflex ccronary artery.Both ends of the thread were passed thruugh a small ments indicating that,on oce hand.it was short crough not vinyl tube to formt a snare that could be tightened to ceclude to induee any ireversible myocardisl injury.and on the and loosened to reperfuse the artery.Body tempemture was other hand,because it was long enough to alter MPI pore momitored via an intraperiloocal thermomer and kept opening in this preparation Prior to this,control rabbits constant hy means ofa beating pod.Heant me and arterial underwent no intervention for 15 min (control groups,C: pressure were monitored contimuously thrughout the ex- #=25)while precanditianed received 5 mim of ischemia periment on a Goald recarder (Gould Inc..Cleveland followed by 5 min of reperfasicn (prcoeditioeed groups. OH).After the surgical procedure,a 15-min stabilization PC:w-25).At the onset of the 30-min cxpcriment.all perod was abserved. rabbits roecived an intrvenors bols of either saline (m n=10,C:为=9，PC:=9),yckssporin A (CsA,10 mg/kg) 2.2.otocol上：Ca induced MPT pore opening in (Sbam CsA C CsA,PC CsA:-8 per group)or cyclo prvendinoed hear sporin 29 (Cs29.10 mg/kg)(Sham-Cs29,C-Cs29.PC- Cs29;-8 per group).At the end of the experimen,bemts Protocol I investigated whether mitochoodria isolated were excised while still beuting,and mitochondria isolaled from preconditioned hearts display a dilferent palter of from the myocardium at risk fo further assessment ofC Candueed MPT pore opening than contrmols and,if so induced MPT pere openng. how it compares to that of cyclsperin-treated hearts. 2.2.2.Preparation of isolaed mitochondria 22.1.Experimental derign Preparation of mitochondria was adapeed from a previ- Seventy-six rablsts were ineluded in this protocol and ously deseribed procedure [16,17].All operations were randomly assigned to one of nine groups (Fig.1).Sham carried out in the cold Heart pieces (0.5-1.0 g)were placed animals underwent no ischemia fie 30 min (n-26).All isolation hulTer A白w或aiig70 mM sicrose,210nM control or preconditioned rahbits underwent a 10-min cor- mannisol,1 mM EDTA in 50 mM Tris/HCI pH 7.4.The onary artery occlusion followed by 5 min of reperfusion tissue was finely minced with scissors and then homogenized The 10-min duration was cbosen according to pilot experi- in the same buffer (10 ml buffer per g tissue).using
cological reproduction of this in vitro alteration might protect the ischemic-reperfused heart from necrosis and apoptosis. 2. Methods 2.1. Surgical preparation The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Male New Zealand White rabbits, weighing 2.2– 2.5 kg were anesthetized by intramuscular injections of xylazine (5 mg/kg) and ketamine (50 mg/kg), as previously described [15]. An intravenous infusion of a mixture of xylazine (20 – 50 Ag/kg per min) and ketamine (40 – 100 Ag/kg per min) was then maintained throughout the experiment. After a midline cervical incision, a tracheotomy was performed and animals were ventilated with room air. A cannula was inserted into the right internal jugular vein for administration of drugs and fluids and into the left carotid artery for measurement of blood pressure. After an intravenous bolus administration of fentanyl (10 mg/kg), a left thoracotomy was performed in the fourth left intercostal space. Despite the fact that opioids can protect the ischemic heart, administration of fentanyl, likely did not alter the results since it was performed in a similar way in all experimental groups. The pericardium was opened and the heart exposed. A 3.0 size silk suture attached to a small curved needle was passed around a marginal branch of the left circumflex coronary artery. Both ends of the thread were passed through a small vinyl tube to form a snare that could be tightened to occlude and loosened to reperfuse the artery. Body temperature was monitored via an intraperitoneal thermometer and kept constant by means of a heating pad. Heart rate and arterial pressure were monitored continuously throughout the experiment on a GouldR recorder (Gould Inc., Cleveland, OH). After the surgical procedure, a 15-min stabilization period was observed. 2.2. Protocol 1: Ca2+-induced MPT pore opening in preconditioned heart Protocol 1 investigated whether mitochondria isolated from preconditioned hearts display a different pattern of Ca2+-induced MPT pore opening than controls and, if so, how it compares to that of cyclosporin-treated hearts. 2.2.1. Experimental design Seventy-six rabbits were included in this protocol and randomly assigned to one of nine groups (Fig. 1). Sham animals underwent no ischemia for 30 min (n = 26). All control or preconditioned rabbits underwent a 10-min coronary artery occlusion followed by 5 min of reperfusion. The 10-min duration was chosen according to pilot experiments indicating that, on one hand, it was short enough not to induce any irreversible myocardial injury, and on the other hand, because it was long enough to alter MPT pore opening in this preparation. Prior to this, control rabbits underwent no intervention for 15 min (control groups, C: n = 25), while preconditioned received 5 min of ischemia followed by 5 min of reperfusion (preconditioned groups, PC: n = 25). At the onset of the 30-min experiment, all rabbits received an intravenous bolus of either saline (sham: n = 10, C: n = 9, PC: n = 9), cyclosporin A (CsA, 10 mg/kg) (Sham-CsA, C-CsA, PC-CsA: n = 8 per group) or cyclosporin 29 (Cs29, 10 mg/kg) (Sham-Cs29, C-Cs29, PCCs29; n = 8 per group). At the end of the experiment, hearts were excised while still beating, and mitochondria isolated from the myocardium at risk for further assessment of Ca2+- induced MPT pore opening. 2.2.2. Preparation of isolated mitochondria Preparation of mitochondria was adapted from a previously described procedure [16,17]. All operations were carried out in the cold. Heart pieces (0.5 – 1.0 g) were placed in isolation buffer A containing 70 mM sucrose, 210 mM mannitol, 1 mM EDTA in 50 mM Tris/HCl pH 7.4. The tissue was finely minced with scissors and then homogenized in the same buffer (10 ml buffer per g tissue), using Fig. 1. Experimental design. Protocol 1: effect of ischemic PC and cyclosporin on MPT pore opening. All groups underwent a 30-min experiment period. Sham underwent no ischemia. Preconditioned and control groups underwent 10 min of ischemia followed by 5 min of reperfusion. PC consisted of one episode of 5 min of ischemia and 5 min of reperfusion. C, control group; PC, preconditioned group; CsA, cyclosporin A; Cs29, non-immunosuppressive derivative of CsA (i.e. specific MPT pore opening inhibitor); C-CsA and C-Cs29, CsA and Cs29-pretreated controls; respectively, PC-CsA and PC-Cs29, CsA and Cs29-pretreated preconditioned, respectively. CsA, Cs29 or saline was injected as an IV bolus at the beginning of experiments (arrows). Protocol 2: effect of ischemic PC and in vivo cyclosporin on infarct size and apoptosis. All groups underwent 30 min of ischemia and 4 h of reperfusion. CsA, Cs29 or saline was injected as an IV bolus at the beginning of experiments (arrows). 116 L. Argaud et al. / Cardiovascular Research 61 (2004) 115–122
L.Argoud et al /Carduurvier Rreari 51 Chy 115-122 117 successively a Kootes tissue grinder and a Potter Elvejem retum of extramitochondrial Ca coocentration to near The homnogenate was centrifuged at 1300 x g for 3 min.The haseline level.Folkwing sufficient Ca loading.extra supernalant was poured through choesocloth amd ocntriligod mitocondrial Ca'oonocntration abruplly inereses indi- m10,川ex装r1 D min.The mitochondrial pellet wa8 cating a massive release of Ca by mitochondris due to tesuspended in isolation bufer B containing 70 mM aucrose MPT pote opening (Fig 2).The amount of Ca nocessary 210 mM maarnitol,(1.1 mM EDTA in 50 mM Tris/HC pH to tripger this mssive Ca release is usod here ns an 7.4.After aliquots were removed for protein measurements indicatoe of the suseeptahility of MPT pore to Caowverload. the mitnchondna (hy alinots off me proeeins)were washed in isolarion buffer B,centrifaeed at f8o0 x g for 1D min and 2 2.4.Elermon micrmenyy stored as pellets on ice prior to MPT pore opening experi- Electron microsoopy was performed either ar the end of ments.Protein content was routinely assayed according to the pre-incubation period (ie.before Ca"loading).or at the Gornall's procedure using bovine serum albumin as a stan- end of the experimen,following either no intervention (to dard [18].Purity and integrity of isolated mitochoedriawere address alteration of the preparaiorCaoveroad(Le. assessed by messuring specific activilies of monoamine after MPT pore openingh Under cuch of these throe exper- oxikase (EC 1.4.3.4)as an ouler membrane marker enyme. imental conditions,samples of mitochondria were fixod lir and cytochrome c oxidase (EC 1.9.3.1),as an inner mem- 2 h in 2%glutaralkhyde.100 M phoephale bulTer,pH 7.4 e marker1eL1920月 and pastfixcd in 1%%osmium tetroxyde.Dehydraation was performed in a series of ethsnol and propylene oxide 2.2.3.Ca"'induced MPT pore opening extractions,before sample embedding in Epon. MPT pure opening was assessed following in vitro Ca+ overload.Isolated mitochondria (6 mg proceins)were ss 2.3.Prorocol 2:cardioprorective effects of MPT pore pended in 100 ul buffer B.and added in 900 ul of buffer C a平g加hibiton识o (150 mM sucrose,50 mM KCl,2 mM KH,PO.5 mM succin acid to 20 mM Tris HCl pH 74)within a Teflon Based on the in vitro results of protocol 1.regarding the chamber equipped with a Ca-selective microelectrode,in respective MPT pore opening profiles of ischemnic PC and conjunction with reference electrode [21.221 Modifications cyclosporin.we perfirmed proocol 2 to compare the anti- of the medium (ie.extm-mitochondrial)Ca"coccentrtion necrotic and antiapoptotic effects of both types of interven- were continuously recorded using a custom made Syn- tion in the in situ mhbit heart. chronie software.Mitochondria were gently stirred for 1.5 min.At the end of the pre-incuhation period,20 uM 2 3.1.Experimental derign CaCl,administration was performed every 60 s.As depicted Fifty -nine rahbits were inclded in this protocol and in Fig.2.each 20 uM Cacl administration was performed randomly assigned to ane of six groups (Fig.1).All animals as a peak of extrammitocbondrial Ca concentration.Ca'is underwent a 30 min prolonged coronary artery occlusion then very rapidly taken up by the mitochondria resulting in a followed by 4 h of reperfusion.Prior to this,rabbits underwen cither no intervention (eontrol groups.=31) or PC by 5 min ischemia fillowed by 5 min of reperfusion Sham (precanditioned groups n=28)All rahhits reeeived an intravenou%olus of cither saline(Cr=1之，and PC =12)cyclsporin A (CsA,10 mg/kg)(C-CsA:n=12 and PCC-CsA:=9)or cyclosporin 29 (Cs29,10 mg/kg)(C- Cx29:n=7 and PC-Cs2gw=T月，13 min hefore the pro longed ischemia In these six groups,hearts were harvested NPT go at the end of the 4-h reperfusion period for further assess. osening ment of infiret sice and myocardial apoplosis (this latker lechnique was performed in seven animals per group) 5n, 2.3.2.Aru af risk wd infurct sic determimation At the end of the 4-h reperfusion.the coreary artery was briefly reoccluded and 0.5 mg/kg Uniperse blue pigment (Ciba-Geigys.Hawthome NY)was anjected intvenously to delineate the in vivo aren at risk (AR) Fig.2.Ca''-induood MPT poce epening.Typical example of a MPT poee as previously described [23].With this technique,the eing nour电it四2h国on onatmd peparatien af mitochondna.In the conrrol mrochondris (10 min of ischemi follwdd by previously non-ischemic myocardium appears blue.where S ma of rp=fioionla C'ovkad uf 180 M(aine pubes ef 20 M) as the previously ischemic myocardium (AR)remains uas negrinnd m induoe MPT pon opmning vs 1230 p'M(6 peloos nf unstained.Anesthetized rabbits were then euthanized by 20 gM Ca i the sham heart.C.comrrol group. an intravenous injection of 4 mEg KCL The beart was
successively a Kontes tissue grinder and a Potter Elvejem. The homogenate was centrifuged at 1300 g for 3 min. The supernatant was poured through cheesecloth and centrifuged at 10,000 g for 10 min. The mitochondrial pellet was resuspended in isolation buffer B containing 70 mM sucrose, 210 mM mannitol, 0.1 mM EDTA in 50 mM Tris/HCl pH 7.4. After aliquots were removed for protein measurements, the mitochondria (by aliquots of 6 mg proteins) were washed in isolation buffer B, centrifuged at 6800 g for 10 min and stored as pellets on ice prior to MPT pore opening experiments. Protein content was routinely assayed according to Gornall’s procedure using bovine serum albumin as a standard [18]. Purity and integrity of isolated mitochondria were assessed by measuring specific activities of monoamine oxidase (EC 1.4.3.4), as an outer membrane marker enzyme, and cytochrome c oxidase (EC 1.9.3.1), as an inner membrane marker enzyme [19,20]. 2.2.3. Ca2+-induced MPT pore opening MPT pore opening was assessed following in vitro Ca2+ overload. Isolated mitochondria (6 mg proteins) were suspended in 100 Al buffer B, and added in 900 Al of buffer C (150 mM sucrose, 50 mM KCl, 2 mM KH2PO4, 5 mM succinic acid to 20 mM Tris/HCl pH 7.4) within a Teflon chamber equipped with a Ca2+-selective microelectrode, in conjunction with reference electrode [21,22]. Modifications of the medium (i.e. extra-mitochondrial) Ca2+ concentration were continuously recorded using a custom made SynchronieR software. Mitochondria were gently stirred for 1.5 min. At the end of the pre-incubation period, 20 AM CaCl2 administration was performed every 60 s. As depicted in Fig. 2, each 20 AM CaCl2 administration was performed as a peak of extramitochondrial Ca2+ concentration. Ca2+ is then very rapidly taken up by the mitochondria resulting in a return of extramitochondrial Ca2+ concentration to near baseline level. Following sufficient Ca2+ loading, extramitochondrial Ca2+ concentration abruptly increases indicating a massive release of Ca2+ by mitochondria due to MPT pore opening (Fig. 2). The amount of Ca2+ necessary to trigger this massive Ca2+ release is used here as an indicator of the susceptibility of MPT pore to Ca2+ overload. 2.2.4. Electron microscopy Electron microscopy was performed either at the end of the pre-incubation period (i.e. before Ca2+ loading), or at the end of the experiment, following either no intervention (to address alteration of the preparation), or Ca2+ overload (i.e. after MPT pore opening). Under each of these three experimental conditions, samples of mitochondria were fixed for 2 h in 2% glutaraldehyde, 100 M phosphate buffer, pH 7.4 and postfixed in 1% osmium tetroxyde. Dehydratation was performed in a series of ethanol and propylene oxide extractions, before sample embedding in Epon. 2.3. Protocol 2: cardioprotective effects of MPT pore opening inhibition in vivo Based on the in vitro results of protocol 1, regarding the respective MPT pore opening profiles of ischemic PC and cyclosporin, we performed protocol 2 to compare the antinecrotic and antiapoptotic effects of both types of intervention in the in situ rabbit heart. 2.3.1. Experimental design Fifty-nine rabbits were included in this protocol and randomly assigned to one of six groups (Fig. 1). All animals underwent a 30 min prolonged coronary artery occlusion followed by 4 h of reperfusion. Prior to this, rabbits underwent either no intervention (control groups; n = 31), or PC by 5 min ischemia followed by 5 min of reperfusion (preconditioned groups; n = 28). All rabbits received an intravenous bolus of either saline (C: n = 12, and PC: n = 12), cyclosporin A (CsA, 10 mg/kg) (C-CsA: n = 12 and PC-CsA: n = 9) or cyclosporin 29 (Cs29, 10 mg/kg) (CCs29: n = 7 and PC-Cs29: n = 7), 15 min before the prolonged ischemia. In these six groups, hearts were harvested at the end of the 4-h reperfusion period for further assessment of infarct size and myocardial apoptosis (this latter technique was performed in seven animals per group). 2.3.2. Area at risk and infarct size determination At the end of the 4-h reperfusion, the coronary artery was briefly reoccluded and 0.5 mg/kg Uniperse blue pigment (Ciba–GeigyR, Hawthorne, NY) was injected intravenously to delineate the in vivo area at risk (AR), as previously described [23]. With this technique, the previously non-ischemic myocardium appears blue, whereas the previously ischemic myocardium (AR) remains unstained. Anesthetized rabbits were then euthanized by an intravenous injection of 4 mEq KCl. The heart was Fig. 2. Ca2 +-induced MPT pore opening. Typical example of a MPT pore opening recording in one sham and one control preparation of isolated mitochondria. In the control mitochondria (10 min of ischemia followed by 5 min of reperfusion), a Ca2 + overload of 180 AM (nine pulses of 20 AM) was required to induce MPT pore opening vs. 320 AM Ca2 + (16 pulses of 20 AM Ca2 +) in the sham heart. C, control group. L. Argaud et al. / Cardiovascular Research 61 (2004) 115–122 117
g 上AdTd/Ga山masealir Beream由A利0fi-2 ecised and cut into five to six 2 mm thick transverse slices. CsA and its non-immunosuppressive derivative [Me-De parallel to the atrioventricular groove.After removing right cyclosporin (cyclosporin 29,Cs29)were used either in ventricular tissue,cach hearl slices was weighed.The basal iv0(10kg1 V)or in vitru0.25.00,1,2,d5 surface of cach slice was pholographed for later measure- uM).CsA and C329 were dissolvod in clhanol and ment of'the AR.Each shce was then incubaled for 15 mim diluted in bulfer C prioe to utilization.in oeder to obtain in a 1%solution of triphenylterazolium chlorde at 37 "C for cach dilution the equivalent of 2.5 pl pure etlanol in to differentiate infaretod (pale)from viable (hrick rod) 1 mil for in vitro experiment.The effeet of this amount myocardial area [24].The slces were then rephotographed of pure ethanol was evaluated in the sme conditioes. Fnlarged projections of these slices were traced for deter- For in vivo use,CsA gnd (s29 were dissolved in a minarion of the boundaries of'the AR and area of'necrosis mixture of Cremophor FI.(polyethoxylnted enstor oil) (AN).Extent of the AR and AN was quantified by with ethanol-91%. computerized planimetry and comrected for the weight of the tissue slices.Total weights of the AR and the AN were 2.5.Smmtistical analysis then calculated and expressed in grams and as per of total keft ventricule (LV),and of the AR weight,respec- Comparisons between groups were performed using one- tively.We decided prospectively that bearts with a risk way analysis of varianoe (ANOVA)and,when a significant regian less than 10%of'the LV weight woukd be excluded fvalue was obtamed.mcans were compared using a from the study. Tuckey's lest.Dulferenoes m the relationship betwoen infaret size and area at risk were evaluated by analysis of covari 2.3.3.Myocardial apoptasts ance (ANCOVA)and post boc Tuckey's test,with infarct The TUNEL assay uses a terminal deoxynucleotidyl size as the dependent variable and area at risk as the transferase (TdT)to label free 3'Oll ends in geromic covariant.All values are expressed as mean+standard emor DNA.and thus localizes and assesses cells undergoing (S.E.M).Statistical sigrificance was defined as a value of DNA fragmentation [25].Freshly frozen non-ischemic and Pc0.05 ischemic myocardial samples were cut (5 um thickness) mounted on silanized glass slides.fixed with 4%parafor maldehyde for 1 h at mom temperature.The soctions were 3.Results washed in PBS.Endogenous peroxide activity was quenched by a 30-min incuhation in 3%hydrogen peroxide One hundred forty-four mhhits were included in the in methanol a room temperature.The heart slices were present study:80 in prutocol 1 and 64 in peotocol 2. washed in PBS,and permeabilized for 2 min at 4 C in Nine rabbits were excluded:four in protocol I,because 0.1%triton X-100 in 0.1%sodiumm citrute (Sigma).Pro- of technical failure during evaluation of MPT pore teins were removed from the tissue sectioes hy incubation opening.and five in protocol 2 (three because of cardio for 30 min at 37 'C in 20 Hg'ml proteinase K (Sigma genic shock during sustained ischemia or uexplained [ONA fraprents in the1 issue 83 ctions were determi司si吧 death during reperlision snd twe because of a smaller in situ oell death detoction kit (Bochringer Mannheim risk region)Results ae then presented for the remainng Switzerland).Afer washing in PRS,sections were incutut- 135 rabhitx od for 60 min at 37 C in a humid chamher with the TUNFI reaction mixtre supplied by the kit amnd containing TdT and .1 Protocnl !Ca"-indsscvl MPT pawre apening in fluorescein-dlTP During this incubation step,TdT cata- pecnndirioed learr lyzes the attachment of fluorescein-dlTP tn the fiee 3'OH ends in the DNA.After washing with PBS,coverslips were .11 Isolated mitachondria preparanom mounted.Slides incubated without TdT were used as In all cases,mitochondrial suspensions exhibited high negative controls Slides were examined with a Leica moncamine oxidase and cytochrome c oxxase spccific fluoresoence microscope.For each slide,10 sparate fields actives (i.e.spccific mitochondrial marker enzymes)(data were analyzed by two independem observers using a x 40 not shown).The quality of'the preparation was firther objeclive.Cardiomyocytes with gromn nuckar loorescence demonstrated by clctron microscopy.Fig.3A depiets were definod as TUNEL positive cells.The mean perocnt- isolated mkochondria suspended in bulTer C.ic.just e of TUNEL positive cardiomyoeytes wis enleulated and before Ca londing Clearly.mitochondris display intact expressod as the number of'TUNEL positive cells relative to membranes and dense matrix space Comparable mor- the total number of'myocytes (nucleik phology wis obsarved in the sham proup at the end of the proocol,indicating that the preparation did not alter 2.4.Chemicals over the time of the experiment (data nat shown).In contrast.following Ca-induced MPT pore opening. Both fomms of cyclospurin used in the present study most mitochondria urderwent dramatic morphological were a generous gift of Novartis (Basel,Switzerland) changes,as we observed large swollen mitochondria
excised and cut into five to six 2 mm thick transverse slices, parallel to the atrioventricular groove. After removing right ventricular tissue, each heart slices was weighed. The basal surface of each slice was photographed for later measurement of the AR. Each slice was then incubated for 15 min in a 1% solution of triphenyltetrazolium chloride at 37 jC to differentiate infarcted (pale) from viable (brick red) myocardial area [24]. The slices were then rephotographed. Enlarged projections of these slices were traced for determination of the boundaries of the AR and area of necrosis (AN). Extent of the AR and AN was quantified by computerized planimetry and corrected for the weight of the tissue slices. Total weights of the AR and the AN were then calculated and expressed in grams and as percentage of total left ventricule (LV), and of the AR weight, respectively. We decided prospectively that hearts with a risk region less than 10% of the LV weight would be excluded from the study. 2.3.3. Myocardial apoptosis The TUNEL assay uses a terminal deoxynucleotidyl transferase (TdT) to label free 3VOH ends in genomic DNA, and thus localizes and assesses cells undergoing DNA fragmentation [25]. Freshly frozen non-ischemic and ischemic myocardial samples were cut (5 Am thickness), mounted on silanized glass slides, fixed with 4% paraformaldehyde for 1 h at room temperature. The sections were washed in PBS. Endogenous peroxide activity was quenched by a 30-min incubation in 3% hydrogen peroxide in methanol at room temperature. The heart slices were washed in PBS, and permeabilized for 2 min at 4 jC in 0.1% triton X-100 in 0.1% sodium citrate (SigmaR). Proteins were removed from the tissue sections by incubation for 30 min at 37 jC in 20 Ag/ml proteinase K (SigmaR). DNA fragments in the tissue sections were determined using an in situ cell death detection kit (Boehringer MannheimR, Switzerland). After washing in PBS, sections were incubated for 60 min at 37 jC in a humid chamber with the TUNEL reaction mixture supplied by the kit and containing TdT and fluorescein-dUTP. During this incubation step, TdT catalyzes the attachment of fluorescein-dUTP to the free 3VOH ends in the DNA. After washing with PBS, coverslips were mounted. Slides incubated without TdT were used as negative controls. Slides were examined with a LeicaR fluorescence microscope. For each slide, 10 separate fields were analyzed by two independent observers using a 40 objective. Cardiomyocytes with green nuclear fluorescence were defined as TUNEL positive cells. The mean percentage of TUNEL positive cardiomyocytes was calculated and expressed as the number of TUNEL positive cells relative to the total number of myocytes (nuclei). 2.4. Chemicals Both forms of cyclosporin used in the present study were a generous gift of NovartisR (Basel, Switzerland). CsA and its non-immunosuppressive derivative [Me-Ile4 ]- cyclosporin (cyclosporin 29, Cs29) were used either in vivo (10 mg/kg, IV) or in vitro (0.25, 0.50, 1, 2, and 5 AM). CsA and Cs29 were dissolved in ethanol and diluted in buffer C prior to utilization, in order to obtain for each dilution the equivalent of 2.5 Al pure ethanol in 1 ml for in vitro experiment. The effect of this amount of pure ethanol was evaluated in the same conditions. For in vivo use, CsA and Cs29 were dissolved in a mixture of Cremophor EL (polyethoxylated castor oil) with ethanol-94%. 2.5. Statistical analysis Comparisons between groups were performed using oneway analysis of variance (ANOVA) and, when a significant F value was obtained, means were compared using a Tuckey’s test. Differences in the relationship between infarct size and area at risk were evaluated by analysis of covariance (ANCOVA) and post hoc Tuckey’s test, with infarct size as the dependent variable and area at risk as the covariant. All values are expressed as mean F standard error (S.E.M.). Statistical significance was defined as a value of P < 0.05. 3. Results One hundred forty-four rabbits were included in the present study: 80 in protocol 1 and 64 in protocol 2. Nine rabbits were excluded: four in protocol 1, because of technical failure during evaluation of MPT pore opening, and five in protocol 2 (three because of cardiogenic shock during sustained ischemia or unexplained death during reperfusion and two because of a smaller risk region). Results are then presented for the remaining 135 rabbits. 3.1. Protocol 1: Ca2+-induced MPT pore opening in preconditioned heart 3.1.1. Isolated mitochondria preparation In all cases, mitochondrial suspensions exhibited high monoamine oxidase and cytochrome c oxidase specific actives (i.e. specific mitochondrial marker enzymes) (data not shown). The quality of the preparation was further demonstrated by electron microscopy. Fig. 3A depicts isolated mitochondria suspended in buffer C, i.e. just before Ca2+ loading. Clearly, mitochondria display intact membranes and dense matrix space. Comparable morphology was observed in the sham group at the end of the protocol, indicating that the preparation did not alter over the time of the experiment (data not shown). In contrast, following Ca2+-induced MPT pore opening, most mitochondria underwent dramatic morphological changes, as we observed large swollen mitochondria, 118 L. Argaud et al. / Cardiovascular Research 61 (2004) 115–122
【.gond et al/Cmiueernier8bi0fi-f护 19 Pig 4.avrlead reqpind fre MPT peee npering in pcandiimnd n cycospori-td hearts Cooverlood squired for MPT pere upering in prpol I perest of sh valoIe he ourirl gruap. Ca''ewerload angrired for MPT pore opming was sgnifkcanty reduced m va alars atimrals PC irxrsed Cr"kad lo neg shmn valsk Minchandri inolated fren animk trealad with cither CcA re Ce2 were partkculry是iston to Ca·overloed.Full ba,n3 enrcormcn eyC-pndc法中sdeC9 priralod nheib.C. cormt PC pecordnane.a5s.mpn形s C pcn形 PC. 3.1.2.Fecd f irchemin-repurferiun o Ca-induved MPT 飞Wwn男 In the sham group.the amount of Caz required to open the MPT pore sernged 3018+20 M.This Caz overload was significantly reduced in the control group,averaging 165+22 uM (Pc0.05 vs.sham).In preconditioned hearts.the Ca overload required for MPT pore opening significantly increased when compared to controls,aver aging 300t34 uM (P<0.05 vs.control,P-ns vs.sham) 1 um (Fig 4). In all groups.in vivo pretreatment by CsA or Cs29 resulted in a significant inrease in the Ca overload Fig.3.Miuchondri momphulngy fulrwing Ca indced MPT pore required to induce MPT pore opening (Fig.4).There wis operig Electon microscogy condmmed the itegrtty and prry of tilocadkiad fractius befuce Cad"ino MPT pore oping (Al. no difference among CsA and Cs29-treated groups. Fellwirg Ca"indeed MPT pone apening mitnchendria appeared wulen with dieppearrce of memhene integrty (H) with disniption of ouer memhrane and disappearance af the cristne (Fig 3B). We evaluated in vitro the effects of increased concen- tratioes of either CsA or its non-immunosuppressive de- tivative Cs29 on Ca'-indaoed MPT pore opening in mitochondria isolated from sham hearts.Exposure to ★ CsA during 1 min before the first 20 M Ca pulse. 2 using concentralions ranging fromn 0.25 to I RM.signili- cantly and dose dependently debyed Ca-induced MPT pore opening when comparod to untrented sham hearts PC C-C8A PC-C8A C-C829 PC-C829 (date no shown).This demonstmted that the abrupt Ca release was indeed due to MPT pore opening.The pres- ence of 2.5 ul ethanol.i.e.the amount required to dissolve percert of AR.As expected.PC sigificantly rediced infarct siae vs.the CsA.did not modify mitochoodrial Ca uptake and curttul proup (CL Siniarly,al ryulopuria-realod pourul (C-Co and C. C)or [C-A and PC-C29)developed 【clease. nfaret Dhan custrdls."Pe005 vs.C
with disruption of outer membrane and disappearance of the cristae (Fig. 3B). We evaluated in vitro the effects of increased concentrations of either CsA or its non-immunosuppressive derivative Cs29 on Ca2+-induced MPT pore opening in mitochondria isolated from sham hearts. Exposure to CsA during 1 min before the first 20 AM Ca2+ pulse, using concentrations ranging from 0.25 to 1 AM, significantly and dose dependently delayed Ca2+-induced MPT pore opening when compared to untreated sham hearts (data not shown). This demonstrated that the abrupt Ca2+ release was indeed due to MPT pore opening. The presence of 2.5 Al ethanol, i.e. the amount required to dissolve CsA, did not modify mitochondrial Ca2+ uptake and release. 3.1.2. Effect of ischemia-reperfusion on Ca2+-induced MPT pore opening In the sham group, the amount of Ca2+ required to open the MPT pore averaged 308 F 20 AM. This Ca2+ overload was significantly reduced in the control group, averaging 165 F 22 AM ( P < 0.05 vs. sham). In preconditioned hearts, the Ca2+ overload required for MPT pore opening significantly increased when compared to controls, averaging 300 F 34 AM (P < 0.05 vs. control, P= ns vs. sham) (Fig. 4). In all groups, in vivo pretreatment by CsA or Cs29 resulted in a significant increase in the Ca2+ overload required to induce MPT pore opening (Fig. 4). There was no difference among CsA and Cs29-treated groups. Fig. 4. Ca2 + overload required for MPT pore opening in preconditioned and cyclosporin-treated hearts. Ca2 + overload required for MPT pore opening in protocol 1 (as percent of sham values). In the control group, Ca2 + overload required for MPT pore opening was significantly reduced vs. sham animals. PC increased Ca2 + load to near sham values. Mitochondria isolated from animals treated with either CsA or Cs29 were particularly resistant to Ca2 + overload. Full bars, no pretreatment; empty bars, CsA-pretreated; striped bars, Cs29 pretreated rabbits. C, control; PC, preconditioned. *P < 0.05 vs. sham. y P < 0.05 vs. C. z P < 0.05 vs. PC. Fig. 5. Effect of cyclosporin and PC on infarct size. AN is expressed in percent of AR. As expected, PC significantly reduced infarct size vs. the control group (C). Similarly, all cyclosporin-treated control (C-CsA and CCs29) or preconditioned (PC-CsA and PC-Cs29) rabbits developed smaller infarct than controls. *P < 0.05 vs. C. Fig. 3. Mitochondria morphology following Ca2 +-induced MPT pore opening. Electron microscopy confirmed the integrity and purity of mitochondrial fraction before Ca2 +-induced MPT pore opening (A). Following Ca2 +-induced MPT pore opening, mitochondria appeared swollen with disappearance of membrane integrity (B). L. Argaud et al. / Cardiovascular Research 61 (2004) 115–122 119
13 L Argaud r al/Ga山awaiir Brreark Al0i-2切 exposed to aCaaverlad.Moreover,ischemic PC and the 14 MPT pore inhibitor cyclosporin,that causes a PC-like in 1 vitro pttem of Ca'-induoed MPT pore opening.prowvide a strong anlinecrolie and amiapoplotie protection when ad- 10 ministered in vivo 4.J Cr'-indaed MPT pore npening in preexwditinned JecFIN We used quantitative pocentiometrie nppronch to ad- dress the ssceptibility of the MPT pore to open following c4C心2生 心4代B2 Caloding.in purified mitocbondria that were directly isolated from in vivo inpurred myocardium [22.26.271.In F￥6C.ocyle upopouis TUNE且pootive can与ecytes in the vitro use of Cs29.that is devoid of the calkineurin-depen- rick regin.Full hix no gretreatnest:empy hex CA-perered dent immmosuppressive action of CsA.but conserves its efeets on mitochondrial cyckphalin D(i.e.highly specifie P5001a.C. fir the MPT pore),confirmed that the abrupt in vitro Caz relesse fiom tsolated mitochondris actually refleets MPT 32.Pn￥2 uref size amd wpumfosiv pore opening [28] Using a isolated rat heart peeparution,Ilausenloy et al. 3.2.1.Infarct size and Javadow et al.recently suggested that suppression of Heart rate and blood pressure were not significantly MPT pore opening may be important for PC [5,29].Others different among the six groups of animal (data not shownk repurted that in the settings of ischemia-reperfusion,MPT AR was compurable among the six groups of rabbits,with pore activity can be modulated by mediators of PC like mean value3 veraging24土3%，27土2%，32土2% mitocbondrial Kchannels,hest shock proteins or protein 29±3%，32±3%，and37±2%of the LV weight.in C kinase C [30-331 The present study is in agreement with C-CsA.C-Cs29.PC.PC-CsA and PC-Cs29.respectively these previous investigations.Yet,here for the first time, (P-ns among the six groups).PC signiticantly roduced mitochondria were directly isolated from in situ rabbit hearts infarct size that averaged 154%of the AR versus that had been preconditioned by in vivo ischemia.and 558%in the control group (P<0.05).Similarly.all features of ischemic PC (ie.limitation of apoptosis and cyclosporin-treated (CsA or Cs29)rahbits developed signif. necrosis)were assessed simultaneously in the same exper- icantly smaller infarcts than controls.AN of C CsA,C. imental preparaticn. C29,PC CsA and PC Cs.29er8d24±4%，26±6% We demonstrated that a single episode of reversible 24±6%，25±5%of the risk region,respectively(Pc0.0l ischemia fi.e.10 min)significantly alters Ca induced vs.control group:P-ns vs.PC group)(Fig.5).ANCOVA MPT pore opening.Ca"-induced MPT pore opening was confirmed this significant difference among icbemic pre- assessod follwing such reversible ischemic insult,in conditioned or eyelosporin-meed hearts on one hand,and order to avoid isolating mitochondrin from hererogeneously control hearts on the other hand.There wns no difference infarcted tissoe with a mixture of dead and still vinhle among preconditioned groups and cyelosporin-treated cardiomyocytes.After 30)min of ischemia and 4 h of groups. reperfusion,surviving candiamyocytes (i.e.mitochondria) come from the least ischemic part of the AR in control 3 2.2.Myocandial apoptasts bearts,but from a lirger and more severely ischemic nisk The peroentage of TUNEL positive cardiomyocytes in regicn in myocardium salvaged by PC:those two popul- the AR was significantly reduced in the PC group tions of milochondria are.therefore,not truly comparable. (1.9+0.9%%)when compared to the control group This problem can only be avoidod by exeising myocardium (118土4.2%)(Fie6).Similarl%,it average1.8±0.7% before my ireversible injury,as was performed in proloool I.8±06%06±05%a08±04%in C-CsA.PCsA 1 of'the present study. C-Cs29 and PC-Cs29 proups,respeetively (P.0.015 vs Mitochondria direetly isolated from preconditioned control) hearts required a significmtly higher Ca loading than controls to open the MPT pore.Cainduced MPT pore opening in mitochondria isolated from preconditioned henrts 4.Diseussion resembled that of mitocbondria isolated from cyclospurin- trealed coctrol bearts,with in both cases a significant delay In the present stody.we report for the first time that of pore opening.This was still true when Cs29.devoid of mitochondria directly isolated from in vivo precanditioned any action on cakineurin but specific for the MPT pore,was rabbit hearts display a delayed MPT pore opening when used.The larger dely observed in cyclosporin-trested
3.2. Protocol 2: infarct size and apoptosis 3.2.1. Infarct size Heart rate and blood pressure were not significantly different among the six groups of animals (data not shown). AR was comparable among the six groups of rabbits, with mean values averaging 24 F 3%, 27 F 2%, 32 F 2%, 29 F 3%, 32 F 3%, and 37 F 2% of the LV weight, in C, C-CsA, C-Cs29, PC, PC-CsA and PC-Cs29, respectively (P= ns among the six groups). PC significantly reduced infarct size that averaged 15 F 4% of the AR versus 55 F 8% in the control group ( P < 0.05). Similarly, all cyclosporin-treated (CsA or Cs29) rabbits developed significantly smaller infarcts than controls. AN of C-CsA, CCs29, PC-CsA and PC-Cs29 averaged 24 F 4%, 26 F 6%, 24 F 6%, 25 F 5% of the risk region, respectively (P < 0.01 vs. control group; P= ns vs. PC group) (Fig. 5). ANCOVA confirmed this significant difference among ischemic preconditioned or cyclosporin-treated hearts on one hand, and control hearts on the other hand. There was no difference among preconditioned groups and cyclosporin-treated groups. 3.2.2. Myocardial apoptosis The percentage of TUNEL positive cardiomyocytes in the AR was significantly reduced in the PC group (1.9 F 0.9%) when compared to the control group (11.8 F 4.2%) (Fig. 6). Similarly, it averaged 1.8 F 0.7%, 0.8 F 0.6%, 0.6 F 0.5% and 0.8 F 0.4% in C-CsA, PC-CsA, C-Cs29 and PC-Cs29 groups, respectively ( P < 0.05 vs. control). 4. Discussion In the present study, we report for the first time that mitochondria directly isolated from in vivo preconditioned rabbit hearts display a delayed MPT pore opening when exposed to a Ca2+ overload. Moreover, ischemic PC and the MPT pore inhibitor cyclosporin, that causes a PC-like in vitro pattern of Ca2+-induced MPT pore opening, provide a strong antinecrotic and antiapoptotic protection when administered in vivo. 4.1. Ca2+-induced MPT pore opening in preconditioned hearts We used a quantitative potentiometric approach to address the susceptibility of the MPT pore to open following Ca2+ loading, in purified mitochondria that were directly isolated from in vivo injured myocardium [22,26,27]. In vitro use of Cs29, that is devoid of the calcineurin-dependent immunosuppressive action of CsA, but conserves its effects on mitochondrial cyclophilin D (i.e. highly specific for the MPT pore), confirmed that the abrupt in vitro Ca2+ release from isolated mitochondria actually reflects MPT pore opening [28]. Using an isolated rat heart preparation, Hausenloy et al. and Javadov et al. recently suggested that suppression of MPT pore opening may be important for PC [5,29]. Others reported that, in the settings of ischemia-reperfusion, MPT pore activity can be modulated by mediators of PC like mitochondrial KATP + channels, heat shock proteins or protein kinase C [30 – 33]. The present study is in agreement with these previous investigations. Yet, here for the first time, mitochondria were directly isolated from in situ rabbit hearts that had been preconditioned by in vivo ischemia, and features of ischemic PC (i.e. limitation of apoptosis and necrosis) were assessed simultaneously in the same experimental preparation. We demonstrated that a single episode of reversible ischemia (i.e. 10 min) significantly alters Ca2+-induced MPT pore opening. Ca2+-induced MPT pore opening was assessed following such a reversible ischemic insult, in order to avoid isolating mitochondria from heterogeneously infarcted tissue with a mixture of dead and still viable cardiomyocytes. After 30 min of ischemia and 4 h of reperfusion, surviving cardiomyocytes (i.e. mitochondria) come from the least ischemic part of the AR in control hearts, but from a larger and more severely ischemic risk region in myocardium salvaged by PC: those two populations of mitochondria are, therefore, not truly comparable. This problem can only be avoided by excising myocardium before any irreversible injury, as was performed in protocol 1 of the present study. Mitochondria directly isolated from preconditioned hearts required a significantly higher Ca2+ loading than controls to open the MPT pore. Ca2+-induced MPT pore opening in mitochondria isolated from preconditioned hearts resembled that of mitochondria isolated from cyclosporintreated control hearts, with in both cases a significant delay of pore opening. This was still true when Cs29, devoid of any action on calcineurin but specific for the MPT pore, was used. The larger delay observed in cyclosporin-treated Fig. 6. Cardiomyocyte apoptosis. TUNEL positive cardiomyocytes in the risk region. Full bars, no pretreatment; empty bars, CsA-pretreated; striped bars, Cs29 pretreated rabbits. C, control; PC, preconditioned. *P < 0.01 vs. C. 120 L. Argaud et al. / Cardiovascular Research 61 (2004) 115–122
上Argoud et al/Carisucurvier Aroari利h0i5-f护 121 versus preconditioned hearts is likely related to a dose afforded comparahle nntinecrotic effect.The ntinecrotic effect,since cyclsporin blocks the MPT pere in a dose. effect of Cs29 clearly indicates that MPT pore opening is dependent mannet.It is possible that a smallr dose of CsA a key event in necrotc cardiomyocyie death.The fact that would still limit cell death and bloek Ca'-induced MPT eyelosporin and ischemic PC did not have any additive pore opening for a value of Ca load much closer to that of effect on infaret size may indireetly suggest that they both the preconditioned group.sach im approach might give a act on MPT pore opening. better estimate of the ivolvement of MPI pore opening in MPT pore opening is known to causse eylochrome e the proteerive effeet of ischemie PC and cyclosporin pre- release which aetivate downstream caspuses to further trearment.In addition,the spocifie role of several factors exccute DNA fragmentation,i.e.apoptotie cell death [11]. that influence MPT pore openine,including memhrane In apreement with previous stadies we report that PC pocential,pl and reuctive axygen species likely play a role dramatically reduces cardiomyocyte apoptosis[2-4].Cs29 道e present stu小y and deserve further investigations. and CsA also attenuated apoptosis.confirming that MPT These observaticns strongly suggest that iscbemic PC pro- pore opening is a key evenit of the death process consecutive tect the mitocbondria by influencing MPT pore opening. to prolonged myocardial ischemia-reperfusion injury [8,401. How ischemic PC might alter MPT pore opening Ischemic PC has been shown to limit production of oxygen- remains to be delermined,although it has boen proposod denved free radicals,increase cxpression of the antiapop- that activation of mitochondrial K chaneeis during lotie protein Be-2.deerease expression of the proapoplotic schemic PC would drop membrane polential.roduce protein Bax,or limit production of the secondary messenger mitochondrial Ca"uptake and thereby limit induction of ceramide,all factors known to directly affect MPT pore MPT pore opening [22,34].Ore might bypothesize that opening [4,39,41,42].Whether modification of one or matrix Ca concentration might be redaced in mitochon several of these factors by ischemic PC may explain the dria isolted from preconditioned hearts before in vitro mcdulation of MPT pare opening and the consecutive Ca loading.Using Indo-I fluorescence in isolated rat attenuation of apoptosis abserved in the present study hearts,Wang et al.reported that ischemic PC decreases requires further investigation.In any case,these in vivo mitochondrial Ca comcentration following 15 min of data closely purallel isolated mitochondria results.and global ischemia and 30 min of reperfusion [35.Whether demorstrate that ischemic PC alters Ca"-induoed MPT this applies to the present stidy is.however.uncertain pore opening and subsequent cnrdiomocyte apoptosis. since following 10 min of ischemia plus reperfusion,cell The resuhs of the present study indicate that PC affects (and mitochoodrial)Ca overload is usually very limited Cainduced MPT pore opening.and that inhihiting pore [36].Inonganic phosphate,protons,K,ATP,radical oxy- opening in vivo strongly protects the ischemic heart against gen species are modulators of the MPT pore,especially both recrotic and apoptotic cell death. under ischemia-reperfusio conditions.There is evidence that PC may preserve mitochondrial oxygen coesumpticn capacity ad ATP production,and limit the peoduction of Acknowledgements oxygen-derived frce radicals at reperfuasion 37-391 Whether modifications of one or several of'these fctors We thank the oCentre de Microscopie Applquee i la n preconditioned mitochondna might explain the delsyed Biologie Universi Claude Bernand.Lyon,Francen for Caindaced MPT pore opening abserved in the present allowing electron micrographs to be perfarmed,and snudy,requires farther investipations Novartiss (Basel,Switzerland)for providine cyclosporin. Technical assistance of Fahienne Lemme for clectme micro- 4.之，ng Ca-didP了one opening prieets she scopy is prtefully ncknowledged. ivo ischexuie B时 Because the pattern of Ca-induced MPT pore opening References in mitochondria isolated from cyclosporin-trealed hearts resembled that of procoeditioned mitocbondria,we docided II】a可yE,ern血s RB.Retmer KA.Pr6 nditoring4 ith ischem度 to investigate whether CsA would mimie in vivo ischemic adelay uf lhal iny in iscbie mpocanlia Cubus PC.i.c.reduee both mlard sine amnd apoplosis.The anti- 18%6T41114-6 2]Gomicb RA.Gradl DL.Zha JY.Eagler RL Paooerdtionnz it rabbut neerotic efleet of eyelsporin has previously beant repor erdiomyocytes.Role of pH vacbe prens ATPae,ad apeptmis tainly in isolared rat henrt peepamations -12)In the J1▣mtg19969T2到1-8 present study,we evtended this ohservation to the in vivo )ntA.adrnahan1.w1WC、Sws,w物le1.<o rabbit model of myocardial infarction.In addition,we report e pooo山ioning doere5 puptoois is rg bout n vivo.C山 that (1)reductian in infarct size was compsrable in pre- i国19房70%：ls03-04 [4 Moulk N.Englenan RM.Rouseo JA.Flck JE Dean D.Dos DK conditioned and cyclosporin-treated rabbits,(2)PC and eic peesonlilicting rlu apuplois by upregulaling asli. cyclosporin did not appcur to have a additive beneficial deatt ome Id-2 Cirreltion 1991:104H1134-75 effect on infirct size reduction,and (3)Cs29 and CsA SngD以kHL,BsG话.Yelon DM.Ithbrirg
versus preconditioned hearts is likely related to a dose effect, since cyclosporin blocks the MPT pore in a dosedependent manner. It is possible that a smaller dose of CsA would still limit cell death and block Ca2+-induced MPT pore opening for a value of Ca2+ load much closer to that of the preconditioned group; such an approach might give a better estimate of the involvement of MPT pore opening in the protective effect of ischemic PC and cyclosporin pretreatment. In addition, the specific role of several factors that influence MPT pore opening, including membrane potential, pH and reactive oxygen species likely play a role in the present study and deserve further investigations. These observations strongly suggest that ischemic PC protect the mitochondria by influencing MPT pore opening. How ischemic PC might alter MPT pore opening remains to be determined, although it has been proposed that activation of mitochondrial KATP + channels during ischemic PC would drop membrane potential, reduce mitochondrial Ca2+ uptake and thereby limit induction of MPT pore opening [22,34]. One might hypothesize that matrix Ca2+ concentration might be reduced in mitochondria isolated from preconditioned hearts before in vitro Ca2+ loading. Using Indo-1 fluorescence in isolated rat hearts, Wang et al. reported that ischemic PC decreases mitochondrial Ca2+ concentration following 25 min of global ischemia and 30 min of reperfusion [35]. Whether this applies to the present study is, however, uncertain, since following 10 min of ischemia plus reperfusion, cell (and mitochondrial) Ca2+ overload is usually very limited [36]. Inorganic phosphate, protons, K+ , ATP, radical oxygen species are modulators of the MPT pore, especially under ischemia-reperfusion conditions. There is evidence that PC may preserve mitochondrial oxygen consumption capacity and ATP production, and limit the production of oxygen-derived free radicals at reperfusion [37 – 39]. Whether modifications of one or several of these factors in preconditioned mitochondria might explain the delayed Ca2+-induced MPT pore opening observed in the present study, requires further investigations. 4.2. Delaying Ca2+-induced MPT pore opening protects the in vivo ischemic heart Because the pattern of Ca2+-induced MPT pore opening in mitochondria isolated from cyclosporin-treated hearts resembled that of preconditioned mitochondria, we decided to investigate whether CsA would mimic in vivo ischemic PC, i.e. reduce both infarct size and apoptosis. The antinecrotic effect of cyclosporin has previously been reported, mainly in isolated rat heart preparations [9 – 12]. In the present study, we extended this observation to the in vivo rabbit model of myocardial infarction. In addition, we report that (1) reduction in infarct size was comparable in preconditioned and cyclosporin-treated rabbits, (2) PC and cyclosporin did not appear to have a additive beneficial effect on infarct size reduction, and (3) Cs29 and CsA afforded comparable antinecrotic effect. The antinecrotic effect of Cs29 clearly indicates that MPT pore opening is a key event in necrotic cardiomyocyte death. The fact that cyclosporin and ischemic PC did not have any additive effect on infarct size may indirectly suggest that they both act on MPT pore opening. MPT pore opening is known to cause cytochrome c release which activate downstream caspases to further execute DNA fragmentation, i.e. apoptotic cell death [11]. In agreement with previous studies, we report that PC dramatically reduces cardiomyocyte apoptosis [2– 4]. Cs29 and CsA also attenuated apoptosis, confirming that MPT pore opening is a key event of the death process consecutive to prolonged myocardial ischemia-reperfusion injury [8,40]. Ischemic PC has been shown to limit production of oxygenderived free radicals, increase expression of the antiapoptotic protein Bcl-2, decrease expression of the proapoptotic protein Bax, or limit production of the secondary messenger ceramide, all factors known to directly affect MPT pore opening [4,39,41,42]. Whether modification of one or several of these factors by ischemic PC may explain the modulation of MPT pore opening and the consecutive attenuation of apoptosis observed in the present study requires further investigation. In any case, these in vivo data closely parallel isolated mitochondria results, and demonstrate that ischemic PC alters Ca2+-induced MPT pore opening and subsequent cardiomyocyte apoptosis. The results of the present study indicate that PC affects Ca2+-induced MPT pore opening, and that inhibiting pore opening in vivo strongly protects the ischemic heart against both necrotic and apoptotic cell death. Acknowledgements We thank the «Centre de Microscopie Applique´e a` la Biologie-Universite´ Claude Bernard, Lyon, France» for allowing electron micrographs to be performed, and NovartisR (Basel, Switzerland) for providing cyclosporin. Technical assistance of Fabienne Lerme´ for electron microscopy is gratefully acknowledged. References [1] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124 – 36. [2] Gottlieb RA, Gruol DL, Zhu JY, Engler RL. Preconditioning in rabbit cardiomyocytes. Role of pH, vacuolar proton ATPase, and apoptosis. J Clin Invest 1996;97:2391 – 8. [3] Piot CA, Padmanaban DD, Ursell PC, Sievers RE, Wolfe CL. Ischemic preconditioning decreases apoptosis in rat heart in vivo. Circulation 1997;96:1598 – 604. [4] Maulik N, Engleman RM, Rousou JA, Flack JE, Deaton D, Das DK. Ischemic preconditioning reduces apoptosis by upregulating antideath gene Bcl-2. Circulation 1999;100:II369 – 75. [5] Hausenloy DJ, Maddock HL, Baxter GF, Yellon DM. Inhibiting miL. Argaud et al. / Cardiovascular Research 61 (2004) 115–122 121
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