Universite de Montreal
Effets protecteurs du diltiazem et du clentiazem
dam le coeur ischhique normal et defaillant
Par Mario Tanguay
DCpartement de pharmacologie
Facultk de medecine
These presentee & la Facult6 des Ctudes superieures en vue de I' obtention du grade de Philosophite Doctor (Ph.D.) en phannacologie
Fbvrier 1996
Mario Tanguay, 1996 Acquisitions and Acquisitions et Bibliographic Services . servtces bibliographiques 395 Wellington Straet 395. rue Welling!on Ottawa ON KIA ON4 Oetawif ON K1AON4 Canada Canada
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Facul t6 des Ctudes supiirieures
Cette thhe intitulee:
Effets protecteurs du diltiazem et du clentiazem
dans le coeur ischhique normal et defaillant
prbsentee par:
Mario Tanguay
a Ct6 Cvaluee par un jury compose des personnes suivantes:
Gilles CailE ...... president du jury
Louis Dumont ...... directeur de recherche
Gilbert Blaise ...... codirecteur de recherche
Rene Cardinal ...... membre du jury
John G. King ma Jr ...... examinateur externe
Jean-Gilles Latour ...... reprbentant du doyen
Thhe acceptke le 12 juin 1996 Mtme si l'hypothemie et la cardioplt5gie procurent une protection significative en chirurgie cardiaque, une mortalit6 peroperatoire plus elevb est observee chez les insuffisants cardkques. Des etudes antirieures sugg5rent que les antagonistes du calcium de la famille des benzothkdpines procurent une protection myocardique B des doses non cardiodtipressives, ce qui pourrait favoriser leur utilisation en presence de defaillance cardiaque. L'objectif general de ce projet de recherche est donc d'ttudier les effets protecteurs du diltiazem et du clentiazem, en ajout i la cardioplkgie, dam des coeurs ischtmiques normaux et d6faillants.
Le diltiazem et son deriv6 chlore plus lipophile, le clentiazem, sont d'abord ttudies dam des coeurs de lapins, isolb et perfuses selon la mCthode de Langendorff. La pression ventriculaire gauche, le debit coronarien et la Mquence cardiaque sont kvaluts avant et aprh une ptriode d' ischtmie globale de 90 min associ6e i uue cardiopltgie froide avec ou sans antagoniste du calcium et LO4 M). L'addition de clentiazem M a la cardioplegie froide entraine une meilleure r6cuptration de la fonction contractile postischtmique alors que l'utiiisation d'une plus forte concentration n'entraine pas de bent fices fonctionnels apparents. L'ajout du diltiazem 5 la cardioplegie froide, ind6pendamment de la concentration utiliske, nn'pporte pas d'amklioration de la fonction cardiaque postischemique. Les effets protecteurs du clentiazem, qui sont observes i une concentration non cardiod6pressive, pourraient Cue li& i ses effets vasodilatateue coronariens. De plus, les proprikt& physicochimiques distinctes du clentiazem pourraient 2tre associh 2 des effets cytoprotecteun directs, permettant la preservation de 1' integrite membranaire.
Les effets protecteurs des benzothiaztpines sont ensuite kvalu& dans des coeurs isoles provenant de hamsters normaux et cardiomyopathiques de la lignke UM-X7.1 5g6s de plus de 200 joun (coeurs dkfaillants). L'ajout du diltiazem ou du clentiazem (loe8M) ii la cardiopl6gie fioide permet une meilleure r6cuperation de la fonction contractile des coeurs normaw suite une pCriode d' ischhie de 90 min. Dans les coeun dbfaillants, I'ajout de ces agents a la cardiopldgie froide n'appone pas de benefices lors de la reperfhion. L'administration de diltiazem ou de clentiazem (10d M) seul dam des coeurs non-ischbrniques rbv&leune atthation des effets vasodilatateurs coronariens en prhence de I' insuffisance cardiaque.
Afm de miewc catact6riser la sensibilite wdiaque et coronarienne a ces antagonistes du calcium en prhence d'insuffisance cardiaque. des courbes dose-reponse sont realistes. Les rbultats obtenus indiquent que I'insufisance cardiaque n'est pas associee une augmentation de la sensibilitk cardiaque (effets inotropes negatifs) au dilitiazem ou au clentiazem mais a une dbensibilisation coronarienne (diminution de leurs effets vaso- dilatateun). L'utilisation concornitante du L-NAMEou de l'indomkthacine suggtre que cette dtsensibilisation coronarie~en'est pas due a une alteration de la synthbe du monoxyde d'azote (NO) mais que des facteun prostanoides vasoconstricteurs pourraient Stre imp1 iques .
En conclusion, bien que les benzothiazepines soient des agents protecteun efficaces en ajou t a la cardioplkgie dam des coeurs ischCmiques normaux, ces molecules s'averent inefficaces en prdence d' insuffisance cardiaque. Nos travaux suggtrent donc que I'utilisation clinique de ces agents en protection myocardique peropBratoire serait moins approprik chez les patients qui presentent une deterioration de leur fonction cardiaque. Puisque la pene d1e€ficacit&du diltiazem et du clentiazem en presence d'insuffisance cardiaque pourrait s'expliquer par une alteration de Ieur activid vasodilatatrice coronarienne, il serait intkressant d'btudier les effets des antagonistes du calcium de deuxieme generation qui sont dot& d ' une plus grande vasost lectivitb. Table des matikes
Sommaire ...... iii
Liste des tableaux ...... ix
Liste des sigles et abkviations ...... xii
PRE- PARTIE: Introduction ...... 1
Chapitre 1: Ischdmie et protection myocardique perop6ratoire
1.1 Introduction ...... 2 1.2 Conskquences biochimiques et cellulaires de l'ischemie ...... 3 1.2.1 Alterations mttaboliques ...... 4 1.2.2 Alttrations ioniques ...... 6 1.2.3 AIterations membranaires ...... 7 1.3 Strategies de protection myocardique peropbratoire ...... 9 1.3.1 Cardioplkgie et hypothermie ...... 9 1.3.2 Additifs B la cardioplegie ...... 10
Chapitre 2: Antagonistes du calcium en protection myocardique
2.1 Introduction ...... 15 2.2 PropriCtks p harmacologiques ...... 16 2.2.1 Classification des antagonistes du calcium ...... 16 2.2.2 Mecanisme d'action ...... 16 2.2.3 Effets vasculaires et cardiaques des antagonistes du calcium . 18 2.2.4 SClectivid tissulaire des antagonistes du calcium ...... 22 2.3 Mbcanismes cardioprotecteurs des antagonistes du calcium ...... 24 2.3.1 Prevention de la surcharge calcique ...... 25 2.3.2 Diminution de la demande metabolique et pr6servation Cnergetique...... 26 2.3.3 Effeu antiperoxydants et prkervation de I'integrid membranaire ...... -27 2.3.4 Effeu sur les neunophiles ...... 29 2.3 .5 Effers vasodilatateurs ...... 30 2.4 Antagonistes du calcium et cardioplegie ...... 32 2.4.1 ~tudesexpCrimentales ...... 32 2.4.2 ~tudescliniques ...... 34 2.5 Choix des benzothiazipines cornme agents cardioprotecteurs ...... 35
Chapitre 3: Protection myocardique dam Ifinsuf€iance cardiaque
3.1 Introduction ...... -38 3.2 Protection myocardique peropCratoire chez les insuffisants cardiaques . 39 3.3 Antagonistes du calcium dans I' insuffisance cardiaque ...... 42
DEUXI~MEPARTIE: Contribution originale ...... 46
Hypothkes de travail et buts du projet de recherche ...... 47
Chapitre 4: Clenliazem. diltiazem and cold canlioplegia in isolated ischemic rabbit hearts: Relalion between additive cardwprotection. phyticochemicd properties. and preservation of myo canliaf lipid components
4.1 R6surnC ...... SO 4.2 Introduction ...... 52 4.3 Materiel et rnkthodes ...... 54 vii
4.4 R&ultats ...... 59 4.5 Discussion ...... 69 4.6 Bibliographic ...... 78
Chapitre 5: Resistance of the failing dystrophic hamster heart to the cardioprotective eflects of dilriazern and clentiizzem: evidence of coronary vascular dysfunctions
4.1 Resume ...... 88 4.2 introduction ...... 94 4.3 Materiel et methodes ...... 96 4.4 Resultzits ...... 100 4.5 Discussion ...... 107 4.6 Bibliographic ...... 114
C hapitre 6: Involvement of the nMc oxide and the cyclooxygenase pathways in the decreased coronary sensitivity to diltiazem and clentiazem in the failing hamster heart
6.1 Resum$ ...... 124 6.2 Introduction ...... 126 6.3 Matiriel et methodes ...... 128 6.4 Resultats ...... 132 6.5 Discussion ...... 143 6.6 Bibliographic ...... 154
TROISI~EPARTIE: Discussion g6n6rale ...... 163
C hapitre 7: Discussion g6n6rale ...... 164
7.1 Aspects m6thodologiques ...... 164 viii
7.2 Discussion de l'ensemble des rkultats ...... 170 7.3 Perspectives ...... 175
Remerciements ...... 206 Liste des tableaux
Tableau 4-1. Effect of clentiaem and diIn'aem added to cold cardioplegia on reperfucion mechanical recovery of isolated rubbit heart subjected to Wmin ischemia ...... 60
Tableau 4-2. Myocardial lipids and proteins at the end of reperfusion ...... 67
Tableau 5-1. Baseline hemdynamic parameters in nonnal and failing CMH hearts ...... 100
Tableau 61. Bml hernodynamic values of normal and failing beans ...... 132
Tableau 6-2. Median diltiazem and clentiaem concenrrarionr for coronary dilution and negative inotropic actions in normal and failing hearts ...... 134
Tableau 6-3. Endorhelid modulation of EC, values for coronary dilation evoked by diitiaem and clentiazem in normal and failing hearts . . 139
Tableau 1-1. Maximal coronary flow increase induced by diltiazem and clentiazem in normal and failing heans ...... 205 Liste des figures
Fgure 2-1. Structure chimique des beruothiidpines ...... 36
Figure 4-1. Effects of ciennnntzernand diltiaern added to cold cardioplegia on repemion recovery of coronary flow in rabbit heart subjected to Wminglobal ischemia ...... 62
Figure 4-2. Effects of cientz'atem and dikiazem added to cold cardioplegia on repemion recovery of heart rate in rabbit hemsubjected to 90- min global ischemia ...... 64
Figure 4-3. Relation between myocsrdic! tirnriruem and diltiuem ievek and postischemic mechanical impaimtent ...... 65
Figure 4-4. Effects of clentiazem and diitiazern added to cold cardioplegia on total myocardial calcium content ...... 68
Figure 5-1. Coronary flow changes following 90rnin global ischemia in noml and foiling CMH heam ...... 102
Figure 5-2. L M)P changes following 90-min global ischemia in normi and failing CMH hems ...... 102
Figure 5-3. Diltiarem and clentiaem added to coid cardioplegia: fleas on postischemic L WPin noml and failing CMH hearts ...... 104
Figure 5-4. DiItiazern and clentiafem added to coid cardioplegia: fleets on coronary flow at repemion in normof and failing CMH hearts ...... 104 figure 5-5. Coronary Md cardiac fleas of a 10 nM injbion of dikazern and clentiazem in noml and failing nunischemic CMH hearts ... 106
Eigure 6-1. Cumulative concentration-response curves of diitiazem and clentiuzem in normal and failing hearts ...... 133
Wgure 6-2. Effect of L-NAME (30 pM) on the coronary flow of normal and failing heans ...... 137
Figure 6-3. Effect of L-NAME on the cumulative concentration-response curves of diLtiazetn and clentiazem in normal and failing heam . . 138
Figure 6-4. Effect of indomethacin (lOpM) on the coronary flow of normal and failing heans ...... 14 1
Figure 6-5. Eflect of indomethacin on the cumulative concentration-responre curves of diln'uzern and clentiazem in normal andfaiiing hearts . . 142 Liste des sigles et abr6viations
AA acide arachidonique ATP adenosine aiphosphate CF coronary flow CMH cardiomyopathic hamster cm centim&res coll . collaborateurs DP diastolic pressure EGO concentration efficace m6d iane EDRF Endothelial-derived relaxing factor fig. figure h heures HR heart rate [Cw concentration inhibitrice mkdiane L-NAME NG-nitro-L-arginine mtthyl ester L-NMMA NG-monomCthy1-L-arginine LVDP l@ ventricular developed pressure LVP left ventricular pressure M molaire mM millirnolaue min minutes Ctm microns nM nanomo Iaire NO monoxyde d 'azote P probab ilitk PAF platelet-a~n*vatingfactor PG I, prostacycline SE, SEM erreur-type TxA2 thrornboxane A, A Sophie Les interventions chirurgicales cardiaques effectuees sous circulation extracorporelle cornportent g6dralement une pkriode d'ischkmie myocardique globale induite par le clampage aortique. Cette ischkmie ainsi que la reperfusion survenant a la fin de la chirurgie peuvent entralner des effets dtlCtkres sur les cellules myocardiques pouvant se traduire par des arythrnies et/ou des ddficits contractiles. Parmi les rnkcanismes irnpliquis dans I'apparition de ces ltsions mentionnons la production de radicaux libres de 1 'oxygene, la diminution des reserves Bnergetiques, I 'atteinte membranaire, la surcharge calcique, la vasoconstriction, etc.
Diverses techniques de protection myocardique furent ddveloppees dam le but de limiter les consequences nkfastes de I ' ischem ie peroperatoire. I1 slag it principalernent de
I'hypothermie, qui vise a abaisser la temperature myocardique. et de l'utilisation de solutions cardiopl6giques, crystalloides ou sanguines, qui permettent 1'arret Clectro- mhique du coeur. Cependant, malgre 1't tude de difftirentes solutions cardiopl6g iques et de divers modes d'administration, la meilleure mtithode de cardioprotection peroperatoire demeure controversb. De plus, les methodes usuelles de protection myocardique peuvent s'av6rer moins efficaces chez les ins~ff7sant.scardiaques. Une mortalit6 perophmire plus Clevk est en effet observtie dam cene population de patients.
11 es t donc necessaire de d6velopper d 'autres stratigies de protection myocardique ou encore d 'arnkliorer les procedures existantes. Plusieua substances ont Cd ajoutk aux solutions cardiopltgiques dmle but d'en ameliorer I'efficacitk. Le concept de I'addition des antagonistes du calcium a ces solutions est n6 du rdle central joud par le calcium dans I'ischbmie myowdique et la reperfusion. Ces agents limitent 1' influx calcique et prkvie~entainsi la surcharge
intracellulaire de calcium. De plus, des travaux andrieurs suggbrent que les antagonistes du calcium peuvent ruuire les dommages myocardiques relib B I'ischtmie et a la reperfusion par d'autres m&anismes: activi~vasodilatatrice coronarienne. effets sur les radicaux libres, effets protecteurs rnembranaires, etc. Cependant, les travaux effectuts jusqu'i maintenant indiquent que les antagonism du calcium amiliorent I'efficacite des solutions cardiopltgiques uniquement en normothermie et que leurs effets inotropes negatifs limitent leur utilisation en presence d' insuffisance cardiaque. L'objectif general de ce travail est de documenter les effets protecteurs du diltiazem et du clentiazem. en ajout a la cardiopltgie froide, dans des coeun ischtmiques normaux et defaillants.
En introduction nous rappellerons quelques notions portant sur I'ischhnie et la protection myocardique peroptratoire et celles de 1' util isation des antagonis tes du calcium dam ce contexte. La complexitt de la protection myocardique peroptratoire en prbence d'insuffisance cardiaque y est kgalement traitie. Le corps de I'ouvrage est constituC de trois articles portant respectivement sur ies effets protecteurs du diltiazem et du clentiazem en ajout B la cardiopltgie froide dans le coeur de lapin isolC, sur les effets protectem de ces agents dam le coeur de hamster normal et dbfaillant et finalement sur les facteurs qui pourraient moduler leur efficacite en prbence d'insuffisance cardiaque. PRE~REPARTIE
INTRODUCTION Chapitre 1
Ischhie et protection myocardique perop6atoire
1.1 Introduction
L1ischt5rnie myocardique se produit gMraIement i la suite d'une sthose coronarienne.
Une reperfusion pr6coce est aiors essentielle afin de rkduire l'etendue de la nCcrose tissulaire et de permeme la rkuptkation du myocarde ischkrnique (Przylenk et coll. 1986,
Reimer et dl. 19n). Ainsi, 11efficaci3de la restauration du debit sanguin myocardique par des techniques comme la thrornbol yse. I 'angioplastie et la revascular isation chirurgicale est bien dhonak (Braunwald 1985. Dewood et coll. 1983, Patel et Kloner
1987). Cependant, bien que la reperfusion soit indispensable & la survie du myocarde ischemique, elle peut paradoxalement entrainer des effets dklttkres dam ce tissu
(Braunwald et Kloner 1985, Hearse 1977).
Les codquences de I'ischemie et de la reperfusion dependent de la dude de l'ischtmie et des conditions dam lesquelles elle se produit. Ainsi une thrombose coronarieme peut arnener l'apparition d'une IEsion idvenible. A l'opposb, la reperfusion effectuk A la suite d' un met cardiopl6gique en chirurgie cardiaque entrdne plut6t des lQions rCversibles comme des arythmies de reperfusion ou le phtnomtne du myoca.de sider6
(myocardiul stunning) (Vaage et Valen 1993). Ce dernier terme dhigne les dysfonctions contractiles qui persistent aprh la reperfusion du myocarde et ce, en depit de I'absence de dommages irr6versibles (Bolli 1992, Braunwald et Kloner 1982). MalgrC l'amtlioration des techniques chirurgicales et des mtthodes de cardioprotection, les dysfonctions contractiles posm@ratoires derneurent Mquentes et contribuent B augmenter la mortalid et la morbidite chez les patients A risque comme les insuffisants cardiaques
(Bolli et coll. 1991, Bolli 1992).
Au cours de ce chapitre nous passerons brikvement en revue les altkrations biochimiques et cellulaires apparaissant Ion du processus d'ischkmie myowdique. Nous porterons egalement une attention particuliere 2 la ICsion de reperfusion survenant lors de chirurgie cardiaque et aux mCthodes de cardioprotection habituellement utiliskes dam ces circonstances.
1.2 Consdquences biochimiques et cellulaires de I' isch6mie
Le developpement de strategies de cardioprotection dcessite la comprkhension des changements biochimiques et cellulaires survenant lors de 1' ischemie et de la reperfusion.
Bien qu ' une s6quence temporelle ait id suggeree, les Cvhements cellulaires apparaissant durant 11isch6miene se produisent pas nkcessairement dam un ordre precis (Hearse et coll. 198 1). I1 est clair cependant que la gravit6 de ces changements s 'accroit en fonction du temps d'ischemie. Les changements pr6coces sont en g6nbraI reversibles. Ainsi une occlusion corowieme de 15 minutes chez le chien n'induit aucun signe de lbions
irrt5versibles (Reimer et coll. 1977). En revanche, une occlusion coronarienne de 40 minutes entraine 1'apparition de &rose dam la region endocardique.
Les cellules myocardiques puisent normalernent leur Cnergie via la phosphorylation oxydative mitochondriale (Mickelson et coll. 1990). Bien que le glucose puisse etre utilise comme substrat, 1'oxydation des acides gras libres predomine puisqu 'elle fournit
60 B 90 % de I'bnergie sous forme d'adhosine triphosphate (ATP) .
Lors de l'ischimie les rdserves en oxygtne du myocarde chutent rapidement. Le mdtabolisrne oxydatif cesse don et la glycolyse anaerobique devient la principale source de phosphates a haute energie (Jennings et coll. 1986). En rnoins d'une minute, la concentration d'acide lactique (lactate) et d'ions hydrogbne forrnie au cours de la glycolyse anaerobique est augmentee. L'accumulation de ces produits peut contribuer aux I6sions tissulaires observk durant 1' ischemic (Neely et Grotyoham 1984). De plus, le lactate et les ions HCenaainent I'inhibition de la glycolyse ana6robique comme source de phosphates B haute hergie (Rovetto et coll. 1975). Cependant, meme si cette voie metabolique pouvait fonctio~ernormdement, la viabiliti du myowde pendant une pdriode d'ischkmie prolongee ne serait pas assuree. La glycolyse ne produit effectivement pas plus de 7% des phosphates ii haute tnergie requis pour un fonctionnement myocardique normal (Reimer et Jemings 1992). Ainsi, I'utilisation des
phosphates & haute tnergie excede tr& rapidement leur production et les rberves d' ATP et de crhtine phosphate diminuent. Les experiences in vitro rhlisks par Iennings et coll. (1981) ont demon& que la concentration en crbtine phosphate chute de 72 % aprb
1 B 3 minutes d'ischdmie seulement. D'autre part, la concentration myowdique en ATP diminue de 65% apres seulement 15 minutes d'occlusion coronarienne chez Ie chien et de plus de 90% aprk 40 minutes (Jemings et coll. 1978). De plus, aprts une occlusion coronarienne de 15 minutes, les concentrations en ATP demeurent rtduites mtme aprh
72 heures de reperfusion (DeBoer et coll. 1980).
Parallelement a ceae deperdition Cnergetique, une augmentation de produits m&iboliques comme I 'adenosine monophosphate, I' inosine, 1' hypoxanthine, la xanthine et 1'ad6 nos he est observke. L'accumulation de cenains de ces produits peut contribuer a la ltsion d ' ischCmie-reperfusion. La voie enzymatique de la xanth ine oxydase a notamment kt6 proposCe comme source majeure de production de radicaux libres de I'oxygkne dam le tissu reperfus6 (McCord 1988). Ainsi au moment de la reperfusion, I'oxydation de
I'hypoxanthine par la xanthine oxydase entraine la formation de I' anion superoxyde. Les radicaux libres de l'oxygene pourraient jouer un r61e important dam la pathogCnke du myocarde s idtrk (BoI li 1992). 1.2.2 AIttkations ioniques
Les alterations m6taboliques obsendes durant l'ischemie sont associks ii des aldrations
ioniques. La premitre modification du milieu ionique B appafit14tre est une perte en potassium du cardiocyte (Kleber 1983). Un autre changement important survenant ensuite est I'augrnentation intracellulaire de l'ion NaC. Cette augmentation peut etre due en partie 5 la stimulation de I'Cchangeur Na+-H' causee par I'accumulation de protons pendant ltischt5mie (Tani et Neely 1989). Une autre explication est I'inhibition
mhbolique de la Na+-Kc-ATPase (Tani 1990). Dam les deux cas, l'augrnentation en
Na' peut entrainer une surcharge intracellulaire en calcium via la stimulation de
I'Cchangeur Nac-Ca+ au moment de la reperfusion. Les rnCcanismes par lesquels la surcharge calcique survient ne sont toutefois pas compl&ernent tlucid8s. En plus des mCcanismes ci-haut mentionnk, cette perturbation de 1 ' horntostasie calcique pourrait egalement provenir d'une diminution de la sequestration du calcium par le r6ticulum sarcoplasrnique (Krause et Hess 1985). Une awe hypothbe est I'augmentation de la perrnWilt6 de la membrance sarcoplasmique associke a la dtplttion des phospholipides membranaires dam le myocarde ischdmique (Chien et coll. 198 1).
Le calcium ag it cornme cofacteur (ou coenzyme) pour plus ieurs systkmes enzymatiques.
La surcharge calcique durant la ptriode d'ischbmie et de reperfusion peut alors entrainer
1'activation d' ATPases avec, comme consequence, une dtplCtion des rherves d ' ATP.
Des prot6ases et des lipases peuvent aussi ttre activees pour ainsi augrnenter non seulement le metabolisme de I'acide arachidonique mais aussi occasionner une
dborganisation des p hospholipides membranaires (Nayler et coll . 1985).
1.2.3 Alt6rations membranaires
L'activation des phospholipases par l'tl6vation du calcium cytoplasmique peut conaibuer aux alterations membranaires observees lors de l'ischemie et de la reperfusion. Les
radicaux libres de l'oxygbne form& lors de la reperfusion peuvent aussi Cue a l'origine de tels dkordres. Parmi les effets toxiques des radicaux libres mentionnons l'oxydation d'enzymes, de protiines de transport membranaires et la peroxydation des acides gras polyinsaturb des phospholipides membranaires (Ambrosio et coll. 199 1. Burton 1988).
Les althtions des phospholipides membranaires peuvent jouer un r6le preponderant dam la pathogenhe des lCions observkes lors de I'iscMmie myocardique (Kaa et Messineo
1981, Post et all. 1995, Sen et coll. 1987, van der Vusse et coll. 1994). De nombreuses
Ctudes font &at d'une diminution du contenu phospholipidique total dam le rnyowde
ischemique (Chien et coll. 1981, Vasdev et coll. 1980, Yanagishita et coll. 1987).
Plusieurs chercheurs ont egalement observe une accumulation de produits de dkgradation des phosp holipides cornme les Iysop hosp hoiipides et les acides gras non es t6r i fies comme l'acide arachidonique, dam le tissu cardiaque ischCmique (Corr et coll . 1987, Schwertz et coll. 1987, van Bilsen et coll. 1989). Les plasmalog&nes sont les constituants phospholipidiques pr6dominants dam le sarcolemme myocardique et les membranes du rkticulurn sarcoplasmique (Scherrer et Gross 1989). Des Ctudes recents demo ntrent que
l'activation d'une phospholipase A, stlective pour les plasmalog&nes est l'une des
althtions biochimiques prbmsurvenant durant I ' ischCm ie myocardique (Ford et coll.
1991, Hazen et all. 1991). De plus, I' ischbrnie et la reperfusion peuvent enmaher ine
accumulation de produits du catabolisme des plasrnalogihm (Davies et coll. 1992. Ford
et Gross 1989). Le r6le prkis des plasmalog&nes demeure encore incertain. Ces
composb pourraient notamment contribuer au rnaintien de la stabilit6 et de la fluidi3
membranaire de mtme qu'8 la stabilisation des systhes enzyrnatiques et des canaux
ioniques B I'intkrieur du sarcolemme (Kako 1986, Scherrer et Gross 1989). Le
cholest6rol est un autre constituant membranaire majeur pouvant Ctre alter6 lors de
1' ischkmie ou en prknce de radicaux libres (Kurryk et coll. 199 1, van der Laarse 1987).
La consequences d'une alteration du contenu en cholesdrol lors d'une ischtrnie ne sont
pas bien connues. Cependant, le cholesttrol pourrait moduler le fonctionnement de pompes ioniques et la permkabilite membranaire (Yeagle 199 1).
Durant une ischdmie. l'accumulation intracellulaire des produits metaboliques de la glycolyse erobique, de la lipolyse et de I' hydrolyse de I'ATP induit une augmentation de I 'osrnolal itk (Garcia-Dorado et Ol iveras 1993). Cette pression osmotique tlevke enaaine une augmentation du volume intracellulaire et des perturbations membranaires.
Cehchercheun ont tmis I' hypothk que I' apparition de lbions irreversibles pourrait
&re lice l'oediime cehlaire et au stress mtcanique impod au sarcolemme (k~ings
1986). 1.3 Strat@es de protection myocardique perophtoire
Une ischemic myocardique globaie est habituellernent induite lors de chirurgie cardiaque sous circulation extracorporelle puisque ce type d' intervention requiert un coeur en asystolie et en Ctat de relaxation. Cette pCriode d'ischtmie entraine cependant des effets dCICt8res sur les cellules myocardiques comme il a deji kt6 rnentio~iiau cours de ce chapitre. Les travawc expiirimentawr visant 5 arntliorer les strategies de cardioprotection peroperatoire se trouvent donc justifib.
1.3.1 Cardioplegie et hypothermie
Divenes techniques de protection myocardique furent dkveloppees dam le but de limiter les consequences nifastes de l'ischimie peroperatoire. L'infusion de solutions cardioplkgiques dam les coronaires peut proteger le myocarde en entrainant un arret diastolique rapide, limitant ainsi la deperdition des rberves CnergCtiques du myocarde
(Hearse et dl. 1974, Hearse et dl. 1981b). Les techniques de cardiopl6gie sont bash su.I'm& tlecao-rnhnique du coeur via l'addition de potassium ou du magn&sium. Le principe d'utilisation de la cardioplegie est d'abord apparu en 1955 (Melrose et coll.
1955). Toutefois la technique de Melrose qui consistait a injecter de fortes doses de citrate de potassium a kt6 associ6e ii de la nkrose mymdique (McFarland et coll. 1960,
Tyers et coll. 1975). La cardiopltgie a td rtinuoduite dam Ies anntes 1970 2 la suite de modifications de I'approche anerieure (Roe et all. 1977). I1 a kt6 alors dimontr6 que 1' hypothermie. qui permet une rUuction de l'activitt5 mitabolique du coeur, peut potentialiser les effets b6n6fiques de la cardioplegie (Hearse et coll. 1980, Rosenfeldt et coll. 1980). Depuis Ion, la cardiopI6gie crystalloide, riche en potassium et hypothermique est devenue l'une des techniques les plus utilisies en chirurgie cardiaque.
Malgre 1'Ctude de differents types de solutions cardiopltgiques et de divers modes d 'administration, la meilleure methode de cardioprotection peroperatoire demeure controversee (Demmy et coll. 1994, Noble and Mazer 1991). Bien que la solution crystalloide ait it6 traditio~ellement utiliste cornme vihicule de cardioplkgie,
I'utilisation de la cardioplkgie sanguine a gag& en popularid ces dernieres annees
(Domelly et Djuric 1991, Krukenkamp 1993). Un consensus n'a pas non plus Cd Ctabli en ce qui cuncerne la ternphture optimale de la solution cardioplCgique (Buckberg 1994.
Yau et coll. 1993).
1.3.2 Additifs P la cardioplkgie
Les mCthodes usuelles de protection myowdique peuvent s'avdrer moins efficaces lors d'une pkriode d'ischhie prolongee ou en presence d'une atteinte myocardique anterieure. I1 a etk ddemontrk, par exemple, que la mortalie et la morbiditi postopkatoire augmentent significativement chez Ies patients soufiant d'insuffisance wdiaque
(Christakis et coll. 1992. Tabone et cull. 1992, Wechsler et Junod 1989). I1 s'avtre donc n&essake de d&elopper d'autres strattigies de protection rnyocardique ou d'amdiorer les prockdures ex is tan tes .
Plusieurs approches ont it6 employ& afin d'ambliorer I'efficacid des solutions cardioplkgiques. L'une d'entre elks consiste a util iser des add itifs rnt tabol iques comrne le glucose, les phosphates B haute tnergie et certains acides amints. L'ajout de ces substances B la cardiopltgie permet d'ameliorer le mBtabo1isme Cnergttique et la rkuphtion myocardique (Robinson et coil. 1987, Svedjeholm et dl. 1995). L'addition d'un beta-bloquant comme le propranolol peut tgalement sfavtrer btnifique en permettant une diminution de l'activite mktabolique durant l'ischbrnie (Kanrer et coll.
1981).
Substances agissant via un micanisme ionique
La prevention des perturbations ioniques induites par I' ischCmie, cornme la surcharge calcique, constitue une autre approche prtconisk dam la recherche d'additifs efficaces
B la cardioplCgie. L'utilisation de substances permenant de limiter I' influx calcique stav&redonc ineressante. Ainsi l'ajout d'antagonistes du calcium B la cardiopl6gie a fait
I'objet de nombreuses etudes dont les resultats sont variables (revue : de Jong 1986). L'extrusion cellulaire du potassium est considkr6e comme un micanisme nature1 de
protection pendant 1' ischemie (Cole et dl. l99L. Wilde et ~011.1990). Les activateua
des canam potasiques sont donc de nouvelles entites chimiques qui prbentent un interit
dans la protection du myocarde en chirurgie wdiaque. L'aprikal im, un reprbentant de
cette classe pharmacologique, a prmd un effet cardioprotecteur additif & la cardioplegie
froide dam le coeur de lapin isole ischemique (Pignac et coll. 1994).
Les anesthtsiques locaux comme la lidocaine et la procaine sont frequemrnent utilisC comme constituants de solutions cardioplCgiques (Fiore et coll. 1990. Heme et coll.
1981~). Les mecanismes cardioprotecteurs de ces agents ne sont pas complttement elucidb. Ces agents ont notamment la capaciti d' induire un arrgt wdiaque rapide via
I ' inhibition de I' influx sodique et de la dkpolarisation membranaire. Leurs proprittb antiarythmiques peuvent aussi contribuer ii prevenir les dCsordres tlectrophysiologiques observks lors de la reperfusion (Fiore et coll. 1990).
Protecteurs mernbranaires
Les radicaux libres de I'oxygkne entrainent une peroxydation des lipides mernbranaires et constituent des mediateurs importants de la lQion d'ischtmie-reperfusion. Toute manoeuvre contribuant a prbvenir leur formation ou leurs effets pourrait s'avbrer intkressante en chirurgie cardiaque. A cet effet, les antioxydants tels que les pidgeurs de radicawc libres (camlase, superoxyde dismutase) ou les substances prevenant la formation des radi~uurlibres, tels que les ch6lateurs du fer (dkferoxamine) ou les inhibiteurs de la
xanthine oxydase (allopurinol), se son t r6v616s b6nefiques lors de travaux expkrimentaux
visant & etudier leur eficacit6 comme additifs a la cardioplCgie (MenaschC et coll. 1988,
Greenfield et coll. 1988, Vinten-lohansen et coll. 1988). D 'autres composks prbentent
6galement un intMt comme protecteurs membranaires. Le mannitol, qui est
traditionnellement ajout6 awc solutions cardiopl6giques pour en augmenter l'osmolarib5
et ainsi prevenir ltced&mecellulaire, serait dot6 de proprittes antioxydantes (Ferreira et
coll . 1989). Les 2 1-arninost&oTdes, des inhibiteurs de la peroxydation lipidique
particulierement efficaces dam I' ischBmie drebrale, ont rkemment demontrk des effets
cardioprotecteurs dam des rnodbles d ' ischhie-reperfusion (Carrea et coll. 1992, Levitt
et coll. 1994).
Aubes voies de protechn myocardique
Plusieurs autres composb aux proprititks diverses ont it6 Ctudib comme additifs ii la cardiopldgie dont les glucocorticost~roides, l'adknosine, l'albumine, l'insuline et
l'oxygene (Hearse et coll. 1981~). La vasodilatation est une voie de protection
myocardique qui a cependant tti peu exploree. I1 a Ct6 dkmontr6 que la prbence d'une
maladie coronarienne peut entrainer une distribution hdterogkne de la solution de cardiop16gie (Becker et coll. 1981, Grondin et coll. 1981, Landymore et coll. 198 1). Des agents vasodilatateun tels que la nitroglyc6rine ou les antagonistes dr? calcium pourraient donc am6liorer la protection offerte par la cardioplegie en assurant une rneilleure
distribution de celle-ci dans le myowde (de Jong 1986. Landymore et coll. 1983). De
plus, des substances vasodilatatrices pourraient pennettre de prevenir le phenornhe
d'hypoperhsion qui a tg observe aprb une ptriode d'ischCmie (Bolli et coll. 1990,
Kloner et Rryklenk 1992). Paradoxdement, la cardiop Egie en elle-meme induirait des
dommages vasculaires impliquant probablement l'endothtlium (Lindal et coll. 1988,
Saldanha et Hearse. 1989). La prksence de vasodilatateue coronariens pourrait
possiblement conner cette atteinte vasculaire.
En resumt, plusieurs substances ont Ctt utilisCes comme additifs i la cardioplegie.
Cependant le dtveloppement de strategies de cardioprotection demeure complexe.
L'approche idble consisterait a utiliser un agent qui pourrait prevenir ou amoindrir tous
les changements associts a 1I' ischbm ie peroptratoire. Parmi les substances CnumCrkes anttrieurement, les antagonistes du calcium sont probablernent les agents possedant la
plus grande diversit6 d'effets cardioprotecteurs. Bien qu'ils aient ett? principalement etudib dam Ie but de preve~la surcharge calcique. les antagonists du calcium peuvent
rkduire les dommages myocardiques relib 1' ischCmie et i la reperfusion par d'autres
mtcanismes: activite vasodilatatrice coronarienne, effets sur les radicaux libres. effets protecteurs membranaires, etc. Chapitre 2
Antagonistes du calcium en protection myocardique
2.1 Introduction
Les antagonistes du calcium, ou bloqueurs des canaux calciques. constituent une classe p harmacologique dont le developpement origine des uavaux de Fleckenstein et coil.
(1967). Ces chercheurs ont observe que la prknylamine et le vkrapamil mimaient les effets cardiaques d'une diminution de la concentration en calcium ionisd et bloquaient le couplage excitation-contraction. De plus, les effets ddpresseurs de ces agents sur la contractilitt cardiaque ttaient renversb par des agents qui entrainent une augmentation du calcium intraceilulaire (activateurs btta-adrdnerg iques, glucosides cardiaques, calcium). D'autres travaux ont appon6 une connaissance approfondie des proprietes pharmacologiques des antagonists du calcium. Ces medicaments sont maintenant largement utilises dam le traitement de plusieurs pathologies cardiovasculaires dont
I ' angine, I' hypertens ion et les arythmies cardiaques d'origine supra-ventriculaire.
Plusieurs Ctudes ont aussi d6montrk leurs effets benkfiques en protection myocardique.
Au cours de ce chapitre nous discuterons du mecanisme d'action des antagonistes du calcium de m6me que de leurs efiets cardiaques et vasculaires. Nous analyserons ensuite les etudes expCrimentales realis& dam le but de ddmontrer le potentiel Wnefique des antagonistes du calcium dam l'ischemie-reperfusion er les diffkrentes hypotheses Cmises pour expliquer leurs effets cardioprotecteurs. Nous porterons une attention particulibre aux controverses concernant I ' util isat ion de ces substances en protection myocardique peroperatoire.
2.2 Proprii3eS pharmacologiques
2.2.1 Classification des antagonistes du calcium
Les antagonistes du calcium constituent un groupe het&ogene tant sur le plan chimique que pharmacologique. 11s se divisent en trois grandes classes, les p hbny la1 kylam ines , les
1.4-dihydropyridines et les benzothiazepines, reprbsentees respectivement par le verapamil, la nifkdipine et le diltiazem. I1 existe Cgalement une quatrieme classe, les diphknylpiphzines (cinnarizine, flunarizine, lidoflazine), mais celle-ci est rnoins etudite au point de we clinique.
Une bl6vtion de la concentration cytosolique en calcium entraine une augmentation de la wntractilite du myocarde et du muscle lisse vasculaire. Cette entr6e de calcium peut
Stre induite par divers stimuli permettant l'activation de canaux ioniques membranaires sp6cifiques au calcium (Bronner et Freslon 1990). Divers types de canaux calciques ont M identifib (Lucchesi 1989, Struyker-Boudier et coll. 1990). Les canawr sensibles au voltage (VOC, vokage-operated channels) s'ouvrent en r6ponse B un changement du potentiel membranaire et permettent ainsi l'entrk de calcium B 11intt5rieurde la cellule.
Les receptor-operated channels (ROC)sont des canaux qui s'ouvrent a la suite de la liaison d'un agoniste spkifique (nor6pinbphrine. histamine, acttylcholine. etc.) A son rtcepteur mernbranaire. Certains seconds messagers tels que ceux form& lors de
I'hydrolyse du phosphatidylinositol mernbranaire entrahent egalement une tlbation du calcium cytosolique via des canaux nommb second-messengeraperated calcium channek
(SMOC). Findement, des canaux actives par I ' Ctirement des cellules musculaires 1isses
(stretch-operatedchannels) ont tte plus rkcemment proposes et seraient imp1 iquts dans le maintien du tonus vasculaire rnyogbnique.
Les antagonistes du calcium agissent pr6fkentiellement sur les canaux sensibles au voltage (VOC). Ces derniers sont divisks en trois sous-types en fonction de leur comportement tlectrophysiologique et de leur sensibilite B divers agents; soient les sow- types "L" (lent). "T"(transitoire) et "Nu(neuronal) (Nowycky et coll. 1985, Schwartz et coll. 1988). Seul le canal calcique de type L est inhibe par les antagonistes du calcium. Ce sous-type de canal est abondant dans le muscle lisse. les tissus cardiaques et le muscle squelettique. Les travaux portant principalement sur le muscle squelettique ont r6vM que les canaux L sont des glycoprotkines constitukes de 5 sous-unit& : a,, a,, a, p et y (Nargeot et Charnet 1994). Chaque classe d'antagonistes du calcium se fote sur des sites distincts de la m&ne sous-unie (a,). tout en interagissant allost&iquement. L'affinit6 des antagonism du calcium pour leur site de liaison respectif s'avbre d'autant plus importante lorsque la cellule est moins polaris6e et que la membrane se dtpolarise plus fi6quernment (Wibo 1989).
2 J.3 Effets vasculaires et cardiaques des antagonistes du calcium
Les canaux calciques de type L sont presents dam les membranes des myocytes cardiaques et squeleniques, de meme que dam Ie muscle lisse et les neurones. Toutefois, les antagonistes du calcium agissent principalement sur les vaisseaux sanguins et le muscle cardiaque B cause notamment de la cinetique de I'interaction entre ces agents et
Ieur sites recepteurs et de 1' importance du calcium extracellulaire dam I 'activit6 contractile de ces tissus. De plus. I'ornnipr&ence du calcium dam I'organisme et son r61e physiologique important ont men6 a la realisation de nombreuses dtudes fondamentales dans divers champs thirapeut iques. Ainsi, les antagonistes du calcium pourraient inhiber le d6veloppement de l'athtroscltrose et posskderaient des proprittb antiplaquettaires et immunosuppressives (Dumont et coll. 1993, Kiyomoto et coll. 1983.
Triggle 1989).
Actions sur le muscle kse vcrscu&kue
La contraction du muscle lisse vasculaire est triburaire d'une augmentation de la concentration cytoplasmique en calcium. Une augmentation du calcium cytosolique peut provenir d'un plus grand passage transmembranaire du calcium extracellulaire ion de
I'ouverture des canaux calciques sensibles au voltage en riiponse 1 une dkpolarisation rnembraoaire. Une augmentation transitoire de la concentration cytoplasrnique en calcium peut egalement &re induite a la suite d'une liberation intracellulaire du calcium en provenance de rhrvoirs intracellulaires (rkticulum sarwplasmique), par exemple lors de la formation d ' inositol trip hosp hate en reponse a I 'activation de rkpteurs membranaires
(Lucchesi 1989). Une entree du calcium en provenance du milieu extracellulaire peut aussi Cue observie lors de I'activation de rwteurs (Putney 1990). Dans le muscle lisse. le calcium libre intracellulaire se lie la calmoduline pour former un complexe qui active la kinase des chaines legkres de la myosine. La kinase activke enuaine une reaction de phosphorylation sur l'une des chalnes IBg5res de la myosine qui se traduit par I 'activation du systkme actine-myosine et par la contraction musculaire (Freslon 1990).
Les antagonistes du calcium induisent une relaxation du muscle lisse vasculaire en inhibant ['entree de calcium via les canaux calciques. L'effet vasodilatateur des antagonistes du calcium varie selon le territoire vasculaire consid6rL I1 s'avere beaucoup plus important sur les meres que sur les veines. L'activitk dilatatrice coronarieme des antagonistes du calcium est Bgalement particuli6rement importante, ce qui en fait des agents de choix dam le traitement de I'angine d'origine vasospastique. La nifkdipine et les autres dihydropyridines poss&dentles effets vasodilatateurs les plus prononcks, suivis par le diltiazem et le v6rapamil. La diminution de la r6sistance vasculaire periphCrique observk avec ces agents explique leur efficacite dam le traitement de I'hypertension drielle. De plus. une sensibilitg accrue B I'effet vasodilamteur des antagonistes du calcium a 6t6 demontree chez l'animal et chez l'homme hypertendu (Robinson et coll.
1982, Sharma et colI. 1984).
Actions sur l'endothe'liurn vasculaire
Les ce1lules endothkliales modulent le tonus vasculaire via la liberation de substances dilatatrices et constrictrices (Furchgott et Vanhoutte 1989). Ltune de ces substances,
I'EDRF (endothelium-derived relaxing factor), peut diffuser et agir sur le muscle lisse sous-jacent pour induire une relaxation. De nombreuses etudes montrent que I'EDRF correspond au monoxyde d'azote ou NO (Moncada et coll. 1991). Le NO est form6 entre autres dam les cellules vasculaires endotheliales a partir de la L-arginine. L'activid de l'enzyrne responsable de cette rhction. la NO synthase, est dependante du calcium
(Palmer et Moncada 1989). La liberation de facteurs consuictifs endothCliaux, tels que
1'endotht line, la thromboxane A, ou d'autres facteurs prostanoides, pourrait egalement nkcessiter une augmentation du calcium cytosol ique (Furchgott et Vanhoutte 1989). I1 a donc Ct6 propost que les antagonistes du calcium pourraient interferer avec la liberation de divers factem endothdiaux (Vanhoutte 1988). Cependant, les etudes r6alisies jusqu'i maintenant pour verifier cene hypothbe ont donne des rhltats contradictoires. Ainsi, plusieun etudes indiquent que les antagonistes du calcium n'affectent pas la rkponse dilataaice rnediee par l'endothtlium (Jayakody et coll. 1987, Miller et coll. 1985,
Rubanyi et coll. 1985, Rubanyi et Vanhoutte 1988). D'autres travaux ont par come ddmontr6 que le diltiazem et le clentiazem B de fortes concentrations dirninuent la production de I'EDRF (Rubanyi et coll. 1988,199 1). A l'oppost, certaines etudes sugg5rent que I'effet vasodilatateur de I'EDRF et des antagonistes du calcium est additif
(Vanhoutte 1988) et que certains antagonistes calciques induisent une vasodilatation en partie dependante de I'endothbliurn (Boulanger et coll. 1994, Vilaine et coll. 1991).
Puisque les effets endoth6liaux des antagonistes du calcium ont kt6 observC de fortes concentrations ou avec des agents plus liposolubles (clentiazem. S-11568). certains auteurs ont suggerk que ces effets seraient uibutaires d'une interaction non sptcifique des antagonistes du calcium avec des sites autres que ceux associes aux canaux calciques
(Rubanyi et Vanhoutte 1988, Vilaine et coll. 1991). De plus, Itexistence des canaux calciques de type L dam les cellules endothCliales n'a pas encore it6 clairement ttablie
(Rubanyi et Vanhoutte 1988, Adams et coll. 1989).
Aclions sur le cardiocyte
Le micanisme impliquC dans I 'excitation-contraction des cellules cardiaques diftPre de celui du muscle lisse. Dans le cardiocyte, la troponine I joue Ie rde d'inhibiteur de
I'appareil contractile. La liaison du calcium a la troponine entraine la levee de cene inhibition pennettant donc une interaction enw I'actine et la myosine et, subsbquemment. une contraction rnusculaire. Les bloqueurs des canaux calciques entrainent donc une diminution de la contractilid myocardique (effet inotrope n6gatif) . Cette diminution de la fonction cardiaque, et consequemment de la demande en oxyghne, est un autre mkcanisrne par iequel ces medicaments exercent un effet anti-angineux. L'arnpleur de
cette action inotrope negative varie selon la classe d'antagonistes du calcium et slCtablit
cliniquement dans 1'ordre suivant : vkrapamil > diltiazem > nifaip ine (Taira 1987).
Actions sur le fissu de conduction
La dtipolarisation dam les noeuds sinusal et auriculo-ventriculaire depend de I'entree de
calcium via les canau calciques. Le verapamil est l'antagoniste du calcium qui posskde
les effets inhibiteurs les plus marques sur le noeud sinusal (effet chronotrope negatif) et
la conduction auriculcwentriculaire (effetdromotrope negatif). Aux doses habituellement
utilistes en clinique, la nifkdipine ne diminue pas la vitesse de conduction et son effet
inhibiteur sur le noeud sinusal est renvene, in vivo, par I'apparition d'une tachycardie
riflexe induite par la stimulation du systeme nerveux sympathique (Triggle 1990). Le
d i 1t iazem, quant a lui, possede des effets intermediaires. Les propr ittks
dleccrophysiologiques du diltiazem et du virapamil permettent leur utilisation dam le
traitement des tachycardies supraventriculaires de m5me que pour diminuer la rkponse
ventriculaire lors de fibrillation ou flutter auriculaire.
2.2.4 Selectivit6 tissulaire des antagonistes du calcium
La s6lectivitk tissulaire est une caractiristique importante des antagonistes du calcium.
Ainsi le muscle lisse intestinal, trachd, bronchique et le muscle squelettique sont relativement insensibles aux antagonistes du calcium (Femi et coll. 1994). Ce phhomhe peut s'expiiquer entre autres par une mobilisation de calcium differente dam ces cellules musculaires, qui impliquerait davantage le pool intracellulaire pludt qu 'extracel lulaire lors de la contraction. D ' autres Fdcteurs peuvent aussi expliquer les effets sdectifs des antagonistes du calcium tels que des differences tissulaires dans les types de canaux calciques ou dam la cinetique de leur ouverture (Triggle 1990).
Tel que mentiom6 prkiidernment, il existe une hCtkrogen6itk marqde entre les uois classes dtantagonistes du calcium en ce qui concerne leur sblectivit6 relative sur le muscle lisse et cardiaque. La nifedipine est plus silective pour le muscle lisse vasculaire que pour le muscle cardiaque, alors que le vCrapamil posskde les effets inotropes negatifs les plus pronon&. Le diltiazem prbente des effets interm6diaires entre les dihydropyridines et les phdnylaikylamines (Wood 1989). A I'indrieur d'une mtme classe, des modifications structurales de la moIecule peuvent induire des changements de la vasostlectivit6. Ainsi, I'activitk vasodilatatrice de la nisoldipine est 50 fois supkrieure
B celle de la nifidipine (Godfraind et coll. 1992) tandis qu'une autre dihydropyridine, la nimodipine, agit preferentiellernent sur la vasculature ckrebrale (Kazda et Towart 1981).
Parmi les benzothiaz6pines, le clentiazem (8-chloro-diltiazern) , un derive plus 1ipophile du diltiazem, possede une vasos~lectividsu@rieure B celle du diltiazem mais comparable
B celle de la nifedipine (Narita et Ginsburg 1990, Narita et coll. 1990). Les propri6& physicochimiques spkcifiques B chaque antagoniste du calcium endnent des differences pharrnacodynamiques et pharmacocin6tiques. Compte tenu de ces diffirences, il est possible que le potentiel cardioprotecteur de ces agents puisse differer selon la classe chimique util*, voire meme selon l'agent stlectionnk a l'inttrieur d'une m2me classe.
2.3 Mtkanhnes cardioprotecteurs des antagonistes du calcium
Plusieun Ctudes, r&Iis&s avec divers modbles exp&imentaux, demontrent l'efficacid des antagonistes du calcium en protection myocardique. Ainsi de nombreux travaux expCrimentaux effect& tmt in vitro qu'in vivo demontrent que les antagonistes du calcium previennent ou attknuent le ph6nomtne du myocarde sidtre (Lamping et Gross
1985, Nayler et coll. 1990, Przyklenk et Kloner 1988, Taylor et coll. 1990). Des resultats positifs ont Bgalement ttt5 rapport& en ce qui concerne la capacite qu'ont ces agents i rkduire la taille de 1' infarctus dans des mod6Ies d'occlusion coronarienne prolongke chez I'anirnal (Higginson et coll. 1991, Kingma et Yellon 1988. Yoshida et call. 1985). Le r6le primordial jout par le calcium dam la Itsion d1isch6mie-reperfusion constitue la motivation premiiire sous-jacente 3 l'utilisation des antagonistes du calcium en protection myocardique. Cependant d'autres hypothbes ont aussi tte emises afin d 'expl iquer les mecanisrnes cardioprotecteue de ces agents. 2.3.1 Revention de la surcharge dcique
La prevention de la surcharge calcique est sans doute le rnbnisme le plus souvent cite pour expliquer les effets cardioprotecteurs des antagonistes du calcium. 11 existe effectivernent des etudes qui dCmontrent que les antagonistes du calcium peuvent limiter
I1t%vation cytosolique de calcium lorsqu' ils sont adminisues avant la pBriode d' ischCmie
(Bourdillonet Poole-Wilson 1982, Nayler et coll. 1985). De plus, Nayler et coll. (1988) ont dimontr6 que le Bay K 8644. un agoniste des canaux calciques, augmente le gain en calcium au debut de la reperfusion, suggCrant que I'entree de calcium par les canawr sensibles au voltage contribue a la surcharge calcique. Du Toit et Opie (1992) ont quant a eux dernontrb une amplification du phhomkne du myocarde sidere lors de i'utilisation du Bay K 8644. 11 existe cependant une controverse quant au r6le jouC par les canaux calciques sensibles au voltage comme voie d'entree du calcium lors de I'isch6mie- reperfusion. De fait, i1 a it6 sugg6r6 que les canaux calciques sensibles au voltage sont inactifs lors de la rCoxyg6nation du myocarde et qu'ils ne sont donc pas irnpliqu& dam la surcharge calcique engendrke par la reperfusion (Murphy et coll. 1988, Tani 1990).
Certains chercheurs n'ont d'ailleurs pas observe de prevention de la surcharge calcique
Iorsque les antagonistes du calcium sont administrk B la reperfusion (Bourdillon et Poole-
Wilson 1982, Watts et coll. 1980). Certaines Ctudes indiquent cependant que les antagonistes du calcium peuvent ameliorer le fonction contractile du myocarde sidere meme lorsqu' ils sont administr6s lon de la reperfusion (Du Toit et Op ie 1992, Przyklenk et Kloner 1988, Przyklenk et coll. 1989). Les rnhismes cardioprotecteurs des antagonistes du calcium pourraient donc differer selon le moment de leu administration. I1 est en effet possible que le mecanisme cardioprotecteur des antagonistes du calcium administrk B la reperfusion ne soit pas la prdvention de la surcharge calcique, car I'administration d'agents inotropes, qui agissent via l'augmenmtion du calcium intracellulaire, ameliore la fonction contractile du
myocarde sidere (Bolli et coll. 1985, Ellis et dl. 1984). Par contre, il ne peut Ptre exclu que l'administration d'antagonistes du calcium avant I'ischemie puisse prkvenir la surcharge calcique par des m6canismes indirects tels qu ' une prkservation des rkerves tnergetiques ou de l'inttigrite membranaire (Watts et coll. 1990).
2.3.2 Diminution de la demande mhtabolique et prbervation Bnerg6tique
Un auue mbnisme cardioprotecteur des antagonistes du calcium serait la prkservation des phosphates i haute Cnergie par le biais de leun effets inotropes et chronouopes nOgatifs (Huizer et coll. 1987, Nakazawa et coll. 1985, Neubauer et Ingwall 1989).
Diverses observations suggtrent nhnmoins que la cardiodipression n'explique pas nicessairement cet effet protecteur. 11 a notamment 6t6 ddemontr6 qu'un antagoniste du calcium peut diminuer la taille de I'infactus dam un modde canin d'occlusion coronarienne et ce, sans affecter la demande rnetabolique (Denniss et coll. 1990). De plus, certaines etudes dkmontrent que des concentrations non wdiod6pressives d'antagonistes du calcium entrainent des effets cardioprotecteurs (Cook et Hof 1985,
Grover et Sleph 1989). De fait, I 'utilisation d' isom&es de certains antagonistes calciques a permis de distinguer deux modes de cardioprotection dont I'un serait independant de la cardiod6pression (van Ams terdarn et coll. 1990).
LRS antagonistes du calcium pourraient toutefois avoir des effets ben6fiques metabol iques qui ne seraient pas relib ii leur action cardiod6pressive. Watts et coll. (1990) ont rapport6 que le diltiazem dirninuait I'utilisation de I' ATP de mame que la production de lactate. Selon ces auteurs, les propriith cardiodtpressives du diltiazem ne peuvent A ella seules expliquer ses effets Mnefques puisque celuici riduit la production de lactate meme dam un coeur asystolique. Le diltiazern et son cong6nkre. le clentiazem. peuvent tauire la glyoolyse et amdiorer la r&u@ration myocardique lors de la reperfusion et ce, a des concentrations qui n'affectent ni la Mquence cardiaque ni la contractilite myocardique (Davies et coll. l9W. Lopaschuk et coll. 1992). Les effets benifiques des antagonistes du calcium seraient donc li& en partie B Ieur capacite de rkduire la glycolyse durant la @ride d'ischtmie. La antagonistes du calcium lirniteraient I'accurnulation de sous-produits de la glycolyse qui ont des effets dCICt&ressur le rnyocarde (Tani et Neely t 989).
2.3.3 Effets antiperoxydants et pr6servation de l'intigritdi membranaire
Les radicaux libres de I'oxyghe sont parmi les facteurs importants impliquh dans la pathog6nhe de la ltsion d' ischtmie-reperfusion. Plusieurs travaux expbimentaux indiquent que les antagonism du calcium inhibent la peroxydation des lipides membranaires induite par les radicaux libres de I'oxyg6ne (Janero et coll. 1988, Koller
et Bergmum 1989). Meme si ces effets ont 6t.6 observh A des doses supkrieures ii celles
habituellement utilisks cliniquement, les antagonistes du calcium auraient la proprikt6
de s'accumuler I'inttirieur des membranes cellulaires oh iIs exerceraient Ieur action
antiperoxydante (Pang et Sperelakis 1984, Taganu et coll. 1985). Outre la possibilid
d'un mCcanisme cytoprotecteur direct, les antagonistes du calcium diminueraient la
production des radicaux libres en provenance de la chafne respiratoire mitochondriale
(McCord 1988). Ces agents pounaient effectivement prberver la fonction de la
mitochondrie en y rduisant I'accumulation de calcium (Ferrari et coll. 1990). La
reduction de l'activite des phospholipases sensibles au calcium constitue un autre
mecanisme protecteur puisque cette voie enzymatique mhe a la formation d'acide
arachidonique qui est une source potentielle de radicaux libres (Karmazyn et Moffat
1985). En revanche, il ne peui ttre exclu que la peroxydation Iipidique soit une
manifestation plut6t qu ' une cause de lQion tissuiaire. Dam ces circonstances. la
reduction de la peroxydation Iipidique observee avec Ies antagonistes du calcium ne
permettrait que de constater une prbervation du tissu myocardique et non un effet
antiperoxydant direct (Koller et Bergmann 1989). Cependant, un effet direct des
antagonistes du calcium sur la peroxydation lipidique demeure possible. I1 a en effet kt6
demonu6 que ces agents protkgent les cellules endoth6liales en culture conme les effets
des radicaux libres (Mak et coll. 1992). Puisque le milieu d' incubation des cellules ne
contenait pas de calcium, les auteurs ont suggirb que les antagonistes du calcium exercent des propridth antioxydantes intrinsbques. Cornme il a bd mentionnii au cum du chapitre prWent, de nombreuses etudes font &at
d'une diminution du contenu phospholipidique total dans le myocarde ischCmique (Chien
et coll. 1981, Vasdev et coll. 1980, Yanagishita et coll. 1987). La plupart des Ctudes
portant sur les effets antiperoxydants des antagonistes du calcium ont kt6 r6alisees en ne
mesurant que le malondialdthyde, un produit de la peroxydation lipidique. D'autres etudes seraient nhsaires afin dievaluer l'effet des antagonistes du calcium sur le contenu phospholipidique du myoarde aprbs une pdriode d ' ischtmie.
2.3.4 Effets sur les neutrophiles
Les neutrophiles s'accumulent rapidement dam un myocarde reperfust apds une ptriode d' ischemic prolongte. L'activation des neutrophiles entraine la libtration d'une variete de muiateurs capables de promouvoir la iksion tissulaire comme des radicaux libres de
I'oxygbne, des enzymes protwlytiques et des autaco'ides Iipidiques tels que le PAF
@latelet-activatingfixtor) et les leucotri5nes B, (Mullane et Young 1992). De plus
1'ad hCrence des neutrophiles aux parois vasculaires exacerbe 1' hypoperfusion observtk lors de la reperfusion myocardique.
Les antagonists du calcium pourraient inhiber I 'activation des neutrophiles et la
I ibtration subsequente d'enzymes proteolytiques, de radicaux libres. de leucotri5nes et de PAF (Azurna et dl. 1986, Jouvin-Marche et coll. 1983, Irita et coll. 1986). De plus, il a ete dkrnontre que l'administration de diltiazem et de clentiazem ii la reperfusion ruuit la taille de I'infarctus par un mbisme qui comprendrait, notamment. une rkduction de
1 'accumulation des neutrophiles dans le myocarde (Rousseau et coll. 199 1, 1994).
Cependant, bien que Ifactivation des neutrophiles sernble jouer un rdle important dam la pathog6nk des lbions myocardiques Mvenibles, sa contribution au dkveloppement du myocarde sidtd demeure controve& (Becker 1991). De plus, les effets bkn6fiques des antagonistes du calcium observb dans des rno&les de coeurs isolQ ne peuvent s'expliquer par une action qu'ils pourraient exercer sur les tlkments circulants puisque les solutions de perfusion den contiement g6kralement pas (Kloner et Przyklenk 1992).
2.3.5 Effets vasodilatateurs
Les effets vasodilatateurs des antagonistes du calcium constituent vraisemblablement un mkcanisme de cardioprotection. Des travaux experimentaux indiquent que les effets btntfiques des antagonistes du calcium lors d'une occlusion coronarienne sont consequents a une augmentation du dkbit sanguin dans la zone ischCmique via l'am~liorationdu dkbit collat6ral (da Luz et coll. 1980, Szekeres et coll. 1985). En revanche certains auteurs ont dCrnontr6 qu'il &it possible de diminuer la gravid de l'ischbmie myocardique avec les antagonistes du calcium sans obtenir d'augmentation parallele du dtbit sanguin (Grover et Parham 1987, Thuillez et dl. 1983). Ces derni8res donnk suggerent que les antagonistes du calcium proegent le myocarde ischtmique sans pour autant agir sur le debit coronarien. Une controverse subsiste donc en ce qui concerne l'importance des effets des antagonistes du calcium sur le debit collatkral durant
I' ischemic.
Les effea vasodilatateurs des antagonistes du calcium slaverent bbnkfiques autrement qu'en agissant sur le debit colladral. Ainsi, les antagonistes du calcium protegent le myocarde lors d'ischemie globale rhlisde dam des moddes de coeurs isolC. Certains chercheurs ont don attribut ces effets benefiques ii leur action vasodilatatrice (Cook et
Hof 1985, van Amsterdam et coll. 1990). La vasodilatation coronarienne produite par les antagonistes du calcium permettrait une meilleure distribution des nutriments
Cnergttiques de meme qu'une tlimination plus rapide des dLhets mttaboliques lon de la reperfusion. Par surcroit, un vasodilatateur coronarien pourrait acdldrer la rCcup6ration myocardique en antnuant le phhombne d'hypoperfusion (low-rqlow) observe lors de la reperfusion (Kloner et Przyklenk 1992).
En chirurgie cardiaque, les effeu dilatateurs coronariens des antagonistes du calcium permettraient une meilleure distribution de la solution wdioplegique (de Iong 1986,
Landymore et coll. 1983). 11s attiinueraient aussi ies effets d6let2res de la cardiopltgie sur la circulation coronarienne (Lindal et coll. 1988, Saldanha et Hearse, 1989). Outre leurs effets dilatateurs coronariens, la presence d'une vasodilatation ptripherique permemait une am6lioration de la rBcup6ration myocardique postisch6mique en reduisant la postcharge. L'agent utilis6 devra cependant ttre suffisamrnent vasostlectif pour tviter
I 'apparition d'effets inotropes nkgatifs. Compte tenu de Ieur potentiel cardioprotecteur, les anagonistes du calcium ont fait I 'objet depuis quinze ans de nombreux travaux visant B 6valuer leur efficacit6 en protection rnyocardique peropikatoire. Nous passerons en revue les Ctudes exptrimentales et cliniques qui traitent de l'association d'un antagoniste du calcium et d'une solution de cardioplbgie dam le but d'optirniser la cardioprotection.
Les etudes expCrimentales consacrtes aux antagonistes du calcium utilises en ajout aux solutions cardiopI6gique.s ont ttk nombreuses mais leur interpretation est compliquke par la diversit6 des modkles et des protocoles expkrimentaux (revue : de Iong 1986). 11 est ntkmnoins possible d'en tirer quelques conclusions. Tout d'abord, la plupart des uavaux exptrimentaux dtmontrent que les antagonistes du calcium amdiorent les effets cardioprotecteurs de la cardioplkgie en normothermie (37OC). Dam de tel les conditions, une meilleure rkuperation de la contractilit6 myocardique de rntrne qu ' une diminution de la liberation de crhtine kinase ont id observis avec le diltiazem, la nifedipine et le verapamil (Chiavarelli et coll. 1986, Yamaxnoto et coll. 1983a. 1983b).
Des doutes subsistent cependant en ce qui concerne l'efficacit6 des antagonistes du calcium en association i la cardioplegie froide (<20°C). En effet, plusieurs Ctudes rapportent que les effets cardioprotecteurs additifs a la cardioplegie n'ont pu ttre observQ dam des conditions d'hypothermie (Chiavarelli et coll. 1986, Fukunami et Hearse 1985,
Hearse et coll. 1984, Hearse et coll. 1985). Diverses hypothhes ont dors et6 proposkes pour expliquer ce ph6nom8ne. L'une d'entre elks est que I'hypothermie entrainerait un changement dam la conformation du canal calcique ou dans les composantes
membranaires avoisinantes, diminuant ainsi la capacid des antagonistes du calcium B exercer leur effet pharmacologique (Hearse et coll. 1985). Une etude rQlis6e sur des myocytes isolb de rat dkmontre d'ailleurs que la suppression des contractions par les anagonistes du calcium nimsitent des concentrations de 10 a 100 fois plus tlev6es i des tempkratures inferieures a 27OC qu'8 37OC (Piper et coll. 1985). Une autre hypothese es t que I'hypothermie agirait par un mCcanisrne cardioprotecteur similaire i celui des antagonistes du calcium, masquant ainsi I'effet de ces derniers (Hearse et coll. 1985). 11 est possible par exemple qufune modification de la conformation du canal calcique par la solution hypothermique entraine une perte de sa capacite de transport rendant superflue toute tentative d ' inactivation pharmacolog ique.
Bien qu'aucun Mnefice n'ait 6d observ6 avec les antagonistes du calcium dam la plupart des ttudes in vim, des travaux r6alisk dam des mod&lesde circulation extracorporelle chez I'animal ont permis de dkmontrer une meilleure r6cuptration de la contractilit6 cardiaque (dP/dt)lors de I'ajout de ces agents B la cardioplegie et ce dam des conditions d'hypothennie (Magovern et coll. 1981, Melendez et coll. 1988). ~tantdonne la controverse entourant Ifefficacit6des antagonistes du calcium en hypothermie, d'aucres ttudes seraient requises afin de mieux caract&iser les effeu cardioprotecteun de ces rnkdicaments dam les memes conditions exp&imentales.
Lots d'une etude clinique randomisee chez des patients soumis a un pontage coronarien. l'addition du diltiazem A la solution cardioplegique fioide s'est uaduite par une meilleure protection myocardique (Christakis et coll. 1986). Dans cette etude, la liberation de crkatine kinase Ctait reduite, indiquant une diminution de la lbion ischtrnique, et les phosphates ii haute energie dtaient mieux prbervb. Cependant. le diltiazem a induit des effets inotropes negatifs de mSme qu'une friquence plus dlevte de blocs auriculo- ventriculaires post-opbatoires. Le diltiazem devrait donc due utilisd avec precaution chez des patients prhentant une altiration importante de leur fonction venuiculaire. A la suite d'une Ctude clinique au cours de laquelle le diltiazem a Ct6 ajoutk ii la cardioplkgie sanguine fioide, Barner et coll. (1987) ont conch que les bbnkfices observh quant a la liberation de crdtine kinase Ctaient faibles comparativement au risque de blocs auriculo- ventriculaires. En dipit d'une absence d'effets cardioprotecteurs, Nomura et coll. ( 199 1) ont quant B eux obsenk une augmentation de la production de prostacycline, une pros taglandine vasodilatatrice, quand le dil tiazem ttait ajoute 5 la cardiopldgie. Cette liberation accrue de prostacycline pennettrait de meilleures conditions de reperfusion. Des etudes cliniques ont aussi kt6 rMis& avec d'aunes antagonists du calcium.
L'utilisation de la nifuipine dam la solution cardioplegique a enaaine une amelioration
des pararnkees h6modynamiques postoperatoires et une d irninu tion de la 1iberation de
crbtine kinase (Clark et coll. 1981, Trubel et dl. 1994). Flameng et coll. (1986) n'ont
cependant pas obtenu d'am6Iioration clinique lon de I'utilisation de cet agent, malgr6 une
perte moins importante des nuclbtides chez les patients dont le myocarde n'avait pu etre
refroidi au-dessous de 2S°C. Finalement, I'utilisation du vtrapamil comme composante de la solution cardiopl6gique n'a pas apportk de benefices chez les patients sournis B de courtes @nodes dta.rrSthypothermique (Gufin et coll. 1986). Le vkrapamil augmentait
plut6t I'incidence de blocs auriculo-ventriculaires et de dtpression de la fonction contractile chez ces patients.
2.5 Choix des benzothiazipines comme agents cardioprotecteurs
Par mi les antagonistes du calcium, les benzothiaz6pines constituent un choix attrayant comme agents cardioprotecteurs. Les benzothiaz6pines possedent des propribtb vasculaires et wdiaques intermediaires entre celles des phtnylalkylamines et des dihydropyridines. Gene caract6ristique pourrait constimer un atout puisqu' une protection myocardique eficace est tributaire de plusieurs mkcanismes, impliquant autant des effets sur le mttabolisme 6nergCtique du myocarde que des effets vasculaires. De plus il a Cd dCmontrC que le diltiazem prot5ge le myocarde ischemique A un niveau de dtpression myocardique moindre que celui engendrC par le vCrapamil ou la nifkdipine (Grover et Sleph 1989, Hamm et Opie 1983). L'efficacid du diltiazem, tout comme celle du veraparnil et de la niedipine, demeure toutefois controvers6e Ion d'une utilisation en ajout ii la cardioplCgie froide.
- . . - . ------. .. - Figure 2-1. Structure chimique des benzothiazkpines
Le clentiazem, le derive chlor6 du diltiazem (fig. 2-I), possbde une plus grande tipophilicitti que le diltiazem, ce qui permet d'accroitre sa retention tissulaire et sa duree d'action (Kikkawa et coll. 1988, Murata et coll. 1988, Narita et coll. 1988). Ces proprieth physicochirniques et pharmacocinCtiques seraient susceptibles d'induire une me illeure wdioprotection meme dans des conditions d' hypothermie. De plus, le clentiazem est plus vasostlectif que le diltiazem (Narita et Ginsburg 1990). I1 pourrait ainsi prothger le myocarde tout en Cvitant les effets cardiodCpresseurs observb lors de l'addition du diltiazem la cardioplegie. Cependant les rQultats mitigb obtenus avec le diltiazem s'expliqueraient par I'utilisation de doses trop Clevees. En effet, tel que nous I 'avons souligne ant6rieurement. il est possible d'obtenu une wdioprotection sans depression myocardique avec le diltiazem.
En conclusion, les antagonistes du calcium prtisentent un grand in&& en protection myocardique. Les mkanismes cardioprotecteurs imp1 iqub sont mu1 tiples et dependent de I'agent utilist et de son mode d'administration. Les effets inotropes nkgatifs des antagonistes du calcium peuvent cependant en limiter I'utilisation puisque les patients candidats i une protection myocardique perophtoire pressentent friquemment une det6rioration de leur fonction cardiaque. Les donnCes actuel les sont insuffisantes pour recommander l'emploi de ces agents dam cette catkgorie de patients. Des ttudes sont requises afin de determiner si les antagonistes du calcium prbentent des effets cardioprotecteurs significatifs en pr6sence d' insuffisance cardiaque. Chapitre 3
Protection myocardique dans l1i,ru&hnce cardiaque
3.1 Introduction
L ' ins ufisance cardiaque est I 'une des maladies cardiovasculaires dont I ' incidence a le plus augment6 au cours des dernieres anndes (Andrews et Cowley 1995). Le nombre d'hospitalkations associb B l'insuffisance cardiaque a plus que uiplt au cours des deux dernibres dtkennies et le vieillissement de la population n'est pas sans contribuer a ce phbnombne. Un nombre grandissant de chirurgies cardiaques, comme Ies revascularisations, sont maintenant pratiquks dam cette population de patients (Baker et
COIL 1994).
Les patients ayant une dysfonction ventriculaire gauche importante sont consid&is comme de mauvais candidats au pontage coronarien (Lip et Metcalfe 1994, Tabone et dl. 1992). Des Ctudes moment en effet une monalid ophtoire importante ( 18 a 33 % ) de mZme qu'un taux Clevt de dkcih postoptratoires dam cette catkgorie de patients
(Manley et coll. 1976, Spencer et coll. 1971, Yatteau et coll. 1974). Cewrci sont gineralement exclus des dtudes randornisks et sont davantage considCrk comme candidats A la transplantation cardiaque. Cependant, la faible disponibilitk des organes de meme que l'am6lioration des techniques ofiratoires ont contribub a augmenter l'int6r6t de la revascularisation coronarienne wmme option therapeutique chez les insufisants
cardiaques soufiant d'une maladie coronarienne. De plus. des etudes plus rkenw
dhontrent que les patients ayant une dysfonction ventriculaire grave ont une meilleure
esphnce de vie aprb un pontage coronarien comparativement ii un traitement maid
(Alderman et coll. 1983, Milano et coll. 1993. Wechsler et Junod 1989). La mortalit6
o@ratoire demeure nhmoins supkrieure chez ce groupe de patients si on la compare a
celle des sujets prksentant une meilleure fonction ventriculaire prdopkratoire. Dam ce
contexte, il apparait nhsaire de dCvelopper de meilleures strategies de protection
myocardique peropkratoue.
3.2 Protection myocardique perop6ratoire chez les insuffsants cardiaques
Selon Christakis et coll. (1992), le type de protection myocardique peut influencer
significativement la mortalit6 optratoire chez les patients dont la fraction d 'ijection
systolique se situe entre 20% et 40%. Ainsi ces auteurs ont obtenu de meilleurs rtsultats
chez des patients A haut risque en optant non pas pour la cardioplegie crystalloide mais
pludt pour la cardiopldgie sanguine. Jegaden et coil. (1994) ont quant B eux obtenu une
reduction de la mortal it6 postop6ratoire et une meilleure survie tardive avec I'utilisation de la wdiopldgie oxygknke. Toutefois, peu d'ttudes ont kt6 rklish dam le but d'haluer spkifiquement les modalit& de protection myocardique chez les insuffisants cardiaques. EChuri et coll. (1988) ont dhontre qu'une perhsion continue de cardiopkgie sanguine offre une meilleure protection que la cardiop lligie crystal loide intermittente lors de remplacements valvulaires effectuk chez des patients presentant une hypemophie venrriculaire gauche. Les patients mt&lors de cette 6tude prkentaient cependant une fraction d'kjection se situant environ 50 % alon qu'il a kt6 dtabli qu'une augmentation significative du kteu de risque correspond davanrage 5 une fraction d'ejection infdrieure
B 35 ou 40% (Alderman et coll. 1983, Baker et coll. 1994, Christakis et coll. 1992).
L1&tudedu CASS a ainsi montrk que la mortaliti operatoire passe de 1.6% a 6,996 lonque la fraction di6jection prbphtoire atinferieure B 35 % (Alderman et coll. 1983).
Diverses raisons expliquent le risque operatoire plus Cleve observe chez les insuffisants cardiaques. Premihrement, contrairement aux patients qui ont une bonne fonction ventriculaire prbpt5ratoire, les sujets ayant dkjj8 une a1 ttrat ion de la fonction ventr iculaire ne poss&dent pas une rberve contractile leur perrnettant de toltrer une protection rnyocardique inadequate (Christakis et coll . 1992). De plus. les insuffisants cardiaques ont souvent une maladie coronarienne etendue. 11 a &ddbmontrk que la presence d'une sthose importante, qui empkhe une distribution homoghe de la solution de cardiopl6gie, est associk B une protection myooudique inadequate (Becker et col I. 198 1.
Grondin et coll. 1981, Landymore et coll. 1981). De msme, un coeur hypertrophique est davantage r6sistant ii la protection myocardique (Rabinov et coll. 1989); I' induction de I'm& par la cardioplegie est retardke et cet anst est plus dificile ii maintenir en prbsence d ' hypemophie. Certains changements m6taboliques observes dans I ' insu fisance cardiaque expliquent
aussi une mob borne toldrance B I'isch6rnie peroptratoire. Des niveaux plus faibles de
phosphates a haute 6nergie ont tt6 rapport& dam le coeur insuffismt ou hypertrophique
(Huddleston et coll. 1992, Sink et coll. 198 1, Meenon 1%9). Ainsi, il a Cd sugg6r6 que
la plus grande wln6rabilitt5 du rnyomrde hypertrophique est explicable par des riserves
tnergktiques moindres iors de I'induction de I' ischemie (Sink et coll. 198 1). Outre ces
altdrations m6taboliquest le coeur defaillant peut kgalement presenter des dysfonctions
coronarie~esqui influenceront la cardioprotection et la rkcuptration postoptkatoire.
L'insuffisance wdiaque a tt6 associQ A une diminution de la perfusion sous-
endocardique (Vamer 1988). Ce phtnornene peut dbs lon contribuer :! 1'amp1 ification
des dommages causC par liisch&nie peropbratoire. Des travaux recents r6alis6s chez
l'animal et I'homme demontrent que dam plusieurs lits vasculaires la dilatation rnedik
par I'endothelium est aMr& en prbence d'insuffisance cardiaque (Drexler et coll. 1992.
Katz et coll. 1993, Kubo et coll. 1991, Ueno et coll. 1994). De plus des niveaux
plasmatiques d'endothkline, un puissant vasoconsuicteur, plus klevb que la normale ont ttt observes dans 1' insufisance cardiaque (Liischer et Noll 1993). I1 est donc possible que Ie phbnomhe d'hypoperfusion du myocarde observe apres une periode d'ischemie so it accentut5 avec 1' insuffisance cardiaque, compromettant aim i une rdcupbration myocardique adequate. Les dysfonctions endothiliales associh a i'insuffisance cardiaque demeurent cependant controversCes puisque certaines etudes demontrent une amplification de la vasodilatation mkdi6e par l'endothdlium et de la production basale de NO dam cette condition pathologique (Habib et coll. 1994, Larosa et coll. 1993,
O'Murchu et coll. 1994).
La tolCrance rauite A I'ischemie perophtoire serait secondaire aux changements
pathophysiologiques apparaissant durant le dkveloppement de I ' insuffisance cardiaque.
De plus, outre I'utilisation d'une borne mCthode de protection myocardique, il est
essentiel de pro&der A une sklection appropriee des patients pouvant bdneficier d'une
chirurgie de revascular isation. Les insufisants wdiaques qui ont une symptomatolog ie
angineuse importante constituent de rneilleurs candidats au pontage coronarien. En effet,
il a tti demontre qu'une dysfonction ventriculaire causb par une reduction du dtbit
coronarien peut etre partiellement ou complBternent rktablie par une revascularisation
coronarienne: il s'agit du phbnomhe du myocarde en hibernation (Rahimtoola 1993).
Des patients sans angine ou avec dyspnk peuvent aussi ttre de bons candidats au pontage
coronarien lorsqu'il y a evidence d'anomalies reversibles de perfusion myocardique observables Iors de la scintigraphie au thallium (Christakis et coll. 1992. Jegarden et coll.
1994).
3.3 Antagonistes du calcium dam llinsuff~ancecardiaque
Les antagonistes du calcium ont fait I'objet de plusieun travaux exptrimentaux visant B tvaluer leur eficacite en protection rnyocardique perop6ratoire. La plupart de ces travaux, qui consistent B ajouter un antagoniste du calcium A la solution cardiopltigique. sont rMisQ sur des coeun qui etaient a priori normaux. Cependant, comme il a etk mentiom5 antkrieurement, les chirurgies cardiaques sont de plus en plus fiequentes chez les patients d6iaillants. I1 est donc souhaitable d'utiliser des mod8les expdrimentaux d ' insufisance cardiaque afin de drifier 1'efficacii et 1' innocuie des antagonistes du calcium en protection myocardique peroperatoire.
Dam une etude recente* un rnodde canin d'hypertrophie ventriculaire a tt6 utilist afin d'etudier les effets de la cardiopl6gie crystalloide normothennique avec ou sans virapamil
(Huddleston et coll. 1992). Bien qu'aucune difference n'ait kt6 observee en ce qui a trait ii la prkrvation d' ATP, le v6raparnil a permis une meilleure rkupkration de la fonction systolique et diastolique apes 1' ischBmie globale. Ce modble d'hypertrophie induite par une sttnose aortique est associ6 P une diminution du contenu en phosphate a haute tnergie, une diminution du debit sanguin endocardique et une reduction de la compliance ventriculaire. 11 s'agit donc d'un modkle adequat afin d'btudier ['impact de l'hypertrophie sur la protection myocardique. Cependant, outre la diminution de la compliance myocardique, les auteurs ne font pas mention d'une alteration de la contractilite myocardique dam ce modble. I1 n'est donc pas possible, a partir de ces travaux, d'btablir un 1ien avec les situations cl iniques dam lesquelles les patients prkentent une dkfaillance cardiaque significative.
Clark et wll. (1983) ont ktudik l'efftcacit6 de la nifkciipine en ajout a la cardioplkgie crystalloide hypothermique chez des patients souffrant d ' insuffisance card iaque: la plupart des patients recnrtb dam cene 6mde etaient classif& en phase IV selon les crieres de
la New York Hem Assmimion. Ainsi, dam cette cattigorie de patients i risque eleve. la
nifedipine a permis d'am6liorer le debit cardiaque et le volume d't5jection systolique et de rCduire la rhistance vasculaire pulmonaire immediatement apr& Ia chhgie cardiaque. La nifuipine a awi rauit la mortalid postoperatoire immediate chez les
insuffints cardiaques (4 % dans le groupe trait6 versus 11 % dans le groupe thoin) en diminuant I ' incidence de dt5faillance aigue. Malgre ces resultats encourageants, les patients souffrantd' insuffisance cardiaque ont gbneralement ttk exclus de la plupart des essais cliniques dont le but Ctait d'ttudier les effets des antagonistes du calcium en protection myocardique peroperatoire (Barner et coil. 1987. Christakis et coll. 1986.
Flameng et coll. 1986. Guffin et coil. 1986, Trubel et coll. 1994).
La effets inotropes et chronotropes nbgatifs des antagonistes du calcium sont les raisons pour lesquelles leur utilisation en protection myocardique chez les insuffisants cardiaques est controvers& (Christakis et coll. 1986, Packer 1989). I1 a 6tt rapport6 que les effets inotropes negatifs des antagonistes du calcium sont amplifies dans certains modkles experimentaux d' insuffisance cardiaque (Ezzaher et coll. 199 1. Porter et coll. 1983).
Cependant, A la revue de la documentation scientifique, d'autres rnoddes de dtfaillance cardiaque n'entrainent pas d'arnplication de leurs effets cardiod6presseurs (Drexler et coll. 1985, Vkniant et coll. 1991). I1 est donc possible que la selection d'une dose appropriee dd'n antagoniste du calcium puisse entrainer une cardioprotection tout en kvitant des effets cardiaques d6ltteres. Les effets d6lCt6res des antagonistes du calcium dam I'insuff'iance cardiaque s'expliquent aussi par une stimulation secondaire des
syskmes mum-hormonaun (Packer 1989). Ce phbnornhe ne devrait =pendant pas poser
un probliime dam le contexte d'une protection myocardique peroptratoire puisque la
stimulation neuro- hormonale es t davantage associh B une utilisation chronique des
antagonistes du calcium. Cependant, il est possible que les dysfonctions vasculaires
associees B I'insuffisance cardiaque modulent I'efficacitt5 des antagonistes du calcium.
Vhiant et dl. (1991) ont d'ailleurs rappod que la reponse vasodilatatrice coronarienne du diltiazem est diminuk dam le coeur de rat difaillant et que ce ph6nombne serait 1% i I'hypertrophie cardiaque. Dans la defaillance cardiaque, le maintien de l'activid vasodilatatrice coronarienne et les bknkfices qui y sont associ6s n'ont pas fait I'objet d' Crudes sp6cifiques.
En conclusion, il apparait justifiC d 'effectuer des ch irurgies wdiaques chez Ies insuffisants cardiaques bien que la mortalit6 et la morbidid peropCratoire demeurent tlevks. Puisque peu d'ttudes ont port6 sur I'utilisation des antagonistes du calcium en protection myocardique perophtoire dam des rnoddes d'insuffisance cardiaque, il nous apparait indressant d'en caracdriser le potentiel cardioprotecteur dam cette situation. DEUYU~MEPARTIE
CONTRIBUTION ORIGINALE Hypothk de travail et buts du projet de recherche
Les antagonistes du calcium poss6dent des effets cardioprotecteurs observables dam
divers rnoddes d'ischkmie-reperfusion. Les effea inotropes nigatifs de ces composb
limitent leur utilisation en prbence d'insufisance cardiaque. Des etudes antkrieures
suggkrent que les antagonistes du calcium de la familles des benzothiazkpines procurent
une protection rnyocardique indipendarnment de leurs effeu cardiod~presseurs. Cette caracteristique favorise l'utilisation du diltiazem et du clentiazem en protection
myocardique perophtoire en prbence de dbfaillance cardiaque. De plus, Ie clentiazem semble possaer des propriktt5s cardioprotectrices distinctes du diltiazem puisqu'il s'agit d'une molCcule plus lipophile et plus vasosClective que son congtn2re.
L'objectif gCn6ral de ce projet de recherche est d'ttudier Ies effets protecteun du diltiazem et du clentiazem, en ajout il la cardiopligie, dam des coeurs ischemiques
normaux et dkfaillants.
La premiere etude consiste principalernent a tvaluer, dans le modble de coeur is016 de
lapin, les effets cardioprotecteurs du diltiazem et du clentiazem utilises en ajout A la cardiopltgie froide. Cette Ctude vise notamment it ttablir les doses eficaces et
~Ccuritairesdes benzothiazkpines et ii 6vaIuer la relation possible entre leurs proprittb physicochimiques et les mkcanismes regissant leurs effets cardioprotecteurs. La deuxieme siirie de travaux vise ii etudier, en presence d'insufisance cardiaque, l'efficacit6 et 1' innocuitt5 des benzothhdpines en ajout B la solution cardioplkgique. Pour ce faire la rkuperation post-ischkrnique est 6valu6e dam des coeurs isolQ de hamsters normaw et de hamsters catdiomyopathiques (souche UM-X7.I). 2gC de plus de 200 jours.
La troisi8me drie d'exp6riences vise B mieux d6finir Ies facteurs qui modulent l'efficacit6 des benzothiazepines en prbence de defaillance cardiaque. Les effets coronariens et cardiaques du diltiazem et du clentiazem sont 6valuks dam le coeur de hamster normal de m8me que dans le coeur defiillant du hamster cardiomyopathique. La contribution du
NO et des produits de la cyclooxyg6nase y est plus spCcifiquement documentee. Clentiazem, diltiazem and cold cardioplegia in isolated ischemic rabbit
hearts: Relation between additive cardioprotection, physicochemical
properties, and preservation of myocardial lipid components
Mario Tanguay, *Guy Lepage, ?Gilbert Blake, $Denis Garceau, and Louis Dumont
Dt!partemnt de phannacologie, Universifkde Montreal; *Service de Gusnoenzt!rologieet de Nutrition,
Centre tie Recherche P4diarrique. Hbpital Ste-Justine, Montrt!ak TDt!panernent d'Aneszht!sie, Hdpital
Notre-Dame, Monrrtat; and $Nordic Merrel Dow Research, Lad, Qukbec.
Journal of Curdiovascidar Pharmacology
Vol. 24, No. 6, pp.950-959, 1994 SUMMARY
D il tiazem is known to exert significant cardioprotection, but its efficacy under hypothermic conditions has not been documented. Because of its lipophilicity and its better tissue penetration, clentiazem, a chlorobenzothiazepine derivative of diltiazem, may offer cytoprotection additive to cold cardioplegia. We investigated the cardioprotective actions of clentiazem and diltiarern (lod and lo4 M) when added to cold cardioplegia
(myocardial temperature of 10°-120C),in isolated rabbit heart subjected to 90-min global ischemia. Functional recovery was assessed by measuring left ventricular developed pressure (LVDP), coronary flow (CF) and heart rate (HR). To explore the potential beneficial mechanisms of these agents, we measured myocardial lipids and total calcium at the end of a 30-min period of reperfusion as well as their myocardial accumulation.
Addition of M clentiazem to cold cardioplegia resulted in significant improvement in mechanical recovery (postischemic LVDP of 88.5 mm Hg with cardioplegia alone vs.
105.5 mm Hg with added clentiazem at 25 mm Hg diastolic pressure, DP). The additive cardioprotection afforded by clentiazem appeared to be concentration dependent since significant cardiodepression (@stischemic LVDP of 79.8 rnm Hg and 18 % reduction in
HR) was observed at a higher concentration (10d M) and these effects were correlated with myocardial accumulation of the drug. The additive cardioprotective effect of clentiazem appeared to be structure related because diltiazem at both and 106 M concentrations offered no benefits in addition to cold cardiopleg ia. These results indicate that the additive cardioprotection observed with 10d M clentiazem could be related to its coronary vasodilator action since it reversed the cold cardioplegia-induced attenuation of hyperemic CF at reperfusion. Other factors must be involved, however, because addition of lod M diltiazem resulted in increased postischemic CF but without improving myocardial recovery. The functional protection offered by 10d M clentiazern was associated with preservation of myocardial lipid components. Myocardial cholesterol content, which is essential for maintenance of membrane integrity, was preserved in that group, whereas a loss was observed in groups treated with cardioplegia alone and in the other treated groups. Total myocardial phospholipids were preserved in groups receiving
M clentiazem plus cold cardioplegia or cold cardioplegia alone. A reduction in plasmalogen content, the predominant myocardial phospholipid species, and an increase in total myocardial calcium were noted only in ischemic hearts that received neither cardioplegia nor benzothiazepines, suggesting that cold cardioplegia is sufficient to prevent irreversible damage. Clentiazem affords cardioprotective benefits additive to cold cardioplegia. Its effectiveness apparently is related to some physicochemical properties, its coronary vasodilator action, and direct cytoprotection through preservation of essential myocardial 1ip id components.
Key Words: Benzothiazepines, cardioplegia, mrdioprotection, coronary flow, myocardial contractility, reperfus ion injury. INTRODUCTION
Cardiop leg ia and hypothen ia provide effective protection during extensive cardiac surgery, but because the incidence of cardiac failure increases during prolonged period of cardiac arrest, particularly in patients with poor preoperative ventricular function, developing better methods of cardioprotection is of vital cl inid relevance. Considerable evidence suggests that calcium overload contributes to ischemia/ reperfusion injury. In this context, use of calcium antagonists in addition to cardioplegic solutions has been proposed. Many experiments have been conducted with diltiazem, nifedipine, and veraparnil (1). Most studies led to the conclusion that these calcium antagonists exert cardioprotective effects when used in normothermic (37OC) cardioplegia. However, these drugs apparently afford no additional protection under hypothermic (r20°C) conditions
(2-5). To explain these findings, the slow calcium channel under hypothermia has been proposed to be unresponsive to calcium entry blockers (2). Hypothermia itself also may act to reduce calcium overload and, as such, mask or render redundant the protective properties of calcium antagonists (6). However, calcium antagonists could exert a protective effect against ischemic injury by mechanisms other than energy sparing andlor direct prevention of calcium overload. Some investigators have reported that calcium antagonists offer cardioprotection independent of cardiodepression (7-9) and have suggested that improved perfusion to the inner layers of the heart muscle might be the mechanism involved. In addition, protection of membrane lipid components such as cholesterol and phospholipids may be the possible target of calcium antagonists against the deleterious consequences of ischemia and reperfusion (10,ll). Plasmalogens, which are the predominant phospholipid components in the myocardium, might play a significant role in membrane enzyme stabilization and ion channel function (1 1).
Among the calcium antagonists, benzothiazepines appear to be particularly attractive as cardioprotective agents. Diltiazem has been shown to protect the ischemic myocardium at a lower functional cost than verapamil or nifedipine (8,12). Clentiazem (TA-3090). a chlorobenzothiazepine derivative, has also been shown to have a beneficial effect on
ATP levels and mechanical recovery at concentrations that do not reduce myocardial recovery (13). Indeed, clentiazem prevented reperfusion injury afier a 90-min coronary occlusion and 72-h reperfusion in dogs (14). Clentiazem may exert its cardioprotective effects because of its tissue retention and long duration of action even under hypothermic conditions (15-17).
We investigated the cardioprotective effects of equimolar concentrations of clentiazem and diltiazem, when added to cold cardioplegia, in isolated globally ischemic rabbit hearts. Furthermore, we explored the potential pathophysiologic mechanisms involved, such as disturbances in myocardial cholesterol, total phospholipids and plasmalogens, as we1 1 as calcium homeostasis. We also examined correlation between the myocardial concentrations of these drugs and their expected cardioprotective effects. MATERIALS AND METHODS
Heart preparations
New Zealand white rabbits weighing 1.8-2.4 kg from Lapins LRonard, St-Scholastique,
Quebec, Canada, were used in all experiments. Animals were cared for in accordance with the principles of the Guide to the Care and Use of &penmental Animals, published by the Canadian Council on Animal Care (1981, 1984). After cervical dislocation, the hearts were cannulated and cooled in sku, quickly excised, and perfused by Langendorff method with a modified Krebs-Ringer buffer consisting (in rnM) of NaCl 119, KC1 4.8,
CaCl, 1.3, KH,PO, 1.2, MgSO, 1.2, NaHCQ 25, and glucose 15. The buffer was continuously gassed with 95 % 02/5% C02(pH 7.4 at 37OC), and perfusion pressure was kept constant at 85 cm H,O. All hearts were maintained at 37OC throughout the perfus ion period in a temperature-controlled chamber.
Coronary flow (CF)was monitored by an ultrasonic flow probe (model T201, Transonic
Systems, Ithaca, NY, U .S. A.) in the perfusion line. Isovolumetric left ventricular developed pressure (LVDP)was measured with a sal ine-filled latex balloon introduced into the left ventricle through the left atrium and connected to a pressure transducer
(model P23AC, Statham Instruments, Hato Rey , PR, U.S. A.). Diastolic pressure (DP) was varied by altering balloon volume. Heart rate (HR) was allowed to change spontaneously and was measured from electrograrn recordings obtained through electrodes attached to the atrium and the apex. All physiologic measurements were recorded on a multichannel recorder (Polygraph model SD, Grass Instruments, Quincy , MA, U. S .A .) .
Myocardial temperature was also monitored continuously by a thermistor (temperature monitor model BAT-10, Physitemp Instruments, Clifton, NJ, U.S.A.) implanted in the
LV wall.
Experimental procedures
After a 30-min equilibration period, a control LV function curve (Frank-Starling curve) was generated by incrementally varying DP (5, 15, and 25 mm Hg). The intra- ventricular balloon was then deflated, and control values of CF and HR were measured after a 5- to 10-min stabilization period. The hearts were then rendered globally ischemic by clamping the aortic line and immediately infusing a cardioplegic solution by opening a line attached to a sidearm of the aortic cannula. St. Thomas' Hospital cardioplegic solution (4OC) was infused at a pressure equivalent to 100 cm H,O until a myocardial temperature of 10°-120C was reached. The cardioplegic solution was composed (in mM) ofNaCl 110.0, KC1 16.0, MgCI, 16.0, CaCl, 1.2, and NaHC03 lO.O(pH7.8and280 mOsm/L) with or without 10d or M clentiazem or diltiazem. It was filtered (0.45 pm) before infusion. Clentiazem and diltiazem concentrations correlated with the plasmatic range observed during previous in vivo studies (18,19). During 90-min global ischemia, the hearts were left at room temperature. At the end of this period, myocardial temperature was -15°-180C. The hearts were then reperfused for 30 rnin. CF and HR were monitored continuously, whereas a LV function curve was assessed after 15-min reperfusion. Isolated untreated ischemic preparations were also used in this protocol.
At its completion, the hearts were frozen in liquid nitrogen for further analyses.
Myocardial samples analysis
Myocardiial ~ie~azernand diIn'uem levek Myocardial clentiazem concentrations were assayed by high-pressure liquid chromatography (HPLC)(19). Myocardial samples (200 mg) were homogenized in 2 m1 sterile water. An internal standard (deacetylated metabolite of diltiazem, 0.1 mg % solution) was added to the myocardial homogenates.
Extraction was performed with 6 ml diethylether and 3 ml phosphate buffer (0.05 M,pH
7.5). After agitation (10 min) and centrifugation (2500 rpm, 10 min), the organic phase was back-extracted with 150 ml 0.05 N H2S04. The acidic phase was collected after centrifugation (2500 rpm, 4 min), and 50 pl was injected into the chromatograph.
Analysis of myocardial diltiazem samples was very similar to that already described except that 0.1 mg% loxapin was used as the internal standard and 6 ml tert-butyl-methylether was used as the extraction solvent (20). The limit of clentiazem and diltiazem detection was 2.5 ng/ml (25 nglg myocardial tissue). Calibration curves were constructed for concentrations ranging 2.5 to 200 nglml , corresponding to 25-2,000 ng/g myocardial tissue. The squared linear regression coefficients (fi were 0.9937 and
0.9922 for clentiazem and diltiazem, respectively. The coefficient of variation never exceeded 10%for any datapoint. Myocardial lipid components. Phospholipids and cholesterol were measured according to methods described in previous publications. Total cholesterol was assessed enzymatically Whringer Mannheim, MontrM, Qudbec, Canada) after lipid extraction from myocardial homogenates (21). Phospholipids were separated by thin-layer chromatography (TLC)(22) and briefly visualized with iodine vapor. The phospholipid band was scraped off and directly tramesterifid with 200 pl acetylchloride. after addition of 250 run01 C13:O as internal standard, in 2 ml methanol-hexane 4: 1 (vol/vol) (23). The resulting fatty acid methyl esters were injected into a 60-m fused silica capillary column coated with SP-2331. Analysis was performed on a Hewlett-Packard 5880 gas chromatograph as described earlier (24). Plasmalogen concentrations were calculated from the nascent dimethylaced peaks after direct transesterification of the phospholipids containing a vinylether bond in the sn-1 position. A11 values are expressed in nanomoles per milligram of protein. Proteins were measured according to the method of Lowry and colleagues (25). with bovine serum albumin as standard.
Total cakiwn content. Myocardial calcium content was quantitated by atomic absorption flame photometry (Perkin-Elmer 2380) according to the wet ashing-oxidation procedure
(26). Approximately 1 g venticular tissue was used for extraction with 8 ml concentrated acid solution: HNO,:HCIO,:H,O (1 :1 :1 [vol/voI]). After being boiled for 10 min, each sample was passed through a 0.8-pm cellulose filter (Millex); 1 ml 10% LaCI, and 1 ml
1% KC1 was then added, and the sample volume was completed to 10 ml with deionized water. Standard calcium solutions of 0.25, 0.5, 1, 2, 3 and, 5 ppm (wt/vol) were prepared, and calibration curves were charted. Results are expressed as nanomoles per milligram of protein.
Statistical analysis
All values are mem f SEM of six isolated preparations. LVDP is expressed as absolute values in mrn Hg, and temporal changes in CF and HR were measured as percentages of preischernic values. We computed multigroup statistics by analysis of variance, using the
LSD (least significant difference) procedure to determine differences between groups.
Multiple comparisons of postischemic LVDP were assessed with baseline values as covariates. Differences were considered statistically significant at p < 0.05.
Drugs and chemicals
Clentiazem (maleate salt) and diltiazem (chlorhydrate salt) were donated by Nordic
Merrell Dow Research, Laval, Qutbec, Canada. Both drugs were dissolved in distilled water and diluted in cardioplegic solution just before the experiments. Plcgisol , the card iopleg ic solution, was purchased from Abbott Laboratories, Monthl, Quebec,
Canada; its pH was adjusted to 7.8 before use by addition of 8.4 56 sodium bicarbonate,
USP, Abbott. This solution was refrigerated and stored no longer than 24 h. RESULTS
No differences among groups were observed either in the volume of cardioplegic solution administered (44 f 2 ml) or in duration of perfusion (73 f 3 s). Temperature in myocardium after infusion of the cold cardioplegic solution was also similar for all groups (11.3 f 0.2O).
Effects of clentiazem and diltiazem, as additives to cold cardioplegia, on postischemic LV fundion
Table 1 shows LVDP recovery values obtained under different DP (5, 15, and 25 mm
Hg) after 90-min ischemia and 15-min reperfusion. These results confirmed the beneficial effects of wld cardioplegia against ischemia since postischemic myocardial
LVDP. at 25 rnrnHg DP. was 88.5 f 3.8 rnrn Hg (80 f 2 % recovery) with cardioplegia as compared with 75.7 4.4 mmHg (72 * 3 % recovery) in the untreated ischemic group (p c0.05). Addition of 10d M clentiazem significantly enhanced the protective effects of cold cardioplegia on LVDP regardless of the stress imposed on the myocardium
(5-25 mm Hg DP). An LVDP reading of 105.5 * 2.4 mrn Hg (89 * 1% recovery) was obtained in the 108 M clentiazem group at a DP of 25 mm Hg @ 79.8 f 5.2 mmHg (70 f 4% recovery) at 25 mm Hg DP. Diltiazem, at both concentrations (1m8 and lo4 M), provided no significant additive cardioprotection to cardioplegia; postischemic LVDP values of 92.8 * 4.2 mm Hg (82 f 1% recovery) and TABLE 1. Efect of clentiazem and dilriazenr added to cold camioplelpia on repemion mecltanical recovery of isolated rabbit heart subjected to Wnlin ischemia" 5 rnm Hg DP 15 mm Hg DP 25 mm Hg DP Be fore After Before After Before After Untreated ischemic group 103.3 f 1.7 68.2 f 4.3b 106.0 f 2,4 73.2 f 4,4b 104.7 f 3.6 75.7 f 4.4b Cardioplegia alone 107.0 * 3.8 11.3 f 2.7 111.0 f 4.5 87.2 & 3.3 110.5 f 4.3 88.5 f 3.8 Cardioplegia + M clentiazem 109.5 f 2.3 94.0 f 4Jb 119.0 & 2.8 103.0 f 3.0b 118.7 f 2.8 105.5 If: 2,4b Cardioplegia + M clentiazem 1 13.5 & 4.4 7 1.5 f 5.4b 116.3 f 3.9 78.2 f 5.Bb 1 15.2 & 4.2 79.8 f 5,Zb Cardioplegia + M diltiazem 109.3 f 4.7 90.5 rt: 3.7 113.0 f 4.7 93.7 * 4.3 112.7 f 4.1 92.8 f 4.2 Cardioplegia + M diltiazem 99.7 f 4.4 78.0 f 4.1 104.3 f 4.6 84.2 & 4.2 102.5 f 4.5 84.0 f 3.7 DP, diastolic pressure " Left ventricular developed pressure (mm Hg) as tbnction of preload (DP), before and 15 min after 90-min global ischemia. Values are mean f SE of six hearts in each group. Significantly different from hearts trealed with cold cardioplegia alone (p < 0.05). 84.0 rt 3.7 mm Hg (82 f 3 % recovery) were recorded for 10-8 and 106 M diltiazem, respectively, at 25 mm Hg DP. Effects of diltiazem and clentiazem, as additives to cold cardioplegia, on postischemic CF and HR CF recovery during the 30-min reperfusion period is shown in Fig. 1. The hyperemia observed early during reperfusion (0-15 min) in the untreated ischemic group was significantly attenuated by cold cardioplegia alone. Addition of 104 M clentiazem to the cardiopleg ic solution provided significantly higher coronary perfusion throughout reperfusion as compared with cold cardioplegia alone (at 30-min reperfision: 110 f 5 % of the preischemic value with 10" M clentiazem vs. 79 & 4% of the preischemic value with cardioplegia alone). However, clentiazem at lo4 M did not cause any change in CF as compared with wld cardioplegia alone (80 f 6 2 of the preischemic value, at 30 min of reperfusion). In comparison, diltiazem had no effect on CF at 10" M (84 k 3% of the preischemic value), whereas a signiticant sustained increase was observed (107 f 6% of the preischernic value) at the lo4 M concentration. 0 Ischemia alone CardiopIegia alone A Cardioplegia + IPM clentiazem Cardioplegia + I Od M clentiazem 3 Ischemia alone Cardioplegia alone Cardioplegia + t O3 M diltiazem Cardioplegia + 166 M diltiazern Reperfusion time (min) FIG. 1. Effects of clentiazem (A) and diltiazem (B) added to cold cardiopIegia on reperfusion recovery of coronary flow in rabbit heart subjected to 90-min global ischemia. Values are mean f SE of six hearts in each group. **p < 0.01 as compared with hem treated with cold cardioplegia alone. HR values at recovery are shown in Fig. 2. The HR increase observed early during reperfusion (10 min) in the untreated ischemic group was significantly attenuated by cold cardioplegia alone (104 f 3 % of preischemic HR in the untreated group vs. 94 + 2 X of the preischemic value with cardioplegia at 10 min of reperfusion). No negative chronotropic effects were apparent with addition of either 1C8 M clentiazem, 10-8 M diltiazem, or 1FM diltiazem to the cold cardioplegic solution. As compared with preischernic values, HR changes at 30 rnin of reperfusion averaged 100 f 4% for lom8 M clentiazem and 97 f 2% and 102 6% for 10-'M diltiazem and lo6 M diltiazem, respectively. However, 106 M clentiazem significantly reduced HR at reperfusion (82 f 4% of the preischemic value at 30 rnin of reperfusion as compared with 94 * 1% with cardiopleg ia alone). Myocardial dent iazem and diItiazem levels The myocardial levels of clentiazem and diltiazem were similar when low concentrations (1W M) were used. In both groups, only one of six myocardial samples had detectable levels (clentiazem 57 ng/g, diltiazem 25 ng/g). However, when high concentrations (lo6 M) were added to cold cardiopleg ia, higher myocardial levels of these benzothiazepines were detected: the clentiazern level being 6.5 times that of diltiazem (728 & 82 vs. 112 f 41 ng/g). Figure 3 shows the relation between myocardial levels of these drugs and post ischemic contractile impairment. With 1pM clentiazem, LVDP reduction was correlated with significant myocardial accumulation. 130 - A O Ischemia alone Cardioplegia alone A Cardioplegia + 108 M clentiazm la, - Cardioplegia + 106 M clcntkern 110 - 100 - 80I B a Ischemia alone Cardioplegia alone A Cardioplegia + 1V M diltiazm Cardioplegia + 1 W M diltiarem 0 10 20 30 Reperfusion time (mitt) FIG. 2. Effects of clentiazem (A) and diltiazem (B) added to cold cardioplegia on reperfusion recovery of heart rate in rabbit heart subjected to 90-min global ischemia. Values are mean * SE of six hearts in each group. Lp < 0.05 and **p <0.0 1 as compared with hem treated with cold cardioplegia alone. FIG. 3. Relation between myocardial clentiazem and diltiazem levels and postischemic mechanical impairment. Myocardial levels of the drugs were measured after 90-min global ischemia followed by 30-min aerobic perfusion. Postischemic left ventricular developed pressure (LVDP)impairment was determined after 90-min ischemia and 15-min reperfusion. Values obtained with 25 mm Hg diastolic pressure are indicated. Values are mean f SE of six hearts in each group, except for myocardial concentrations obtained after 108 M treatments, in which only one of six samples had detectable levels. Myocardial lipid components Table 2 shows the myocardial levels of cholesterol, total phospholipids, plasmalogens, and proteins of the different groups. Ischemia tended @=0.11) to decrease myocardial cholesterol, whereas cardioplegia significantly reduced it further. Moreover, 106 M clentiazem as well as both concentrations of diltiazem accelerated the loss of myocardial cholesterol. On the other hand, this sterol was preserved when 10' M cclntiazem was added to cold cardioplegia. Total phospholipids were significantly reduced in the untreated ischemic group, with 10d and 10d M diltiazem as well as with 104 M clentiazem. In contrast, cardioplegia or addition of 10d M clentiazem preserved total phospholipids. Plasmalogens, which accounted for 27 % of myocardial phospholipids, were significantly decreased by ischemia, whereas no significant changes were observed in the other groups. Myocardial edema was detected only in the untreated ischemic group, since protein content was significantly reduced in that group. Total calcium content Total myocardial calcium content is shown in Fig. 4. Total myocardial calcium was significantly increased in the ischemic group as compared with the controls (10.54 * 1.7 1 vs. 7.34 & 0.47 nrnol/mg protein), whereas no significant change was observed with cardioplegia alone (7.58 f 0.42 nmol/mg protein), with clentimm (7.36 it 0.23nmoIlmg protein) or diltiazem (6.52 -+ 0.58 nmol/mg protein) when added at a concentration of A4 to the cardioplegic solution. U control ischemia alone cardioplegia- - alone pa cardioplegia + 1 0'8 M clentiazem cardioplegia + 1 0-8M diltiazem FIG. 4. Effects of clentiazem and diltiazem added to cold cardioplegia on total myocardial calcium content. Hearts were perfused for 30-45 rnin under aerobic conditions, followed by Wmin global ischemia and 30-min aerobic reperfusion. Control hearts were frozen after 30-45 min of aerobic perfusion (stabilization period). Values are the means f SE of six hearts in each group. *p < 0.05 as compared with control nonischemic group. DISCUSSION Our results demonstrated additive cardioprotection with a calcium antagonist (clentiazem) during hypothermic crystalloid cardioplegia in an isolated heart preparation. The mechanisms of cardioprotection apparently are related to the physicochernical properties of clentiazem , its coronary vasodilator effect, and to its direct cytoprotective actions preserving membrane lipid components. Several experimental models have been used to investigate the beneficial effects of calcium antagonists additive to cardioplegia in ischemialreperfusion (1). In the present study. the isolated perfused rabbit heart model allowed us to examine the direct protective act ions of benzothiazepinel ike calcium antagonists on the myocardium, thereby eliminating the contribution of their peripheral effects, such as a reduction of afterload (27) or their impact on blood components (28-31). Furthermore, the duration of ischemia and the mode of administration of the cardioplegic solution closely reproduced clinical conditions (32,33). Most of the experimental studies performed on isolated hearts involved use of a large volume of cardioplegic solution (20-30 ml/g myocardium) whereas 500-1,000 ml of the solution is usually administered clinically, corresponding to 2-4 mllg myocardium (3434). In the present investigation, duration of cardioplegic solution administration was determined according to myocardial temperature. The infusion was discontinued as soon as myocardial temperature reached lo0- 12OC. This method resulted in a mean infusion volume of 6.4 ml/g myocardium, which more closely approximates the volumes used clinically as compared with that in previous isolated heart studies. Many agents have been included in cardioplegic solutions in an attempt to enhance cardioprotection (35). One strategy consisted of use of calcium antagonists. Experimental s~diesof isolated perfused heart have shown that these agents exert significant cardioprotection when administered as adjuncts to normothermic cardioplegia (1). Somewhat less clear is their beneficial effect when combined with hypothermic card iopleg ia. Nifedipine and diltiazem have been reported to enhance the cardioprotection offered by cold cardioplegia during cardiopulmonary bypass in animals, as demonstrated by better recovery of LV dWdt (36,37). However, most isolated heart studies performed under hypothermic conditions have demonstrated no additional protection by calcium antagonists (2-5). To explain this absence of cardioprotection at low temperatures, it has been suggested that under hypothermia the slow calcium channel may be unresponsive to calcium antagonists (2). Hypothermia and calcium antagonists have been postulated to afford cardioprotection through a common mechanism, with the former masking the protective mechanisms of the latter (6). Moreover, the suppressing effect of calcium antagonists on generation of contractions in ventricular myocytes, as determined by the maximal possible contraction frequency under electrical stimulation, is reduced at < 27OC, so that 10- to 100-fold higher concentrations are needed for the same relative action as that which occurs at 37OC (38). Hypothennia has been speculated to induce conformational changes of the calcium channel or surrounding membrane, as a consequence of temperature-dependent membrane lipid phase transition (6) which may affkct membrane incorporation of calcium antagonists during hypothermia. Our results indicate that additive cardioprotection was obtained with a molecule exerting better tissue penetration (clentiazern). Amounts below the detection limit have been disclosed with both 1pM clentiazem and diltiazem , but the experimental design (30-min reperfusion) did not allow us to measure the concentration present during ischemia and at onset of reperfusion. Several mechanisms have been proposed to explain the audioprotective effects of calcium antagonists. These compounds could exert their beneficial actions by reducing myocardial contractility, thus preserving high-energy phosphate and decreasing 0, consump tion (39). but they may afford cardioprotection in the absence of cardiodepressive effects (7-9). The coronary vasodilating properties of calcium antagonists may explain their beneficial actions (9). Other mechanisms of cardioprotection have been suggested, such as prevention of calcium overload, reduction of lipid peroxidation, and preservation of cell membrane integrity (4O,4 1). The beneficial effects of clentiazem and diltiazem on the ischemic myocardium have also been suggested to be due to decreased energy demand and reduced glycolysis during ischemia (13,42). In the present study, clentiazem M significantly enhanced the beneficial impact of cold cardioplegia on LVDP recovery. This beneficial action was not observed with either 108 or 106 M diltiazem. Because clentiazem is more lipophilic than diltiazem, our data suggest that this physicochernical property influences the degree of cardioprotection. Based on previous data obtained in our laboratory as well as those from other groups of investigators, a 10d M concentration of clentiazem is below the level that could induce cardiodepression (13,43,44). Thus, the beneficial effect of clentiazem probably is not due to a negative inotropic action, and cardioprotection appears to be independent of cardiodepression. Indeed, Murashita and coworkers (45) demonstrated that addition of diltiazem, at 1% or IC, negative inotropic concentrations, to St. Thomas 's Hospital card iopleg ic solution does not improve postischemic recovery in isolated rabbit heart. Because improved postischemic CF was observed with addition of loa8M clentiazem to cold cardioplegia, coronary vasodilation could accelerate removal of detrimental ischemic byproducts and provide a better supply of nutrients (9). Therefore, increased CF appears to be an important factor contributing to cardioprotection. Other factors must be involved in cardioprotection, however, since addition of lo4 M diltiazem to the cold cardioplegic solution enhanced postischemic CF but did not improve myocardial recovery. Membrane incorporation of calcium antagonists may influence their ability to preserve specific membrane components during ischemia/reperfusion. In the present protocol, add it ion of 10d M clentiazem to cold cardioplegia preserved myocardial cholesterol, whereas cholesterol loss was observed in the other groups studied. An adequate cholesterol level in the membrane contributes to decreased conformational flexibility of the hydrocarbon chains of lipids, leading to a reduction in membrane permeability to small uncharged molecules (46). In addition, cholesterol regulates the activity of membrane proteins critical to cellular function, such as Naf -K+-ATPase (46). The acetylcholine receptor is another example of membrane proteins that apparently require cholesterol to function properly (47). Moreover, sarcolemmal cholesterol depletion has been observed in rat heart cell cultures used as an "ischemic modelwdeprived of O2and metabolic substrates (48). Therefore, the cardioprotection afforded by clentiazem may be related to preservation of membrane integrity through an interaction in myocardial cholesterol concentration. Alterations in membrane phospholipid metabolism may also play an important role in the pathogenesis of ischemic myocardial cell injury (49). Depletion of total phospholipids has been reported previously in dog heart after ischemia (50-52). Studies of isolated hearts have shown an accumulation of 1ysophosphoi ipids and nonesterified fatty acids, such as arachidonic acid, after 1-h ischemia (5354). The untreated ischemic hearts in the present investigation had reduced tissue levels of phospholipids, whereas cold cardioplegia pre~e~edphospholipid content. In contrast, membrane phosphol ipids in lo6 M clentkm- and diltiazem-perfused hearts appeared to undergo spontaneous hydrolysis, as evidencsd by their decreased levels. Integrity of phospholipids was preserved with A4 clentiazem. These observations corroborate previous findings of Otani and associates (55) in an in situ pig heart model subjected to left anterior descending coronary artery occlusion followed by 60-min hypotherm ic potassium cardioplegic arrest and 60-min reperfusion. Otani and associates (55) reported a reduction in total membrane phospholipids after reperfirsion in the occluded area, when cardioplegia was not applied, whereas no change was noted in the protected ischemic area of the myocardium that had been exposed to cold cardioplegia. Our results suggest that cold cardioplegia may prevent myocardial phospholipid loss and that the additive cardioprotective effects of clentiazem are not related to preservation of phospholipids. Plasmalogens are predominant phospholipid constituents of the myocard id sarcolemma and sarcoplasmic reticulum membranes (56). A recent study showed that activation of plasmalogen-selective phospholipase A, is one of the earliest biochern ical alterations in ischemic myocardium. Results demonstrated that membrane-associated , calcium-independent plasmalogen-selective phospholipase A, activity increased > 400 % during 2-min global ischemia in Langendorff-perfused rabbit heart (57) and remained increased throughout the ischemic episode and was rap idly reversed during reperfusion. In our study, significant plasmalogen reduction was noted in the untreated ischemic group, but no significant change was noted in those receiving cold cardioplegia, with or without calcium antagonists. The precise role of this phospholipid class is uncertain, but possibilities include stabilization of membrane enzyme systems and ion channels in the sarcolemma and sarcolemmal protection from free radical attack (1 1). Our results indicate that cold cardioplegia with or without benzothiazepinelike calcium antagonists are effective plasmalogen protective agents. Because differential myocardial recovery was obsenred among groups, factors other than plasmalogen preservation must be implicated in cardioprotection. A certain degree of myocardial contractile dysfunction is required to evoke discernible changes in plasmalogen species. In addition, the alterations in plasmalogen levels observed in our study were correlated with tissue edema, confirming the possible influence of these phospholipid species on membrane fluidity and stability (58). Previous investigations support the pathogenetic role of calcium overload in myocardial stunning after global ischemia in isolated heart (59). In the present study. total myocardial calcium was increased only in the untreated ischemic group. This significant increase paralleled the other significant alterations recorded, i-e., the LV functional impairment, tissue edema. and reduced cholesterol, phospholipid, and plasmalogen levels. Cold cardioplegia appears to be sufficient to prevent calcium accumulation although we cannot rule out that addition of calcium antagonists may contribute to lower cytosolic free calcium levels, as reported by other researchers (59.60). The dose range of calcium antagonists is an important factor to consider both in their use as cardioprotective agents as well as for the sake of safety. Indeed, clentiazem has a narrower therapeutic range than diltiazem. In the present study, only the addition of lo4 M clentiazem to the cardioplegic solution resulted in reduced LVDP recovery as compared with cardiopleg ia alone. Myocardial depression was correlated with an increased myocardial concentration of clentiazem; 6.5 times higher than that with diltiazem used at an equivalent dose in cold cardioplegia. These results are in accordance with results of previous studies demonstrating that clentiazem is less easily washed from myocardial or vascular tissue than diltiazem (43,44,61). Even if the findings of the present investigation indicate that a single dose of clentiazem (10d M) is safe, its safety when administered in rnultidose cardioplegia, which is common clinical practice, is not known (62). Therefore, we conducted six experiments to verify myocardial recovery after 90-rnin ischemia associated with repeated administration (every 30 min) of cold cardioplegia with 1P M clentiazem. LVDP was improved (87 f 1% recovery) with detectable myocardial clentiazem concentrations averaging 57 f 10 ng/g (data not shown). Moreover, in these experiments, repeated administration of cold cardiopleg ia with clentiazem induced no negative chronotropic effects. Our observations confirm the safety of M clentiazem as an additive to cold cardioplegia. The clinical efficacy of calcium antagonists as cardioprotective agents has been investigated. Their potential beneficial effects may derive from improvement of cardioplegia delivery by coronary vasodilation (63). These agents may reduce postoperative arrhythmias (64) and prevent postoperative coronary spasm (65). In some studies, addition of diltiazem to cold cardioplegia decreased perioperative ischemic injury, as assessed by postoperative creatine kinase-MB activity, but resulted in conduction abnormalities, indicating the complexity of its therapeutic use (66,67). Although our results demonstrate a cardioprotective effect of clentiazem additive to cold cardioplegia, no data are available on its efficacy in cardiac disease models. Indeed, clinically, to balance the riskhenefit ratio, clentiazem dose-response correlations must be carefully established before it is used as an adjunct to cardioplegia. Clentiazem, but not diltiazem, appears to be an effective cardioprotective agent as an adjunct to cold crystalloid potassium cardiopleg ia. Its beneficial actions may be related to coronary vasodilation and direct cytoprotection through preservation of essential myowdial lipid components. ACKNOWLEDGMENTS: This work was supported by grants from the Canadian Heart and Stroke Foundation and Nordic Merrel Dow Research. M.T. was the recipient of a studentship award from Nordic Merrel Dow Research and FCAR. We thank Nancy Ronco from H6pital Ste-Justine, Judith Pignac, Depanement de pharmacologie, U nivers it6 de Monu&l, and Gilles Degagnk, Faculd de Pharmacie, Universitk de MontrM, for invaluable technical assistance. REFERENCES De Jong JW. Cardioplegia and calcium antagonists: a review. Ann Thorac Surg 1986;42:593-98. Chiavarelli M, Chiavarelli R, Macchiarelli A, Carpi A, Marino B. Calcium entry blockers and cardioplegia: interaction between nifedipine, potassium, and hypo- thermia. Ann Thorac Surg l986;41535-4 1. Fukunami M, Hearse DJ. Temperature-dependency of nifedipine as a protective agent during cardioplegia in the rat. Cardiovasc Res 1985; 19: 95- 103. Heane DJ, Yamamoto F, Shattock MJ. 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Chapitre 5 Resistance of the failing dystrophic hamster heart to the cardioprotective effects of diltiazem and clentiazem: evidence of coronary vascular dysfunctions Mario Tanguay, Gatitan Jasmin, Gilbert Blaise and Louis Dumont Dt?panemenrsde phamcologie, de puihologie er d'anesthesie, Facufrt?de Medecine Universitt?de Morrtrkal, Montrkal, Q C, Canada Canadian Journal of Physiology and Pharmacology Vol. 73, pp. 1108-1117, 1995 ABSTRACT Although hypothermia and cardioplegic cardiac arrest provide effective protection during cardiac surgery, ischemia of long duration, poor preoperative myocardial function, and ventricular hypertrophy may lead to heterogeneous delivery of cardioplegic solutions. incomplete protection, and impaired postischemic recovery. Calcium antagonists are potent card ioprotect ive agents, but their efficacy in the presence of cold card iopleg ia is still controversial, especially in heart failure, since it is often believed that failing hearts are more sensitive to their negative inotropic and chronotropic actions. However, recent data have demonstrated that the benzothiazepine-like calcium antagonists diltiazem and clentiazem, in selected dose ranges, elicit significant cardioprotection independently of intrinsic cardiodepression, thus lending support to their use in cardioprotective maneuvers involving the failing heart. We therefore evaluated the cardioprotective interaction of diltiazem, clentiazern, and cold cardioplegia in both normal and failing ischemic hearts. Hearts were excised from 200- to 225day-old cardiomyopathic hamsters (CMHs) of the UM-X7.1 line and age-matched normal healthy controls. Ex vivo perfusion was performed at a constant pressure (140 cmH,O; 1 cm H,O = 98.1 Pa) according to the method of Langendorff. Heart rate, left ventricular developed pressure (LVDP), and coronary flow were monitored throughout the study. Global ischemia was produced for 90 min by shutting down the perhate flow, followed by reperfusion for 30 min. Normal and failing CMH hearts were either untreated (control) or perfused at the onset of global ischemia with one of the following combinations: cold cardioplegia alone (St. Thomas' Hospital mrdioplegic solution, 4"C, infused for 2 rnin), cold cardioplegia + 10 nM d iltiazem, or cold card ioplegia + 10 nM clentiazem. The cardiac and coronary dilator properties of 10 nM diltiazern and 10 nM clentiazem alone were investigated in separate groups of isolated preparations. Failing CMH hearts had lower basal LVDP (42 f2 vs. 77i2 mmHg for norrnal hearts, p < O.O5), while coronary flow was only slightly reduced (5.6f 0.2 vs. 6.2 f0.2 mL1min for normal hearts). Following 90 rnin global ischemia, coronary flow was increased in both groups, but the peak hyperemic response declined only in failing CMH hearts (+SO& 17% vs. +82* 17% in normal hearts). In normal hearts, LVDP virtually recovered within 5 min of reperfusion but steadily decreased thereafter (-37f 4 % at 30 min). In contrast, in failing CMH hearts, LVDP significantly decreased early during reperfusion but improved over time (- 19& 7 % at 30 min). In normal hearts, the addition of diltiazem or clentiazem to cold cardioplegic solutions resulted in improved postischemic contractile function for the duration of reperfusion (85 f4 76 vs. only 7 1f 6 56 for cardioplegia, p < 0.05). The postischemic increase in coronary flow was similar in all groups. In failing CMH hearts, the addition of diltiazem or clentiazem afforded no significant contractile benefit at reperfusion. In nonischemic norrnal hearts, infusion of diltiazem or clentiazem (10 nM) alone increased coronary flow (+6f 1% for diltiazem and +24f 3 % for clentiazem) without significant negative inotropic or chronotropic effects. In nonischemic failing CMH hearts, infusion of diltiazem or clentiazem did not elicit cardiodepression. By contrast their coronary dilator actions reverted to vasoconstriction (diltiazem) or were significantly attenuated (clentiazem). From these experiments we can conclude that, compared with the normal heart, the failing CMH heart adapted differently to global ischemia. In addition to potential alterations in membrane integrity and changes in calcium hand1 ing , attenuation of the coronary dilator response to diltiazem and clentiazem rather than an increased sensitivity to their intrinsic cardiodepressant actions appears as a potential contributor to the lack of cardioprotection by these calcium antagonists in the failing CMH hearts. Key words: heart failure, cardioplegia, diltiazem, clentiazem, calcium antagonists. coronary flow, contractility. Meme si I'hypothermie et la cardiopltgie procurent une protection significative en chirurgie cardiaque, une pdriode d'ischbmie prolong&, une atteinte myowdique andrieure aussi bien que I'hypertrophie ventriculaire sont associQs ii une distribution hethogene des solutions de cardioplkgie et ii une protection et une rkuperation cardiaque incompkte. Les antagonistes du calcium sont considbrh comme de pu issants cardioprotecteurs mais leur effxcacitb en ajout ii la cardiopldgie fioide est discutable en prhnce de d6hillance cardiaque puisque Ie weur dbfaillant serait plus sensible aux effets inotropes et chronovopes negatifs de ces substances. Cependant des donnk recentes dtmontrent que les antagonistes du calcium de type benzothiazkpine comme le diltiazem et le clentiazem offtent, B certaines concentrations, une cardioprotection significative independante de leurs effets cardiodCpresseurs potentiels, ce qui suggererait leur utilitd dam les mtthodes cardioprotectrices du coeur dbfaillant. Nous avons entrepris d 'tvaluer l'interaction entre le diltiazem, le clentiazem et la cardioplkgie froide dam le coeur normal et defaillant. Les coeurs ont kt6 prklevts dans des hamsters cardiomyopathiques de 200-225 joun de la lignk UM-X7.1(coeurs difaillants) et dam des hamsters normaux du meme ige (coeurs normaux). Une perfusion ex vivo a 6tt5 faite 5 une pression constante de 140 cmH,O (1 cmH,O = 98.1 Pa) selon la methode de Langendorff. La frequence cardiaque, la pression ventriculaire gauche dtveloppk (PVGD)et le debit coronarien ont kt6 enregistrh de fapn continue. Une ischtmie globale de 90 min a Cti produite par occlusion de la ligne de perfusion, suivie d'une reperfusion de 30 min. Les coeurs normaux ou defaillants ont kt6 soumis, lors de l'initiation de la periode ischemique, A une des combinaisons suivantes : absence de cardioprotection, card iopleg ie froide (solution du St. Thomas' Hospital B 4°C infusee pendant 2 rnin). cardioplCgie froide + diltiazem 10 nM, ou cardioplkgie fioide + clentiazem 10 nM. Les effets cardiaques et coronariens du diltiazem et du clentiazem ont aussi 6d 6tudi6s sans cardioplkgie. Les coeurs de hamsters cardiomyopathiques 6taient dkfaillants (PVGD = 42*2 vs. 77*2 mmHg pour les coeurs normaux, p<0.05) alors que Ieur debit coronarien Ctait ltg&rementdiminut @,6f 0,2 vs. 6.2 f0-2 mL/min pour les coeurs normaux). A la reperfusion le debit coronarien Ctait plus Elev6 dans les deux groupes mais la reponse hyperemique maximale hitrnoins prononh dam les coeurs dkfail lants (+SO* 17 vs. + 82k 17%pour les coeurs normaux). La valeur de la -PVGD des coeurs normaux, qui etait presque normale & 5 min de reperfusion, montre un d6clin constant pendant la reperfusion (-37f 4% a 30 min). Pour les coeurs dtfaillants, la PVGD Ctait d iminuk en debut de reperfusion alors qu'elle s'arntliore pendant la reperfusion (-19f7% 5 30 min). Dam Ies coeurs normaux, l'addition du diltiazem ou du clentiazem i la solution de cardioplegie froide amtliore la rkupbration de la fonction contractile postisch6rnique (85 f4 vs. 71 f6 % pour la cardiopkgie seule, p <0.05). L'augmentation du dkbit coronarien ttait sernblable pour les groupes de coeurs normaux @eu de difference entre les traitements). Dam les coeurs defaillants, l'ajout du diltiazem ou du clentiazem il la solution de cardioplegie froide n'apporte pas de ben6fices contractiles lors de la reperfusion. Dans les coeurs normaux non-ischbmiques, 1' infusion de dil tiazem ou de clentiazem induit une augmentation du debit coronarien (+6f 1% pour Ie diltiazem et +Mf3% pour le clentiazem) sans manifestation d'effets inotropes ou chronoaopes nkgatifs. Dmles coeurs dtfaillants non-ischemiques, 1'administration de diltiazem ou de clentiazem n'est pas associk B une cardiod6pression. Par contre l'effet dilatateur coronarien du diltiazem est convenie en vasoconstriction alors que celui du clentiazem est a&nue significativement. Ces rhultats sugg5rent que Ie coeur defaillant du hamster cardiomyopathique s'adapte diff6rernment ii une ischdmie globale et prhente une resistance aux effets cardioprotecteurs du diltiazern ou du clentiazem en ajout a la cardiopltgie froide. En plus de possibles aldrations de Iiint6gritt membranaire et de I'horn6ostasie calcique, l'atthuation des effets dilatateurs wronariens du diltiazem et du clentiazem plut6t qu u'ne amplification de leurs effets card iod6presseurs intrinskques apparait cornme un facteur pouvant expliquer l'absence de cardioprotection de la part de ces antagonistes du calcium dans la dtfaillance cardiaque. Mots cICs : defaillance cardiaque, cardiopltgie, diltiazem, clentiazem, antagonistes du calcium, dkbit coronarien, contractilitk. INTRODUCTION Effective cardioprotection during limited periods of global ischemia can be achieved with cold hyperMemic cardioplegic solutions (Harlan et al. 1978; Hearse et al. 1981). However, long periods of ischemia, poor preoperative ventricular function, and coronary stenosis as well as ventricular hypertrophy lead to heterogeneous delivery of cardioplegic solutions, incomplete protection, and impaired postischemic recovery (Hilton et a1 . 1979; Fremes et al. 1985; Christakis et ai. 1986a; Rabinov et al. 1989). Under these conditions, the cause of greater myocardium sensitivity to intraoperative damage despite hypothermic cardioplegia is not clear. Optimal cardioprotection remains a challenging problem and requires the development of new therapeutic modalities. The central role played by calcium in the pathophysiology of myocardial ischemia and reperfusion, as demonstrated by its excessive intracel1ula.t accumulation, raises the hypothesis that calcium antagonists might be effective cardioprotective agents. Indeed. both experimental and clinical studies have established that among this pharmacological class of compounds, the benzothiazepine-like calcium antagonists diltiazem and clentiazem are wdioprotective under various ischemic conditions (Watts et al. 1986; Christakis et al. 1986b; Rousseau et al. 1993). Their effectiveness has also been demonstrated when they are added to cardioplegic solutions, although as for other calcium antagonists, their protective efficacy has been questioned when they are used with cold cardioplegia (Vouh6 et al. 1982; Yamamoto et al. 1983; Hearse et al. 1985; Prasad and Bharadwaj 1987; Melendez et al. 1988; Rebeyka et al. 1990). The cardioprotective properties of benzothiazepine-like calcium antagonists can be ascribed to either vascular or myocardial mechanisms. Their selective coronary dilator properties can improve perfusion to the inner layers of the myocardium (Warltier et al. 1981; Szekeres et al. 1985; Grover and Sleph. 1989). and an energy-sparing mechanism involving intrinsic negative inotropic and chronotropic effects may contribute to their cardioprotective repercussions (Lavenchy et al . 1986; Watts and Maiorano 1987). Other mechanisms, such as prevention of calcium overload, reduction of lipid peroxidation. preservation of lipid components of the sarcolemma, and more efficient metabolic substrate utilization, have been proposed as contributing factors (Cavero et al. 1983; Koner and Bergmann 1989; Watts et al. 1990; Davies et al. 1991). In the presence of depressed myocardial function, the additive effects of calcium antagonists and cold cardioplegia are controversial. Coronary dilation would translate into benefits, while negative cardiac effsts might exacerbate already impaired myocardial mntfactile function (Clark et al. 1983; Tchirkov and Just 1983; Jamieson et al. 1983; de Jong 1986). The demonstration by our group and others (Grover and Sleph 1989; Davies et al. 1991; Tanguay et al. 1994) that benzothiazepine-like calcium antagonists, in selected dose ranges, elicit significant cardioprotection independently of intrinsic cardiodepression lent support to their use in the failing heart. In the present study, we evaluated the efficacy of diltiazem and its more lipophilic congener, clentiazern, added to cold St. Thomas' Hospital cardioplegic solution on postischemic myocardial recovery. Experiments were performed in isolated normal hearts as well as failing cardiomyopathic hamster hearts (Jasrnin and Proschek 1982). MATERIALS AND METHODS Healthy normal Syrian hamsters (supplied by Charles River Canada Inc., St-Constant, Que.) and cardiomyopathic hamsters (CMHs)of the UM-X7. I 1ine (Jasmin and Eu 1979) were used in this study. Housed under similar conditions with free access to Purina laboratory chow and tap water, the animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care (1993). All CMHs were older than 200 days and had evidence of severe heart failure. Necrotic muscular lesions were apparent at autopsy in form of randomly distributed white streaks, along with enlargement of the 1iver and pulmonary congestion. Isolated hearts Following cervical dislocation and chest opening, the heart was cooled, cannulated in situ, quickly excised, and retrogradely perfused according to the method of Langendorff with modified Krebs-Ringer buffer consisting (in mM) of NaCl, 119; Kcl, 4.8; CaCI,, 1.3; KH,PO,, 1.2; MgSO,, 1.2; NaHC03,25; and glucose, 15. The buffer was continuously gassed with 95% 4 - 5% C4,pH was maintained at 7.4, and perfusion pressure was kept constant at 140 cm H,O (1 cmH,O = 98.1 Pa). All hearts were maintained at 37'C by placing them in a temperature-controlled chamber. Coronary flow was monitored continuously using an ultrasonic flow probe (model T201, Transonic Systems Inc., Ithaca, N.Y.) placed in the perfusion line upstream to the heart. Isovolumetric left ventricular developed pressure (LVDP) was measured with a saline-filled latex balloon introduced into the left ventricle through the left atrium and connected to a pressure transducer (model P23AC, Statham Instruments Inc., Hato Rey, Pueno Rim). The balloon was filled to obtain a diastolic pressure of 0-5 mmHg and its volume was unaltered for the duration of the study. Heart rate was allowed to change spontaneously and was measured from the LVDP tracing. All physiological measurements were registered on a multichannel recorder (polygraph model 5D, Grass Instruments, Quincy , Mass.). Experimental procedures After an equilibration period of 30 min, baseline values of heart rate, LVDP, and coronary flow were recorded. The heart was then rendered globally ischemic by clamping the aortic line for a Wmin period, followed by 30 min of reperfusion. During the 90 min of global ischemia the hearts were left at room temperature. Normal and failing hearts underwent the following experimental protocols. In protocol 1, the effects of 90-min global ischemia without cardioprotection were evaluated. In protocol 2, isolated hearts were administered cold St. Thomas' Hospital cardioplegic solution with or without diltiazem or clentiazem (10d M) . The cardioplegic solution was perfused in the coronary bed at the onset of ischemia by opening a line attached to a side arm of the aortic cannula. The St. Thomas' Hospital cardioplegic solution (4°C) was infused at a pressure equivalent to 100 cmH,O for 2 min. The cardioplegic solution was composed (in mM) of NaCI, 110; Kcl, 16; MgCl,, 16; CaCI,, 1.2; and NaHCO,, 10 (pH 7.8 and 280 mosm/L). Before use, the cardioplegic solution was passed through a membrane filter of 0.45-pm pore width. The duration of infusion (2 min) was determined in preliminary experiments on CMH hearts (n=3), in which myocardial temperature was monitored by means of a thermistor (model BAT-10, Physitemp Instruments Inc., Clifton, N.J.) implanted in the left ventricular wall. With this technique of myocardial cooling, temperature reached 15'C by the end of infusion, indicating that failing hearts were not underdosed with cardioplegic solution. The concentrations of diltiazern and clentiazem added to the cardioplegic solution were based on previous experiments in which 1W8 M elicited significant cardioprotection without cardiac depression (Tanguay et al. 1994). In protocol 3, the inotropic, chronotropic, and coronary dilator effects of a 2-min infusion of 1C8 M diltiazem or clentiazem alone were studied in normal and failing nonischemic hem. Statistical analysis All values are expressed as means f SEM. Each experimental group consisted of six isolated hems. Absolute values of hernodynamic parameters as well as percent changes were computed. Multigroup statistics were evaluated by the analysis of variance (ANOVA)using the LSD (least significant difference) procedure to determine differences between groups when appropriate. Differences were considered statistically significant atp < 0.05. Drugs and chemicals Clentiazem (maleate salt) and diltiazem (chlorhydrate salt) were generously donated by Nordic Marion Merrell Dow Research Inc., Laval, Que. Both drugs were dissolved in distilled water and diluted in cardioplegic solution just before the experiments. Cardioplegic solution (Plegisol) was purchased from Abbott Laboratories Ltd., Montrbl, Que. Its pH was adjusted to 7.8 prior to its use by the addition of 8.4% sodium bicarbonate. USP, Abbott (Abbott Laboratories Ltd., Montrkl, Que). The prepared cardioplegic solution was refrigerated and stored no longer than 24 h. RESULTS In total, 48 hamsters were used in these experiments. The mean body weight of healthy control hamsters was 153 f 2 g, with a slight reduction in the CMH group (129 f 2 g). The baseline hernodynamic parameters of both normal and failing CMH hearts are given in Table 1. Heart rate, LVDP, and rate-pressure product were significantly decreased in failing CMH hearts, while baseline coronary flow was only slightly reduced. TABLE 1. Baseline hernodynamic parameters in normal and failing CMH hem. Nomal hem Failing CMH hearts Heart rate (beatsf min) 190 k 7 159 f 8* Left venuicular pressure (mmHg) 77 * 2 42 f 2* Coronary flow (mL/min) 6.2 * 0.2 5.6 f 0.2 Rate-pressure product 147 f 7 (beats mid mmHg) x 100 Note: Values are means * SEM (n = 24 per group). * p c 0.05 versus normal hearts. Global myocardial ischemia and reperfusion All isolated hearts that underwent ischemia alone recovered from the Wmin global ischemia. In normal and failing hearts, coronary flow was increased at reperfusion (Fig. 1). The peak coronary hyperemic response was observed following 4-5 rnin of reperfision in failing CMH hearts and consisted of a 50% rise in coronary flow. A variable coronary response was seen in failing CMH hem; in three instances, instead of the usual rapid increase, coronary flow was reduced early during reperfusion. In normal control hems, the peak hyperemic response averaged +82f 17 % of preischemic values. The increase observed in both groups abated rapidly (in less than 10 min), and thereafter coronary flow returned towards baseline preischemic values. Following 90-min global ischemia, recovery of contractile function was different between normal and failing CMH hearts. In normal hearts, LVDP had almost fully recovered at 5 min of reperfusion but steadily declined thereafter (Fig. 2). In failing CMH hearts, LVDP was significantly impaired at 5 min of reperfusion (-36f 11 1)but improved over time. After 30 min of reperfusion, LVDP impairment was more pronounced in normal compared with failing CMH hearts (-35 f4 1 vs. -19f 7 1). Changes in heart rate were minimal during reperfusion with no significant difference between normal and failing CMH hearts (- 13 f2 5% for normal vs. - 15 f2 56 for failing CMH hearts ai 30 rnin of reperfusion). Normal hearts Failing hearts Fig. 1. Coronaxy flow changes following 90-min global ischemia in normal and failing CMH hem. Data are expressed as percent change from preischemic values (mean 1 SEM). Normalhearts Failing hearts --- 5 10 15 20 25 30 REPERFUSION (min) Fig. 2. LVDP changes following 90-min global ischemia in normal and failing CMH hearts. Data are expressed as pemreduction from baseline preischemic value (mean * SEM). *p < 0.05 vs. preischemic basehe value within each group. Cardioplegia with or without diltiazem and clentiazem Cold St. Thomas' Hospital cardioplegic solution was infused for 2 min into the coronary bed at the onset of global ischemia. Cardiac arrest was instantaneous and myocardial temperature reached approximately lS°C by the end of infusion. No variation in time to achieve complete mechanical arrest was noted between normal and failing CMH hearts. The volume of cold cardioplegic solution infused was l4.9kO.6, 13.8 ~kO.7,and 15.5k0.6 mL in normal hearts and l2.9f 1.1, l3.6f 0.7, and 12.9k0.6 mL in failing CMH hearts treated with cardioplegia alone, wdioplegia + 10 nM diltiazem. and cardioplegia + 10 nM clentiazem, respectively. The addition of diltiazern or clentiazem to cold cardioplegic solution resulted in a sustained improvement of postischemic LVDP in normal hearts (Fig. 3). In failing CMH hearts, the addition of diltiazem or clentiazem did not ameliorate left ventricular function. The postischemic coronary flow changes observed in the presence of cold cardioplegia alone or combined with diltiazern or clentiazem are presented in Fig. 4. In both groups, a significant increase in coronary flow was evident at 5 and 10 min of reperfusion: thereafter, coronary flow returned towards baseline preischemic values. Diltiazern or clentiazem did not affect the increase in coronary flow observed at reperfusion in normal and failing CMH hearts. At 30 min of reperfusion, heart rate was not significantly affected in normal hem treated with cardioplegia (-9f 5 56) alone or cardioplegia with diltiazem (+3f 7%) or clentiazern (-7f 7 56). In failing CMH hearts neither cardioplegia alone (+8* 11 56) or combined with diltiazem (-8f 6%)or clentiazem (-2f 8 96) significantly affected basal heart rate values . NORMAL HEARTS Fig. 3. Diltiazem and clentiazem added to cold cardioplegia: effects on postischemic LVDP in normal and failing CMH hearts. Data are expressed as percent of preischemic values, where 100% = basehe (mean 4 SEM,n = 6 per group). *p < 0.05 vs. cardioplegia alone. NORMAL HEARTS FAILING HEARTS I O Control Dihiazetm I i I Clentiazem REPERFUSION (min) R.EFERFUSION (min) m.4. DiltintPm and ~lmthmadded to cold cardioplegia: effects on coronaxy flow at reperfision in nod and failing CMH hearts. Data are expressed as means SEM (n = 6 per group). *p < 0.05 vs. baseline preischemic value within each group. Effects of diitiazem and dentiazem in nonischemic hearts To determine changes in myocardial and coronary sensitivity to diltiazem and clentiazem in the presence of heart failure, normal and failing CMH hearts were exposed to a 2-min lo-' M infusion of diltiazern or clentiazern. The inotropic and chronotropic effects of diltiazem and clentiazem were minimal in both groups (Fig. 5). Surprisingly, dil tiazem-induced coronary dilation in normal hearts was converted to vasoconstriction in failing CMH hearts. Clentiazem, which is a more potent vasodilator than diltiazem, elicited weaker coronary dilation in failing CMH hearts. Heart rate (A%) Left ventricular pressure (A%) Coronary flow (A%) -10 -1 A NORMAL FAIILING FIG. 5. Coronary and cardiac effects of a 10 nM infusion of diltiazem and clentiazem in normal and failing non-ischemic CMH hearts. Data are expressed as percent change from baseline (Mean f SEM). Hatched columns = normal hearts; filled columns = failing CMH hearts. NS = non significant. DISCUSSION The present study demonstrates that diltiazem and clentiazem afford significant additive cardioprotection to cold St. Thomas' Hospital card iopleg ic solution in the normal hamster heart. In contrast, the failing CMH heart does not benefit from the addition of either diltiazem or clentiazem to this cold cardioplegic solution. In addition to potential alterations in membrane integrity as well as changes in mlcium handling in CMH hearts, attenuation of the coronary dilator response to diltiazem and clentiazem rather than increased sensitivity to their intrinsic cardiodepressant actions appears as a potential contributor to the lack of cardioprotection by these calcium antagonists in the failing CMH hearts. Since heart involvement is a major feature in polymyopathic hamster, this animal has become widely accepted as a model for research into congestive heart failure. In the UM-X7.1 line (a subline of BIO 14.6) developed at the Universid de Montrhl in the early 1970s (Jasmin and Bajusz 1973; Jasmin and Eu 1979), myocardial necrosis reaches maximum severity in 90-day-old animals. Subsequently, myocardial hypertrophy and interstitial fibrosis develop, and the animals eventually die of circulatory failure. The average life-span of the UM-X7. I strain is about 250 days. An excessive buildup of cellular calcium, biochemical abnormalities, deterioration of membrane structure and function, and microvascular spasms contribute to progression of the disease (Jasmin and Roschek 1984; Makino et al. 1985; Sonnenblick et al. 1985; Hano and Lakatta 1991). Isolated heart preparations from this model of congestive heart failure have been used to determine the contractile abnormalities present in different stages of the disease as well as the protective effects of various drugs (Rouleau et al. 1982; Camacho et al. 1988; Wikrnan-Coffelt et al. 1991; Haleen et al. 1991). Since the failing CMH heart resembles the failing human heart in its biochemical and mechanical dysfunctions, this model can be considered valuable for the assessment of improved cardioplegic solutions. A different pattern of recovery following 90-min global ischemia was observed in normal and failing hearts. In normal hearts, contractile dysfunction was steadily aggravated during reperfusion, whereas in failing CMH heam the contractile deficits were maximal early during reperfusion but improved significantly until the end of reperfusion. The relatively well preserved contractile function of normal hem early during reperfusion (0-10 min) might be related to the inotropic action of intrinsic catecholamines released from the ischemic hearts and to the parallel increase of coronary flow (repayment flow). Previous studies have reported this so-called "erectile" effect, where increasing coronary perfusion pressure resulted in augmented wall thichess, mechanical stretch, and left ventricular pressure (Salisbury et al. 1960; Vogel et al. 1982). Bouchard et al. (1987) have demonstrated that changes in coronary perfusion have less influence on the contractile function of CMH hearts because of reduced myocardial elasticity. The reduced susceptibility of the failing CMH heart to prolonged ischemia also suggests natural preconditioning in this model of heart failure since preexisting intermitlent vasospastic conditions have been suggested in this model of heart failure (Factor et al. 1982; Sonnenblick et al. 1985). The failing CMH heart may also recover differently from global ischemia through mechanisms involving differences in calcium handling or differences in the contractile response to calcium changes (Makino et al., 1985: Panaglia et al. 1986; Whitmer et al. 1988). Metabolic differences may also be implicated in these differences. In failing CMH hearts, mitochondria1 activity is reduced and the myocardium is no longer able to maintain its [Ca2+],homeostasis. This could occur through inhibition in glyoolysis, inadequate delivery of substrate to the mitochondria, and defective oxydative phosphorylation (Proschek and Jasmin, 1982: Wikrnan-Coffelt et al. 1991). It has been previously shown that diseased hearts are more resistant to the induction of arrest, and larger volumes of cardioplegic solution have been advocated (Matsuda et al. 1986; Takahashi et al. 1988; Rabinov et al. 1989). In the present study, the 2-min infusion of cold St. Thomas' Hospital cardioplegic solution resulted in a significant reduction of myocardial temperature (37'C to 15%). As suggested by others, the volume of cardioplegic solution delivered ( > 2 mL/g) and the time of infusion (2 min) utilized in our study appear to be appropriate for rapid cooling and arrest of the heart (Takahashi et al. 1988; Rabinov et al. 1989; Matsuda et al. 1986). Since inadequate protection might occur during mld cardioplegia, the addition of calcium antagonists has been advocated on the basis of their anti-ischemic effects (Cavero et al. 1983; Watts and Maiorano 1987). However, expectations of increased negative cardiac effects have limited their use in the presence of poor left ventricular function. In this situation, diltiazem and clentiazem may offer significant advantages over other calcium antagonists since they appear to be less cardiodepressant and preferentially dilate coronary over systemic arteries (Millard et al. 1983; Boucek et al. 1984; Schwinger et al. 1990; Deisher et al. 1993). Indeed, the cardioprotection afforded by diltiazern and clentiazem has been shown to be independent of cardiodepression (Grover and Sleph 1989; Davies et al. 1991 ; Tanguay et al. 1994). Our results indicate that in the normal hamster heart, diltiatern and clentiazem provide significant additive cardioprotection to cold St. Thomas' Hospital cardioplegic solution. These benefits may implicate a vascular and (or) a myocardial mode of protection. Through their coronary dilator properties, these calcium antagonists may improve transmural delivery of the cardioplegic solution at the onset of ischemia or enhance the production of an endogenous cardioprotective substance such as prostacyclin (Guyton et al. 1983; Nomura et al. 1991). However, the cardioprotection provided by diltiazem and clentiazem does not seem to be related solely to their coronary dilator properties since a similar increase in postischemic coronary flow was noted in the presence of cold cardiopleg ia alone. In addition, the sustained cardioprotective effects of dil tiazem and clentiazem on contractile function were not paralleled by sustained increase in coronary flow. The beneficial effects of diltiazem and clentiazem may also originate from a myocardial mode of protection either through prevention of calcium overload, reduction of 1ip id peroxidation and glycolysis, or preservation of membrane 1ip id components (Koner and Bergmann 1989; Watts et al. 1990; Lopaschuck et al. 1992; Tanguay et al. 1994). In failing CMH hearts, diltiazem and clentiazem did not restore contractility. Several mechanisms may be proposed to explain the absence of cardioprotection by diltiazem and clentiazem in the presence of heart failure. Firstly, there is a completely different postischemic recovery pattern of the left ventricular function in failing CMH hearts. which could be relared to differences in calcium handling. Such differences could result in attenuation of calcium antagonist action on calcium overload. Secondly, as previously stated. the cardioprotective effects of diltiazem and clentiazem may be related to preservation of membrane lipid components (Lopaschuk et al. 1992; Tanguay et al. 1994). Since the cardiocytes of failing CMH hearts have already gone into marked sarcolemmal alterations (reduction in phospholipids and (or) cholesterol content), the above-mentioned mechanism of protection may become obsolete (Okumura et al. 1991). Alterations in diltiazem or clentiazem intrinsic properties may influence the lack of significant cardioprotection observed in failing CMH hearts. One mechanism may originate from exacerbation of their intrinsic negative inotropic effects in the presence of heart failure. However, as suggested by experiments carried out in nonischemic hearts where a 2-min 10 nM infusion of diltiazem or clentiazem was given, we must rule out an increased negative inotropic or chronotropic response to diltiazem or clentiazem. In the presence of either diltiazem or clentiazem, the reduction in LVDP averaged only 1 % while heart rate decreased by less than 5 % in normal hearts and failing CMH hem. Finkel et al. (1992) failed to observe any differences in the inotropic response of myopathic muscles to the calcium antagonists nifedipine and diltiazem or the agonist Bay K 8644. These findings also argue against the presence of a functional defect in the L-type calcium channel in the myopathic hamster. Furthermore it has been previously reported that, at a similar concentration range, diltiazern and clentiazem are devoid of significant negative inotropic or chronotropic effects in the diseased myocardium (Schwinger et al. 1990; Euaher et al. 1991). Since exacerbation of diltiazem or clentiazem intrinsic negative inotrop ic effects is unlikely, another potential contributing factor has been identified in the present study. Results obtained in nonischemic isolated hearts demonstrate that the coronary dilator effects observed in normal hearts are significantly attenuated (clentiazem) or even reverted to vasoconstriction (diltiazem) in failing CMH hearts. Therefore the observed reduced coronary sensitivity may provide an add itiod explanation for the absence of significant cardioprotection by diltiazem and clentiazem in the presence of heart failure. In conclusion, diltiazem and clentiazem, when added to cold St. Thomas' Hospital cardioplegic solution, afforded significant cardioprotection in normal hamster hearts. In contrast, failing CMH hearts were resistant to the cardioprotective effects of diltiazem and clentiazem. In addition to potential alterations in membrane integrity as well as changes in calcium handling in CMH hearts, lack of cardioprotection with diltiazem and clentiazem may implicate an altered response of the coronary vasculature rather than exacerbation of their intrinsic negative cardiac actions in the presence of heart failure. Acknowledgments This study was supported in part by Fondation du Quebec des maladies du coeur. The authors are grateful to Elizabeth Per& for artwork. Mario Tanguay is the recipient of a studenship from Fonds pour la Formation de Chercheurs et 1'Aide 5 la Recherche (FCAR) . Boucek, R.J., Shelton, M., Artmanm M., Mushlin, P.S., Starnes, V.A., and Olson, R.D. 1984. Comparative efTetcs of veraparnil, nifedipine, and diltiazem on contractile function in the isolated immature and adult rabbit heart. Pediat. Res. 18: 948-952. Bouchard, A., Watters. T.A., Wu, S., Parmley, W.W., Stone, R.D., Botvinick, E., Sievers, R., Jasmin, G., and Wikrnan-Coffelt, J. 1987. 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Comparison of the protective effects of verapamil , diltiazem , nifedipine, and buffer containing low calcium upon global myocardial ischemic injury. J. Mol. Cell Cardiol. 18: 255-263. Watts, J.A., and Maiorano, I. 1987. Effects of diltiazem upon globally ischemic rat hearts. Eur. J. Pharmacol. 138: 335-342. Watts, J.A., Norris, T.A., London, R.E., Steenbergen, C., and Murphy, E. 1990. Effects of diltiazem on lactate, ATP, and cytosolic free calcium levels in ischemic hearts. J. Cardiovasc. Pharmacol . 15: 4449. Whiner, J.T., Kumar, P., Solaro, R. J. 1988. Calcium transport properties of cardiac sarcoplasmic reticulum from cardiomyopathic Syrian hamsters @I0 53.S8 and BIO 14.6): Evidence for a quantitate defect in dilated rnyopathic hearts not evident in hypertrophic hearts. Circ. Res. 62: 8 1-85. Wikman-Coffelt, J., Stefenelli, T., Wu, S.T., Parmley, W.W., and Jasmin, G. 1991. [Ca2+Iitransients in the cardiomyopathic hamster heart. Circ. Res. 68: 45-5 1. Yamamoto, F., Manning, A.S., Braimbridge, M.V.,and Hearse, D.J. 1983. Calcium antagonists and myocardial protection: Diltiazem during cardioplegic arrest. Thorac. Card iovasc. S urg . 3 1: 369-373. Involvement of the nitric oxide and the cyclooxggenase pathways in the decreased coronary sensitivity to diltiazem and clentiazem in the failing hamster heart Mario Tanguay, Gaetan Jasmin, Gilbert Blaise and Louis Dumont Dbpanemem de phamcologie, tie pathologie et d 'anesthttsie, Fadtk de Mkdecine, Universitt? de Montrkal, Munntfal, Qc, Canada Soumis ii Jouml of Pharmacology and Erpen'mntal Therapeutics ABSTRACT Venfricular and vascular dysfunction observed in heart failure may modulate the efficacy of calcium antagonists given in this condition. Therefore, the goal of this study was to evaluate the effect of heart failure on the coronary and cardiac responses to the benzothhzepine calcium antagonists dil tiazem and clentiazem. Left ventricular developed pressure (LVP) and coronary flow were assessed in isovolumically-beating, perfused, Wing hearts from cardiomyopathic hamsten (CMH)and in normal hamster hearts. The concentration-response cwes for the negative inotropic action of diltiazem were similar in both normal and failing hearts (the IC, of LVP was 1191 * 224 nM and 2354 f 844 nM, respectively). Clentiazem, which is more lipophilic than diltiazem, induced similar cardiodepression in isolated hearts from both groups (the ICMwas 677 f 63 nM and 734 * 150 nM for normal and failing hearts, respectively). However. the coronary dilator effects of diltiazem and clentiazem were reduced in failing hearts compared to normal hearts. The EC, for the coronary response to diltiazem increased from 90 f 11 nM in normal hearts to 1087 f 525 nM in failing hearts (P<0.01). Although clentiazem is a more potent vasodilator than diltiazem, its coronary dilator effects were also reduced in failing hearts (the EC, was 15 * 3 nM and 56 f 9 in normal and failing hearts, respectively; P < 0.0 1). To better characterize the decreased coronary sensitivity to diltimrn and clentiazem observed in heart failure, we evaluated the possible contribution of nitric oxide (NO) and cyclooxygenase by-products by using the specific NO synthase inhibitor NG-nitro-L-arginine (L-NAME,30 pM), and the cyclooxygenase inhibitor indomethacin (10 pM). Diltiazem concentration-response curves were not modified by the presence of L-NAME in both normal and failing hearts. Even if coronary dilation by clentiazem was slightly reduced by L-NAME,this effect was similar in both normal and failing hearts. Indomethacin infusion normalized the coronary response to diltiazem in failing hearts. The reduction of coronary sensitivity to diltiazem may then be attributed to the simultaneous release of a cyclooxygenase by-product with constricting properties. However, the diminished coronary sensitivity to clentiazem observed in failing hearts did not recover in the presence of indomethacin, suggesting that the physicochemical properties of the molecule are linked to a more complex action on coronary vessels. In conclusion, heart failure was not associated with amplified negative inotropic and chronotropic effects of diltiazem and clentiazem but their coronary dilator sensitivity was significantly reduced. This latter action may be related to the simultaneous release of constricting factor(s) originating from the cyclooxygenase pathway. These fidings may provide a better understanding of the inefficacy of calcium antagonists in hean failure and suggest that new vasoselective calcium antagonists might be more appropriate for the treatment of this pathology. INTRODUCTION Clinical trials have shown that fmt generation calcium antagonists were deleterious to patients in an advanced state of heart failure (Elkayam n aL, 1990; Goldstein et al., 1 99 1; Multicenter Diltiazem Postinfarction Trial Research Group, 1988). The unfavorable clinical effects of calcium antagonists during chronic hean failure may be attributed either to their cardiodepressant action or to excessive activation of the neurohumoral system (Packer, 1989). However, no clear evidence has been found of amplified negative inotropic actions of calcium antagonists in heart failure. Although some studies have reported an increased negative inotropic effect of the benzothiazepine calcium antagonist diltiazem in experimental heart failure induced by pressurelvolume overload (Ezzaher et al., 1991; Porter et al., 1983), no amplification of the drug's negative inotropic impact could be observed in other models of heart failure (Drexler a al., 1985; VCniant et al., 1991). Therefore, other factors may be involved in the lack of efficacy of calcium antagonists in heart failure. Reduced vascular sensitivity to calcium antagonists in the presence of heart failure is an attractive hypothesis. The vascular dysfunction identified in heart failure may modulate the efficacy of calcium antagonists. In addition, such vascular dysfunction may originate from the endothelium. Impairment of endotheliumdependent vasodilation has been documented in heart failure (Drexler et al., 1992; Kubo et al., 199 1; Treasure et al., 1990), whereas the basal production of nitric oxide (NO), which accounts for the biological activity. of endotheliumderived relaxing factor (EDRF) (Palmer er a[., 1987), may be enhanced in these patients (Drexler et al., 1992; Habib et al., 1994; Winlaw et al., 1994). Moreover, it has been suggested that calcium antagonists may interact with the vascular endothelium (Vanhoutte, 1988). Rubanyi et d. (1988, 1991) have reported that diltiazem and clentiazem can suppress the production of EDRF in endothelial cells by a specific benzothiazepine-dependent mechanism. Conversely, the vasorelaxant effect of calcium antagonists and of EDRF seems to be additive (Vanhoutte, 1988). and the new dihydropyridine calcium antagonist S 11568 produces endothelium-dependent relaxation (Vilaine et al., 199 1). However, the interaction between endothelialderived NO and the vasodilating effects of calcium antagonists has not been studied in coronary resistance vessels. Since endothelial function may be altered in heart failure, it is possible that this pathophysiological condition influences the vascular effects of calcium antagonists. This hypothesis may also be explained by interference of calcium antagonists with arachidonic acid metabolism and/or cyclooxygenase by-products (Seeger et of., 1987). In this respect, production of cyclooxygenase-dependent vasoconsaicting factors has been reported in patients with heart failure (Katz et al., 1993). The present study was undertaken to evaluate the influence of heart failure on the coronary and cardiac responses to diltiazem and its more Iipophilic derivative clentiazem, using the cardiomyopathic hamster (CMH)model of heart failure. We also examined the contribution of NO and cyclooxygenase by-products to these responses by means of the means of the specific NO synthase inhibitor Na-nitro-L-arginine methyl ester &-NAME), and the cyclooxygenase inhibitor indomethacin. METHODS Normal healthy Syrian hamsters (supplied by Charles River Canada Inc., St-Constant, Qc, Canada) and CMH of the UM-X7.1 strain (Jasmin and Eu, 1979) were used in this study. All animals were housed under similar conditions with free access to Purina laboratory chow and tap water. They were cared for in accordance with the guidelines of the Canadian Council on Animal Care (1993). All CMH were older than 200 days and showed evidence of severe heart failure (pulmonary and 1iver congestion, peritoneal edema). Isolated heart preparation Following cervical dislocation, the heart was cooled and cannulated in situ, quickly excised and retrogradely perfused according to the method of Langendorff with modified Krebs-Ringer buffer consisting (in mM) of NaCl 119, KC1 4.8, CaCl, 1.3, KH,PO, 1.2, MgSO, 1.2, NaHC03 25 and glucose 15. The buffer was constantly gassed with 95 % O2and 5 % C4(pH 7.4 at 37°C). and perfusion pressure remained unchanged at 140 cm H,O. All hem were maintained at 37°C throughout the study period by placing them in a temperature-controlled chamber. Coronary flow was monitored continuously with an ultrasonic flow probe (Model T201, Transonic Systems Inc., Ithaca, NY) placed in the perfusion line upstream to the heart. Isovolumetric left ventricular developed pressure (LVP) was measured with a saline-filled latex balloon introduced into the left ventricle through the left atrium and connected to a pressure transducer (Model P23AC. Statham Instruments Inc., Hato Rey, Puerto Rico). The ventricular balloon was filled to obtain a diastolic pressure of 5 mm Hg and its volume was unaltered for the duration of the study. Heart rate was allowed to change spontaneously and was measured from LVP tracing. All physiological measurements were registered on a mu1 tichannel recorder (Polygraph Model 5D. Grass Instruments, Quincy , MA). Experimental procedures After an equilibration period of 25-30 min, baseline values of LVP, coronary flow and heart rate were recorded. Then, normal and failing hearts were investigated following these experimental protocols. In the first set of experiments, cumulative concentration- response relationships for diltiazem and clentiazem (lW9 to M) were assessed. The drugs were infused at each concentration for at least 2 min to ensure a plateau response, and a period of 1 min was allowed to elapse between each stepwise increase in concentration. In the second and third set of experiments, the concentration-response curves were determined in the presence of L-NAME (30 pM) or indomethacin (10 pM). Infusion of these inhibitors was started 30 min prior to the cumulative concentration- response cuwe of the caicium antagonists. All experiments were completed within 120 min. The dosage of L-NAMEwas based on a previous study (Smith et al.. 1992) and on preliminary experiments from our laboratory showing a maximal reduction of basal coronary flow at 30 PM. In addition, the coronary vasodilating effect of acetylcholine (0.1 pM) after 1 min of infusion was totally abolished or even reversed to vasoconstriction in the presence of L-NAME (30 pM), whereas the vasodilating action of sodium nitroprusside was not attenuated (Tanguay et al., 1994a). Indomethacin has been demonstrated to inhibit cyclooxygenase at 10 pM (Okada, 199 1; Yang et af., 1993). Drugs and chemicals Clentiazern (maleate salt) and diltiazem (chlorhydrate salt) were generously supplied by Nordic Merrell Dow Research Inc. (Laval, Qc, Canada). LNAME and indomethacin were purchased from the Sigma Chemical Co. (St. Louis, MO). All drugs were dissolved and diluted in distilled water, except for indomethacin which was first dissolved in ethanol and further diluted in Krebs-Ringer solution. A11 drugs were infused in the aortic line at a rate of 11100 of coronary flow, and their concentrations are expressed as final molar amounts in the assays. Data analysis All values are expressed as means f S.E. of 5 isolated preparations. The effect of diltiazem and clentiazem on LVP is represented as a percentage of the pretreatment baseline value, and their action on coronary flow as a percentage of maximal vasodilation. The IC, for inotropic actions and the EC, for vasodilator effects were determined for each heart by the linear regression of probits of response vs. log dose, using the computer program PHARMIPCS Version 4.2 (Tallarkla and Murray, 1987). This study was designed to compare diltiazem and clentiazem concentration-response curve parameters in the absence or presence of L-NAME or indomethacin in normal and failing hearts. Statistical comparisons were then performed by the general linear model (GLM)for analysis of variance of SAS program Version 6.04 (SAS Institute Inc, Cary. NC), using a factorial design where disease (normal and failing hearts) and drug treatment were the main facton. Differences were considered to be statistically significant at P <0.05. RESULTS Totally, 60 hamsters were used in these experiments. Table 1 presents the baseline hemodywnic parameters of both normal and failing hamster hearts. Coronary flow and LVP were significantly reduced in CMH as compared with normal hamsters, but heart rate was not different between the two groups. TABLE I Basal hernodynamic values of normal and failing hearts Coronary flow, left ventricular developed pressure (LVP), and heart rate were measured after a 30- min stabilization period. Values are the means * S .E.of 30 hem in both groups. Coronary flow LVP Hem rate ml/mrh Hi? beats/min Normal hearts 6.3 f 0.2 70.0 =t 1.5 182 f 4 Failing hearts 5.1 & 0.2" 47.7 f 1.8* 176f 7 *The difference between normal and failing hearts is statistically significant (P < 0.01). Effeds of heart failure on the coronary dilator response to diltiazem and clentiazem The concentration-response curves of diltiazem and clentiazem in terms of their coronary vasodilating effects were both shifted to the right in failing hearts (Fig. 1, top) with a significant increase in their EC, values flable 2). As a coronary dilator, clentiazem was about 6 times more potent than diltiazem in normal hem and about 19 times more potent in failing hearts. Negative inobopism, Coronary dilation, % change in LVP % of maximal coronary flow increase $84&b I'~T-~---l--r-- Effects of heart failure on the cardiac response to dWazem and clentiazem Heart failure had no significant effects on the negative inotropic actions of diltiazem and clentiazem (Fig. 1, bottom): there was no significant change in their IC, values in failing hearts (Table 2) . Clentiazem was about 2 times more potent than diltiazem in normal hearts and about 3 times more potent in failing hearts. Negative chronotropic effects were observed only at high concentrations ( > 1 ,uM).The slight reduction in heart rate induced by dildm was similar in normal and failing hearts (maximal effect at 10 rM: -14.9 * 9.4% in normal vs -17.4 & 10.5% in failing hearts, P>0.05). Clentiazem induced significant decreases of heart rate at high concentrations which were similar in normal and failing hearts (maximal effect at 10 pM: - 19.9 * 9.9 % in normal vs -28.1 * 4.6% in failing hearts, P > 0.05). Effeds of heart failure on vascular selectivity Selectivity ratios (IC&CJ represent the selectivity of calcium antagonists for coronary dilation versus depressant effects on the heart. They were calculated using their median concentrations from the concentration-response curves obtained for each heart. The vasoselectivity of the two agents was significantly reduced in failing hearts (Table 2). As shown in the table, clentiazem was also more vasoselective than diltiazem in both normal and failing hearts. Effects of LNAME on the concentmtion-response curves to diltiazem and dentiazem Infusion of L-NAMEresuhed in sustained vasoconstriction in all hearts studied. Basal coronary flow reduction was similar in normal and failing hearts after 30 min of infusion (-46.8 & 1.9% vs -44.3 f 3.01, respectively, Fig. 2). After 30 min of infusion, L- NAME also induced a slight reduction in LVP which was comparable in normal and failing hearts (-16.4 f 1.6% vs -19.3 f 3.O%,respectively). L-NAME had no significant effect on the diltiazem concentration-response curve for coronary dilation (Fig. 3A): no significant change in EC, values was observed in normal and failing hearts (Table 3). The clentiazem concentration-response curve for coronary dilation was affected by L-NAMEin both normal and failing hearts (Fig. 3B). In normal hearts, although the EC, remained unchanged (Table 3). the coronary dilator effects of clentiazem at lower concentrations were significantly decreased by L-NAME (EC,, = 12.0 f 1.6 nM in L-NAME-pretreated hearts vs 4.2 * 1.3 nM in untreated hearts. P < 0.01). In failing hearts, the ECI, (32.1 2.8 nM in L-NAME-pretreated hearts vs 14.4 * 2.7 nM in untreated hearts, P ~0.01)and EC, (Table 3) of clentiazem were significantly increased by L-NAME. Pretreatment with L-NAME had no significant effects on the cardiac response to diltiazem and clentiazem in normal hearts but slightly augmented their ventricular sensitivity in failing hearts (diltiazem IC, values were 9 10 * 277 nM in L-NAME-pretreated hearts vs 2354 f 844 nM in untreated hearts, P c0.05; clentiazem IC, values were 370 f 33 nM in L-NAME-pretreated hearts vs 734 f 150 nM in untreated hearts, Pc0.05). Norma1 hearts Failing hearts I I I 1 I 1 i 0 5 40 15 20 25 30 Infusion time (min) Fig. 2. Effect of GNAME (30 pM) on the coronary flow of normal and failing hew. Values are the means f S.E. of 5 experiments . TABLE 3 Endothelid modulation of EC, values for coronary dilation evoked by diltiazem and clentiazem in normal and failing hearts. EC,, diltiazem or clentiazem concentration (in nM) inducing 50% of maximal coronary dilation obtained in the absence (control) and presence of L-NAME(30 pM) or indomethacin (1 0 pM). Values are the means * S. E. of 5 experiments. Diltiazern Clen hem Control L-NAME Indornethacin Control L-NAME Indomethacin Normal hearts 90k I1 200 f 16 150 f 31 15 f:3 38 f 3 32 & 2 Failing hearts 1087*525* 471f37 217f30+ 56 * 9* 104 f 12t 61 18 *The difference between normal and failing hearts is statistically significant (PC0.01); ?The difference between L-NAME- pretreated and control hearts is statislically significant (P 4 0.05); $The difference between indomethacin-pretreated and control hearts is statistically significant (P < 0.05). Effects of indomethacin on the concentration-respow curves to dikiazern and dentiem Indomethacin infusion induced a slight but significant increase in basal coronary flow in both normal and failing hearts (Fig. 4). This increase was attenuated more rapidly in failing hearts: 7.8 * 1.7% vs 13.2 f 1.7% in normal hearts after a 30-min infusion (P CO.05). Indomethacin had no effect on LVP in either normal or failing hem. Pretreatment with indomethacin had no significant impact on the diltiazem concentration- response curve for coronary dilation in normal hearts (Fig. 5A). However, in failing hearts, it resulted in a left shift of the diltiazem concentration-response curve (Fig. 5A): indeed, the EC, was significantly decreased by indomethacin in failing hearts (Table 3). The clentiazem concentration-response curve for coronary dilation was not affected by indomethacin pretreatment (Fig. 5B, Table 3). The negative inotropic actions of diltiazem was not significantly influenced by indomethacin in either normal (IC,= 686 * 57 nM in indomethacin-pretreated hearts vs 1 191 * 224 nM in untreated hearts) or failing hearts (IC, = 1156 f 330 nM in indomethacin-pretreated vs 2354 f 844 nM in untreated hearts). The negative inotropic effects of clentiazem were unchanged by indomethacin in normal hearts (IC, = 616 f 58 in indomethacin-pretreated vs 677 k 63 nM in untreated hearts). However, indomethacin significantly reduced the clentiazem IC,, in failing hearts (378 * 99 nM in indomethacin-pretreated vs 734 f 150 nM in untreated hearts, P < 0.05). 0 Normal hearts Failing hearts Infusion time (min) Fig. 4. Effect of indomethacin (10 @M) on the coronary flow of normal and failing hearts. Values are the means f S. E. of 5 experiments. *P < 0.05 vs normal hearts at 30 min. DISCUSSION The present study demonstrates that coronary vascular sensitivity to the bemthiazepine- like calcium antagonists diltiazem and clentiazem is decreased in failing CMH hearts, while no difference is evident in their negative inotropic effects. This reduction in vascular sensitivity does not appear to be related to an alteration in the NO pathway in failing hearts even if NO can modulate the coronary vasodilating effects of clentiazem. However, the reduced coronary sensitivity to diltiazem observed in heart failure seems to be mediated by the release of a prostanoid vasoconstricting factor. The CMH has become widely accepted as a model for research into congestive heart failure; this paradigm is particularly useful for the study of subceliular pathological processes and for evaluation of vasodilator drugs (Rouleau er al., 1982; Smith and Nuttall, 1985; Trippodo et al., 1993; Wikman-Coffelt ez al., 1991). In the UM-X7.1 strain, myocardial lesions are predictable and become histologically evident at 40 days of age: the necrotizing process reaches maximum severity in 100day-old animals (Jasm i n and Proschek, 1982). Subsequently, myocardial hypertrophy and muscular fibrosis develop, and heart failure begins at 175-200 days of age. More than 50% of the animals die of heart failure before 250 days of age. Progression of the disease has been ascribed to intracellular calcium overload, increased adrenergic tone, basic genetic defects of membrane structure and function, and microvascular spasms (Hano and Lakatta, 1991; Makino et al., 1985; Sole et al., 1977; Sonnenblick et al., 1985). Besides chronic in viva studies, isolated heart preparations from this model of congestive heart failure have been used mostly to evaluate the effect of various drugs on myocardial contractility @user et al., 1990; Camacho et al., 1988; Haleen et al., 1991; Stefenelli et al., 1989). However, very few investigations have focused on the basal coronary flow changes induced by vasodilators. Our study is the fust to specifically assess the coronary sensitivity of failing CMH hearts to calcium antagonists. Most isolated heart investigations, including the present one, demonstrate that a basal smte of coronary vasoconstriction seems to be present in failing CMH hearts (Buser et al., 1990; Camacho et al., 1988; Haleen et a[., 1991; Stefenelli et al., 1989). Our data indicate that basal coronary flow is reduced by 20% in failing hearts. Although the coronary dysfunction of the failing hamster heart have been poorly explored, the reactivity of other vascular beds has indeed been investigated. An increased ~a~~~~nstrictiveresponsiveness of aortic strips and cremaster muscle arterioles to norepinephrine and 5-hydroxytryptamine has been observed in the young CMH during the active stage of necrosis (Conway et al., 1987; Hunter and Elbrink, 1983). Recently, heightened vasoconstrictive responsiveness to arginine-vasopressin was also repod in the coronary ~sculatureof young non-failing hamster hearts (Conway et al., 1994). Overall data on the vascular and myocardial dysfunction of CMH indicate that there are many common features in the Syrian hamster model of heart failure and the ischemic congestive heart failure associated with obstructed coronary arteries in humans (Sonnenblick er al., 1985). Since the failing hamster heart resembles the failing human heart in its biochemical, mechanical and coronary dysfunction, this model is suitable for assessing the effect of heart failure on the coronary and cardiac properties of calcium antagonists. Calcium antagonists are vasodilators and thus might be expected to be beneficial in patients with congestive heart failure for whom vasoconstriction contributes to limit cardiac performance (Colucci et al., 1985). However, most clinical studies with first generation calcium antagonists have documented detrimental effects in patients with chronic heart failure (EIkayam et d., 1990; Goldstein et al., 199 1; Mu1 ticenter Diltiazem Postinfarction Trial Research Group, 1988). The untoward effects observed after short- term therapy are believed to result from the negative inotropic properties of these agents (Packer, 1989; Parameshwar and Pooie-Wilson, 1993). Nevertheless, conflicting results have been reported regarding the increased negative inotropic sensitivity of calcium antagonists in experimental models of heart failure. Augmented negative inotropic effects of diltiazem and verapamil have been recorded in an experimental model of heart failure induced by pressure and volume overload (Emher a d.,199 1). Similarly, in dogs with chronic volume overload produced by an aortu-caval fistula, diltiazem has been shown to exert depressant effects on left ventricular function at concentrations devoid of cardiac actions in normal animals (Porter et al., 1983). In contrast, in other studies, no amplification of the negative inotropic effects of calcium antagonists was found in experimental models of heart failure. In a conscious rat preparation of chronic heart failure induced by myocardial infarction, diltiazem infusion did not result in overt precipitation of congestive heart failure while the hernodynamic status of the animals was even ameliorated by the drug, as revealed by increased stroke volume and improved mrdiac output during exercise (Drexler et al., 1985). Other results obtained in vivo as well as in isolated hearts indicate that the negative inotropic effixts of diltiazem were not potentiated in the presence of heart failure (V6niant et al., 199 1). Likewise in the present study, neither diltiazem nor clentiazem induced increased cardiodepression in failing hearts. Differences in experimental models of heart failure might provide an explanation for the discrepancies of inotropic sensitivity to calcium antagonists. Hemod ynam ic changes in pressure overload models are acute compared to those seen in humans (Smith and Nuttall, 1985). Conversely, chronic development of heart failure is observed in the hamster model. Another factor that could account for the differing results obtained in pressure/volume loading studies may be the stage of heart failure. Even if Ezzaher and coworkers (1991) noted left ventricular hypertrophy with associated signs of peripheral congestion, left ventricular pressure was not depressed in their "failing" group. Porter and coworkers reported that left ventricular function (dP/dG was increased in their experimental model of volume overload. Conversely, basal myocardial contractility was decreased in all experimental studies reporting no enhancement in the negative inotropic effects of calcium antagonists. Indeed, in the present investigation, in addition to signs of peripheral congestion and myocardial hypertrophy, LVP was reduced by 33%, indicating an advanced stage of heart failure. A previous study using left ventricular papillary muscles isolated from normal and failing hamsters (4-6 months of age), a model in which circulatory components and factors such as coronary flow and heart rate exert no influence on myocardial contractility, showed that the negative inotropic effects of diltiazem, nifedipine, verapa.mil and gallopamil were similar in both groups (Finkel et al., 1992). Finally, in human papillary muscle strips, there was also no significant difference in the negative inotropic effects of diltiazem and other calcium antagonists between moderate (New York Heart Association Class 11-111) and severe (Class IV) heart failure (Schwinger et al., 1990). Most explanations attribute the absence of beneficial effects of calcium antagonists in heart failure to their negative inotropic actions or neurohumoral activation occurring after chronic adm histration. Since heart failure is frequently associated with vasoconsuict ion, this pathophysiological state may also modify coronary sensitivity to calcium antagonists. Very few studies have outlined the coronary response to calcium antagonists in experimental or clinical heart failure. Drexler and coworkers (1985) reported, in a rat model of heart failure, the effects of diltiazem on regional blood flow, including the renal, splanchnic and right ventricle circulation. The authors mentioned that basal blood flow to the right ventricle was similar in normal and failing animals and that diltiazem increased right ventricle blood flow in both groups. Analyzing their data, we found that diltiazem induced a 53% increase in right ventricle blood flow in normal rats compared to a 29% elevation in failing animals. Funhermore, it has recently been reported that diltiazem and Ro #5%7 elicit a reduced coronary response in isolated failing rat hearts (Vkniant et al., 199 1). According to this investigation, the observed decrease in coronary sensitivity of failing hearts to calcium antagonists may be related to a coronary vasodilator reserve diminished by cardiac hypertrophy and ligation of one coronary artery. In our study, coronary sensitivity to diltiazem and clentiazern as well as their vascular selectivity were reduced in failing hem. The altered coronary vasodilating properties of diltiazem and clentiazem were not related to an impaired coronary reserve or to a aecrease in smooth muscle responsiveness in this model of heart failure. Indeed, the coronary peak dilating responses to clentiazem and diltiazem were not diminished in failing hearts (data not shown). In addition, our laboratory has previously reported that the coronary dilating effects of sodium nitroprusside were similar in normal and failing CMH hearts (Vtronneau et al., 1994). Moreover, unpublished observations from this laboratory have shown that peak hyperemic flow after 30-sec coronary occlusion, which might be considered a good indicator of coronary reserve, is not diminished in the failing heart. Indeed, the peak hyperemic response was even amplified in these animals ( 170 f 15% in failing vs 105 f 6% in normal hearts, P~0.01;n=15). Since interactions between calcium antagonists and the endothelium have been reported previously (Rubanyi et al., 1988, 1991; Vanhoutte, 1988; Vilaine et ai., 1991), the observed coronary desensitization may be related to endothelial dysfunction. The endothelial modulation of coronary vascular tone could be altered in the presence of heart failure (Drexler et al., 1992; Kubo et al., 199 1; Treasure et al., 1990; Winlaw et al., 1994). The present study shows that basal production of NO, which accounts for the biological activity of EDRF (Palmer et al., 1987, is preserved in failing hearts, since infusion of L-NAME (an inhibitor of NO synthase) resulted in a similar decrease in coronary flow in normal and failing hearts. These data corroborate previous clinical findings showing that the reduction of forearm blood flow induced by the NO synthase inhibitor NO-monomethyl-Larginine (L-NMMA) in patients with congestive heart failure was comparable to that in normal subjects @rexler et d.,1992; Kubo et al., 1994). The vasoconstrictor effect of LNMMA on hindquarter resistance vessels of rats with chronic heart failure was also similar to that seen in normal controls (DrexIer and Lu, 1992). Thus, it appears that, in heart failure, reduced coronary sensitivity to diltiazem and clentiazem does not originate from altered basal NO production. However, our findings suggest that inhibition of NO synthesis by L-NAME reduces coronary sensitivity to clentiazem. This apparent endothelial modulation of the wronary dilation effects of clentiazem is unlikely to be involved in the decreased coronary sensitivity to this drug in the presence of heart failure since L-NAME affected the clentiazem wronary dilator response in both normal and failing hearts. We did not investigate why, in contrast to clentiazem, the coronary dilator effects of diltiazem were not altered by L-NAME,but such heterogeneity among calcium antagonists has been reported previously. The dihydropryridine calcium antagonist S-11568 caused, unlike nifedipine, endothelium- dependent relaxation in canine femoral arteries that was prevented by methylene blue, hemoglobin and L-NMMA (Vilaine et al., 1991). It has been suggested that the endothelium-dependent effect of S-11568 is due to an interaction with non-calcium channel binding sites on endothelial cell membranes or to some direct intracellular actions. In our case, the higher liposolubility of clentiazern and its better tissue penetration may explain the mechanism underlying endothelial modulation of its coronary dilating effects (Narita and Ginsburg, 1990; Tanguay et al., 1994b). Since NO does not seem to be involved in the reduced coronary sensitivity to calcium antagonists observed in our model of hart Wure. other fktors such as prostanoids might be considered. Release of prostanoid constricting factors in heart failure has already been suggested by studies showing that acetylcholine dilation was improved by administration of indomethacin in clinical and experimental heart failure (Kaiser et al., 1989; Katz et d. 1993). In the present investigation, indomethacin elicited a slight increase in coronary flow in both nonnal and failing CMH hearts, suggesting a predominant constrictor effect of cyclooxygenase by-products in the regulation of basal coronary tone. Interestingly, indomethacin normalized the impaired coronary sensitivity of failing hearts to diltiazem. Thus. the reduced coronary sensitivity to diltiazem appears to be due to the simultaneous release of constricting substance(s) originating from the cyclooxygenase pathway. Evidence from the literature may be relevant in defining the mechanisms involved in diltiazem-induced synthesis of cyclooxygenase products. It has been shown that cardiac alterations may accelerate phospholipid degradation and lead to an enlarged arachidonic acid (AA) pool. In the failing hamster heart, lower levels of the major phospholipids phosphatidylcholine, phosphatidy lethanolam ine and cardiol ip in have been reported when compared to normal hamster hearts (Okumura er al., 1991). In the ischemic canine myocardium, it has been shown that myocyte injury is associated with an accumulation of AA; the authors suggested that defective reacylation of arachidonate in phosphatidylcholine may contribute to the depletion of membrane phospholipid during myocardial ischemia (Chien et al., 1984). In addition, in isolated cells, high innacellular calcium levels are associated with increased AA release through the stimulation of phospholipase A, activity (Chakraborti et al., 1989). AA release was also shown to be markedly enhanced by norepinephrine in cardiocytes from CMH (Kawaguchi et af., 199 1). Since myocyte injury, elevated levels of circulating norepinephrine, and intmcellular calcium accumulation are common features of CMH, it is likely that in the present study the failing heart manifested an accumulation of AA. A previous investigation has suggested that in the presence of an increased AA pool, diltiazem favors the release of a potent vasoconstrictor (Seeger et al., 1987). In the isolated rabbit lung, the AA-induced release of thromboxane A, -3 was augmented in the presence of diltiazem while prostaglandin I, (PGIJ release was decreased. Moreover, diltiazem was nearly ineffective in inhibiting the increase in pulmonary artery pressure induced by exogenously-appl ied AA. Hence, in the present study, diltiazem may have elicited the release of a constricting &tor such as TXA, only in the failing heart, an effect that could be suppressed by the cyclooxygenase inhibitor indomethacin. However, our findings do not exclude the possibility that diltiazern evokes the release of a similar cyclooxygenase constricting factor in normal hearts as well, but the coronary vascular smooth muscle of failing hearts would be hypersensitive to such constricting substances. The latter is unlikely since the concentration-response curve to diltiazem was not affected by indomethacin in normal hearts. In contrast, the loss of coronary sensitivity to clentiazem, observed in failing hearts, was not improved by indomethacin. These divergent observations may be due to different physico-chemical properties, such as lipophilicity, or some specific actions of the molecule. We recently reported that clentiazem, but not diltiazern, improved the cardioprotection provided by cold crystalloid cardioplegia in isolated ischemic rabbit hearts (Tanguay m al., 1994b). The cardioprotective properties of clentiazem were associated with the preservation of membrane lipid components (cholesterol and phospholipids), an effect that was not shared by diltiazern. Moreover. nifedipine and nisoldipine have been shown to inhibit phospholipase A, activity through a mechanism unrelated to their effects on slow calcium channels, while diltiazem had no such effect (Chang et al., 1987). Thus, it is possible that clentiazem could have membrane- stabilizing properties that prevent phospholipid breakdown and increased AA levels. Another explanation might help to understand the divergent effect of clentiazem in relation to AA metabolism. Seeger and coworkers (1987) have shown that verapamil and nimodipine, but not diltiazem, increased the AA-induced release of PGI,, which may then counterbalance the vasoconstrictive action of TXA, that was released simultaneously. These results have been confumed by another study demonstrating that, in contrast to dil tiazem , nifedipine and verapamil enhanced AA-stimulated PGI, synthesis (Gerr itsen m al., 1987). Like verapamil and dihydropyridines, clentiazem may therefore interfere with the synthesis of cyclooxygenase by-products in a different way than diltiazem. The fact that the reduced coronary sensitivity to clentiazem was not improved by indornethacin suggests that other yet undefined factors are involved. In conclusion, in the failing CMH heart, we have demonstrated reduced coronary sensitivity to diltiazem and clentiazem with no change in their inotropic or chronotropic effects. L-NAME experiments indicate that the NO pathway is not involved in the reduced coronary sensitivity to calcium antagonists in the presence of heart failure. The release of vasoconstricting substance(s) from the cyclooxygenase pathway may explain why coronary dilator sensitivity to diltiazem is altered in heart failure. In contrast, other yet undefined fhors mediate the reduction of coronary sensitivity to clentiazem. These fdings may provide a better understanding of the lack of efficacy of calcium antagonists in heart failure. They suggest that new vasoselective calcium antagonists may be more appropriate for treatment of this pathology. ACKNOWLEDGrnNTS We gratefully acknowledge the statistical expertise provided by Dr. Richard Moisan and the technical assistance of Chrystine Lacail!e, Judith Pignac, Pierre Duguay and Marc Vtronneau. This work was supported by a grant from the Canadian Heart and Stroke Foundation. M. Tanguay was the recipient of a studentship from Funds pour la Fonnution de Chercheurs et ['Aide a la Recherche. REFERENCES Buser, P.T.,Auffermann, W., Wu, S.T., Jasmin, G., Pannley, W.W. and Wikman- Coffelt, J.: Dobutamine potentiates amrinone's beneficial effects in moderate but not in advanced heart failure. 31P-MRS in isolated hamster hearts. Circ. Res. 66: 747-753, 1990. Carnacho, S.A., Wikman-Coffelt, J., Wu, S.T., Watters, T.A., Botvinick, E.H.. Sievers, R., James, T.L.,Jasmin, G. and Parmley, W.W.:Improvement in myocardial performance without a decrease in high-energy phosphate metabolites after isoproterenol in Syrian cardiomyopathic hamsters. Circulation 77: 7 12-7 19, 1988. 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M. : Combined inhibition of neutraI endopeptidase and angiotensin converting enzyme in cardiomyopathic hamsters with compensated heart failure. J. Pharmacol . Exp. Ther. 267: 108-116, 1993. Vanhoutte, P. M. : Vascular endothelium and W+antagonists. J. Cardiovasc. Pharmacol . 12 (Suppl 6): S2bS28, 1988. Vtniant, M., Clozel, J.P., Hess, P. and Wolfgang, R.: Ro 40-5967, in contrast to d iltiazem, does not reduce left ventricular contractility in rats with chronic myocardial infarction. J. Cardiovasc. Pharmacol. 17: 277-284, 199 1. V&omeau, M., Jasmin, G. and Dumont, L. : Altered endothelium and smooth muscle regulated coronary dilation in the failing heart of cardiom yopathic hamsters. Cl in. Invest. Med. 19 (Suppl): B10, 1994. Vilaine, J.P., Biondi, M.L.,Villeneuve, N., Feletou, M., Peglion, J.-L.and Vanhoutte, P.M.: The calcium channel antagonist S 11568 causes endothelium-dependent relaxation in canine arteries. Eur. J. Pharmacol. 197: 4 1-48, 1991. Wikman-Coffelt, J., Stefenelli, T., Wu, S.T.,Parmley, W.W. and Jasmin, G.: [Ca2+Ii transients in the cardiomyopathic hamster heart. Circ. Res. 68: 45-5 1, 1991. Winlaw, D.S., Smythe, G.A., Keogh, A.M., Schyvens, C.G., Spratt, P.M. and Macdonald, P.S. : Increased nimc oxide production in heart failure. Lancet 344: 373-374, 1994. Yang , B. C., Nichols, W. W. and Mehta, J. L. : Cardiac effects of acetylcholine in rat hearts: role of endotheliumderived relaxing factor and prostaglandins. Am. J. Physiol . 264 (Heart Circ. Physiol. 33): H 1388-H 1393, 1993. Chapitre 7 Discussion g6n6rale 7.1 Aspects m&hodologiques Les antagonistes du calcium, dont les bemthbzkpines, sont des agents qui posskdent des effets cardioprotecteurs dam divers modkles d ' ischkmie-reperfus ion. Cependant certaines etudes ont mis en doute I'eficacitk des antagonistes du calcium lorsque ces deniers sont ajoutes i la cardioplegie froide. De m*me, les effets inotropes negatifs des antagonistes du calcium peuvent limiter leur utilisation en pr6sence d'insuff~sancecardiaque. Notre projet de recherche visait a evaluer les effeu protecteurs du diltiazem et du clentiazem, en ajout B la cardioplegie, dans des coeurs ischemiques normaux et dtfaillants. Dans le contexte de protection myocardique perop6ratoire. il s 'avbre nkessaire d ' utiliser un modele experimental qui nous permet de mimer les conditions cl iniques . Les chirurgies cardiaques ttant de plus en plus pratiquees chez des patients dCfaillants cardiaques, il importe egalement de reproduire exp6rimentalement l'aldration initiale de la fonction cardiaque . Mod& de coeur isole' Plusieurs modbles ont & propost% afin d'etudier les effets cardioprotecteurs des antagonistes du calcium en ajout B la cardioplegie (De Jong 1986). Dans le present projet, des coeurs kol& selon la mtthode de Langendodf (1895) ont ttk utilisb. Ce modtle expCrimental a CtC largement utilisd dam le pass6 afin d'evaluer les effeu de Ifisch&nieet les benefices d'interventions pharmacologiques en protection myocardique (Galifianes et Hearse 19%). 11 offre plusieua avantages tels que la reproductibilite des resultats et un faible cost wmparativement aux moddes in viva Ce modele permet en outre un bon contr6le des conditions exp6rimentaIes et des param&tresqui modulent le dkbit coronarien et les fonctions inoaopes et chronotropes. I1 est ainsi possible d'evaluer divers param&tresfonctionnels et m6tabol iques indtipendamment de I ' influence du sys3me nerveux autonome et des effets cardiaques ethu vasculaires de certains tl6ments circulants. On reconnait certaines Iimites B I 'utilisation de ce modble exptrimental . Premibrement, il peut y avoir des variations importantes entre les espkes en ce qui concerne la vuldrabilitk du coeur B I ' ischemie et Ies b&n&kes associts aux strategies de protection myocardique (Galifianes et Hearse 1990b). Lfesp&e qui serait la plus representative de I 'homme pour utilisation dans un modele d' ischhie peropthtoire n 'est ma1 heureusement pas connue. D'autre part les coeurs utilisb proviennent a& souvent d'animaux jeunes et en sand alors qu'il a Ct6 dkmontrk que la prhence de certaines pathologies influence significativement la reponse myocardique I'ischbmie (Hearse et coll. 1978). 11 est cependant possible de rkoudre ce probleme par l'utilisation de rnodbles pathologiques. tel que nous l'avons fait dans le cadre du prhent projet de recherche : utilisation du hamster cardiomyopathique en phase de dtfaillance cardiaque. La 1imite temporelle peut constituer un autre dbavantage inherent ;i l'utilisation du modele de coeur isolb. En effet, bien que les coeurs isok puissent maintenir une activit.6 contractile pendant plusieurs heures, I 'absence des 6lhents sanguins et des stimuli hormonaux de mkme que I'apparition d'cedkme entrafnent t5ventuellernent une instabilitk du modble (Galiiianes et Hearse 19Wb). Ces wnsidt5rations font qu'il n'est pas possible d'kvaluer la rkupkration post-ischhique d'une fawn prolongt5e avec ce modkle. Lors du present projet la stabilid des coeurs &it d'environ 2 heures, ce qui permet une Cvaluation adQquatede la rkupt5ration postischhique. L'absence des dlkments circulants tels que les neuuophiles et les plaquetks clans les mod8les de coeun isolQ peut constituer un avantage si I'objectif est d'ttudier les effets cardioprotecteun directs des antagonistes du calcium. I1 est alors possible d'exclure les effets des antagonistes du calcium sur les plaquettes ou les neutrophiles comme wmposante participant B leurs effets cardioprotecteurs (Jouvin- Marche et coll. 1983. Rousseau et wll. 1991). I1 en est de mbme pour les effets periphiriques des antagonistes du calcium telle que la rduction de la postcharge (Stone et COIL 1980). Dans le cadre de ce projet, le mod2le de coeur is016 constituait donc un choix adequal puisqu'il permettait de mesurer les effets cardiaques directs des antagonistes du calcium tout en contr6lant les param&tresfonctionnels wdiaques. Protocole exptfn'mentul Le protocole exp6rimentaI devait reproduke le plus fidblement possible les conditions d'une ischtmie peropkratoire associk la chirurgie cardiaque. Ainsi, la duree de l'ischemie (90 minutes) de meme que le mode d'administration de la cardioplbgie Ctaient reprbentatifs des conditions prkentes en chirurgie wdiaque humaine (Flarneng et coll. 1986, Piwnica et Menasche 1989). En chirurgie cardiaque. 500 B 1000 ml de solution cardioplkgique sont habituellement administr6s. ce qui correspond a 2 a 4 mllg de myocarde (Takahashi et coll. 1988). La plupart des travaux rappones dam des rnodtles de coeurs isolts ont impliquk l'utilisation d'un plus grand volume de solution de cardioplegie, soit de 20 & 30 ml/g de myocarde (Fukunarni et Hearse 1985, Takahashi et coll. 1989, Yamarnoto et all. l983a). De fapn a se rapprocher davantage des volumes de solution de cardiopltgie utilisb en clinique, nous avons determine au cours de ce projet la quantitk de solution cardiopi6gique B infuser en fonction de la temptrature myocardique. L'infusion Ctait en effet cesste des que la temptrature myocardique atteignait 10 B 12 OC. Comparativement aux etudes experimentales anterieures, cette mCthode a enmane l'utilisation de volumes dtinfusion qui se rapprochaient davantage de ceux habituellement utilish en clinique (6 mllg de myocarde en moyenne lors de la premiere serie de travaux). En mnclusion, dmle prbent projet, la durk de ItischCmie de meme que les modalit& d'utilisation de la cardioplegie (tempbrature, volume de solution, durk, etc.) se comparent awc conditions cliniques. k protocole experimental utilisC nous semble donc approprie pour evaluer les effeu cardioprotecteurs des antagonistes du calcium de type benzo th iazkp he. Mod2le d'insuffisunce cdiaque Plusieurs mod6les exptrimentaux d'insuffisance cardiaque ont Ctk dkveloppb (Smith et Nunall 1985). Ces modPles permettent dt&udier cenains aspects associis 2 l'insuffisance cardiaque et fournissent de I' information qu'il serait difficile d'obtenir chez Ithornme. Exptrimentalement I'insuffisance cardiaque peut &re induite en utilisant diverses mtthodes : surcharge volumique (ex. altkration de la valve aonique), augmentation de la pression syst6rnique (ex. constriction de 1 'aorte), infarctus du m yocarde (ex. 1igature coronarienne) et enaalnement elecaique rapide du coeur (Armstrong et coll. 1986, Smith et Nuttall 1985). Toutes ces approches sont jugCes valables dans la documentation scientifique. Le protocole exptrimental et les parambues qui doivent &rekvaluts influenceront cependant le choix du modkle d'insufisance cardiaque. Certains de ces modeles nhssitent notamment des manoeuvres qui peuvent limiter ultkrieurement I'utilisation du mod6Ie de coeur isole. AM, une des methodes utiliskes afin d'entrafner une surcharge volumique est I'altkration de la valve aortique (Ezzaher et coll. 1991). Cette deficience valwlaire cornpromet la validid des r6sultat.s obtenus dans un coeur is016 puisqu'il est impossible d'haluer le d&it coronarien ou encore d'obtenir une perfusion coronarienne adequate. D'autre part, les procidures chirurgicales majeures que nkcessitent plusieurs modbles dtinsuffisance cardiaque constituent un dkavantage puisqu'elles entrainent une mortalit6 plus 61evb et un faible rendement. Findement, les changements hkmodynamiques observ& avec des modbles tels que la surcharge volumique et l'augmentation de la prwion syst6mique sont de nature aigui3 contrairement au developpement chronique de I'insuffisance cardiaque observe chez l'homme (Smith et Nuttal 1985). Chez le hamster cardiomyopathique, le modkle utilid dans le prbent projet. la defaillance cardiaque se d6veloppe progressivement. Ainsi chez la souche UM-X7.1. des lQions myocardiques sont evidentes P 40 jours de vie et le processus de ntcrose atteint son maximum vers 100 jours de vie (Jasmin et Proschek 1982). L'hypertrophie cardiaque et la fibrose se developpent par la suite et I'insuffisance cardiaque est observable entre 175 et 200 joun de vie. Le hamster dWehabituellement d'insuffisance cardiaque avant 250 jours de vie. Divers facteurs contribuent au dkveloppement de la cardiomyopathie du hamster dont une surcharge calcique intracellulaire. une augmentation du tonus sympathique, des anomalies gknktiques des membranes et des spasmes des microvaisseaux (Hano et Lakatta 1991. Makino et coll. 1985, Sole et coll. 1977. Sonnenblick et coll. 198s). Chez 1'homme, I 'ischhie myocardique est la muse d' insufisance wdiaque dont la prkvalence est la plus tlev6e (Dhalla et coll. 1993). Bien que Ie hamster UM-X7.1 prbente plutdt une cardiomyopathie idiopathique, il existe des similitudes enne ce modble expkimental et la pathogenke de I'insuffsance cardiaque chez I'homme. En effet, les vasospasmes coronariens semblent contribuer au developpement de la maladie tant chez I'homme que chez le hamster cardiomyopathique (Somenblick et coll. 1985). Des ktudes ont Cgalement dkrnontre une reduction du d6bit coronarien basal chez le hamster cardiomyopathique de mEme qufune plus grande sensibilitk vasculaire B divers agents vasoconsPictews tels la nor6pirRphrine, la &rotmine et 1'arg inine-vasopressine (Conway et coll. 1987, Conway et coll. 1994, Haleen et coll. 1991, Hunter et Elbrink 1983). Le hamster cardiomyopathique est maintenant largement accept6 comme rnodde d ' insuffisance cardiaque. I1 s' avbre particulibrement utile pour I 'etude de divers processus pathophysiologiques associCs au developpement de I 'insuffisance cardiaque de meme que pour 1'6valuation de mbdicaments (Rouleau et coll. 1982. Smith et Nuttal 1985, Trippodo et coll. 1993, Wikman-Coffelt et coll. 1991). Des preparations de coeurs isolk provenant de hamsten cardiornyopathiques ont aussi permis d'etudier les effets de plusieun m6dicaments sur la contractilitt5 myocardique (Buser et coll. 1990, Camacho et coll. 1988, Haleen et coll. 1991. Stefenelli et coll. 1989). Bref, le hamster cardiomyopathique de souche UM-X7.1 est un modble de d6veloppement chronique de 1' insufisance wdiaque qui a dkji Cte valid& 7.2 Discussion de I'ensemble des dsdtats La premiere serie de travaux de notre projet de recherche, rhlisb dam des coeurs isolCs de lapin, dCmontre que le clentiazem possMe des effets cardioprotecteurs additifs a la cardioplkgie froide. Bien que I'efficacie des antagonistes du calcium dam des conditions d'hypothermie ait anterieurement Cd remise en question (Hearse et coll. 1985). des benbfices significatifs sont rapport& avec un antagoniste du calcium de la farnille des benzothidpines, le clentiazem. Les propriktks cardioprotectrices de cette substance semblent lib sa structure molkulaire puisqu'elle est plus lipophile et plus puisante que le diltiazem. La dose utilisk est dgalement un facteur d'efficacite et d'innocuite important B considerer en cardioprotection. Les effets bt nefiques du clentiazern sont en effet observ& & une concentration non cardiod~pressive M) suggtrant ainsi que le clentiarem possge un profil d 'utilisation skcuritaire (Davies et coll. 1991, Narita et Ginsburg 1990). Les mkmkmes impliqub dans cette card ioprotection sont multiples. 11s pourraient ttre lies aux effets vasodilatateurs de ces molkules, ce qui permettrait une meilleure distribution de la solution cardioplt5gique et une attenuation du phenomkne d ' hypoperfusion associee ii la reperfus ion. La plus grande lipophilicite du clentiazem pourrait aussi expliquer ses effets cardioprotecteurs intrinskques. Une preservation de certains constituants phospholipidiques a Ct6 observh, ce qui milite en faveur de ces effets intrinstques. Bien que les benzothiazkpines soient dotks de proprittis protectrices a des doses non cardiod6pressives, leur profil d'efficacitk et d'innocuitk en prhence de dtfaillance cardiaque n'a pas 6t6 ktabli. La deuxi&meskie de travaux visait B verifier ces parambtres dans un modble d ' insufisance cardiaque chronique, soit le hamster cardiom yopathique UM-X7.1 de plus de 200 jours. Contrairement aux coeurs de hamsters normaux, les coeurs dtfaillants provenant de hamsters cardiomyopathiques sont rbistants aux effets card ioprotecteurs des benzothiaz6pines en ajout B la cardiopltgie. Cene observation suggkre que la d6faillance cardiaque al3re I 'eficacit6 du diltiazem et du clentiazem. Divers facteurs expliquent cette absence d'effets cardioprotecteurs dam le coeur dCfaillant dont les changernents de I ' hom6ostasie calcique observes chez le hamster cardiornyopathique (Makino et coll. 1985, Panaglia et coil. 1986. Whitmer et coll. 1988). De telles althtions pourraient entrainer une attenuation de I'effet prkventif des antagonistes du calcium sur la surcharge calcique. Une aune possibilid est que les aneintes membranaires observees en phase de dtfaillance cardiaque rendent inutile toute tentative de pr6servation des constituants membranaires lors d'une pCriode subsequente d' ischhie-reperfusion (Okumura et coll. 199 1). Des etudes anterieures suggkrent que les effets inonopes nkgatifs des antagonistes du calcium sont amplifib en prbence d ' insufflsance cardiaque (Ezzaher et col 1. 1991, Porter et coll . 1983). La presence d'une cardiodepression pounait alors rnasquer les effets cardioprotecteurs des benzothiaz6pines. Cependant, nos r6suitats indiquent que le clentiazem et le diltiazem n'entrainent pas d'effets inotropes nkgatifs significatifs dam des coeurs normaux et dtifafaillants B une concentration 10 nM (concentration identique a celle utilisCe dam la solution de cardioplegie). Toutefois, la vasodilatation wronarienne induite par ces molkules est atrknu& dans les coeurs defaillants. Ainsi une reduction de la sensibilitk vasodilatatrice wronarieme au diltiazem et au clentiazem constitue une hypothhe attrayante pouvant expliquer I'absence d'effets cardioprotecteurs dans cene condition patholog ique. La troisi8me sCrie de travaux wnsistait mieux cafact&iser la sensibilitk cardiaque et coronarienne du diltiazem et du clentiazem en prbence d'insuffisance cardiaque. Les courbes dose-r6ponse rMis&s dam des coeurs isolCs de hamsters normaux et defaillants indiquent que l'insufisance cardiaque n'est pas associee ti une augmentation de la sensibilite ii leurs effets inotropes nkgatifs intrins6ques. Ces rksultats confirment donc que la riisistance du coeur defaillant de hamster aux effets cardioprotecteurs de ces agents n'est pas explicable par une amplification de leurs effets inotropes nkgatifs. La rbistance aux effets cardioprotectem des benzothiazepines en prksence de dtfaillance cardiaque serait tributaire d 'une algration de leurs effets vasodilatateurs coronariens. En effet, la sensibilit6 vasodilataaice coronarienne au diltiazem et au clentiazem est d iminde dans les coeurs defaillants. De plus cette altkration de la sensibilite coronarienne constitue une nouvelle hypothh expliquant I'absence de Wntfices associ& A I'utilisation des antagonistes du calcium de premihre gtnhtion dam le traitement de I ' insuffisance cardiaque (Elkayam et cull. 1990, Goldstein et coll. 1991). L' influence possible de certains facteurs endothUiaux (NO et prostaglandines) sur la r6duction de la sensibilit6 coronarienne aux benzothiaApines a pu ttre 6vaIuik i l'aide de courbes dose-reponse realis& en prbence d'un inhibiteur de la NO-synthase (L- NAME) et d'un hibiteur de la cyclooxyghase (indornt5thacine). Nos resuiats indiquent que la skr6tion basale de NO n'est pas alter& chez le hamster cardiomyopathique en phase de d6faillance cardiaque puisque le L-NAME induit une vasoconstriction similaire dans les coeurs normaux et dkfaillants. Les effets vasodilatateurs coronariens du diltiazern ne sont pas affect& par le L-NAMEalors que ceux du clentiazem sont attdnues tant pour les coeurs normaw que d6faillants. La voie de synthbe du NO ne semble donc pas &re impliquk dam la rtkiuction de sensibilite coronarienne aux benzothiaztipines en presence d ' insufisance cardiaque. La liMration de facteurs prostanoides vasoconstricteun pourrait expl iquer la d i rninut ion de la rkctivitk coronarienne au diltiazem dam ce modble d' insuffisance cardiaque. En effet, la courbe dose-rkponse du diltiazern est normalisee en presence d'indomethacine. Cependant, la perte de sensibilid coronarienne au clentiazem n 'est pas amClior6e par I'indomethacine. Ces r~sultatsdivergents peuvent s'expliquer par des propriCt6s physicochimiques distinctes des molecules Ctudihs qui influenceraient de manibre spicifique la synthhe de derives de la cyclooxygtnase (Seeger et coll. 1987). Les facteurs qui modulent la perte de sensibilit6 coronarienne au clentiazem observable dam la dkfaillance card iaque demeurent inconnus. En conclusion, bien que les benzothiazkpines soient des agents protecteurs efficaces en ajout B la cardioplegie dam des coeurs ischCmiques normaux, ces molkules s'av6rent d'un moindre interet en prksence d'insuffisance cardiaque. La pene d'efficacid cardioprotecnice du diltiazem et du clentiazem dans l'insuffisance cardiaque s'expliquerait par une altkration de leur action vasodilatatrice coronarie~eet non pas par une amplification de leur effets cardiod6presseurs. De plus des facteurs endothCliaux, tels que des dCrivh de la cyclooxyg~nase,participent i la modulation de leurs effets vasodilatateun coronariens dam la defaillance cardiaque. 7.3 Perspectives La cardioprorection demeure un phtnombne complexe. Outre Itefficacit6 intrinskque de l'agent cardioprotecteur utilist, on se doit de considtrer tous Ies facteurs qui peuvent moduler son activitb. Dam le present projet, I ' utilisation d'un modde d' insuffisance cardiaque nous a permis de demontrer la rkistance des cwurs defaillants aux effets cardioprotecteurs du diltiazem et du clentiazem. 11 s'averera donc tres important dam le d tveloppement futur d'agents cardioprotecteurs d ' util iser des modeles qui miment les pathologies sous-jacentes telles que I 'hypertension, I ' insuffsance cardiaque. I 'ath6roscl&ose, etc. Les r6sulrat.s obtenus ouvrent egdement de nouveaux horizons de recherche. Nous avons mis en hidem qu'en prkence d ' insufflsance cardiaque I' absence d ' une cardioprotection significative par le diltiazem et clentiazem n'est pas attribuable ii une hypersensibilitk i leurs effets inotropes negatifs intrinskques . Une dbensibilisation B leun effets vasculaires coronariens semble en cause et el le serait tributaire d ' une 1iberation de facteurs endothkliaux. Compte tenu de I'importance de la protection vasculaire dam I'ischemie et la reperfusion (Hearse et coll. 1993), une caract6risation plus complete des rntkanisrnes impliqub dans cette dbensibilisation est souhaitable. Afin d'obtenir une vasodilatation coronarienne significative en presence d'insuffisance cardiaque, il est possible qu'il faille augrnenter les doses d'antagonistes du calcium de premiere g6nhtion ii un seuil critique de cardiod$ression. I1 serait intkressant d'evaluer si la sensibilitk coronarienne aux antagonistes du calcium de deuxitme generation, qui sont do& d'une plus grande vasosklectivitt5, est preservb dans le coeur dkfaillant (Little et coll. 1995). Finalement, en plus d'expliquer leur inefficacite comme agents cardioprotecteurs, la d&ensibilisation aux effets vasodilatateurs coronariens des antagonistes du calcium peut servir d'hypothbe B I'absence de bbnkfices lors de leur utilisation dans le traitement chronique de 1' insuffisance cardiaque (Elkayam et coll. 1990, Goldstein et coll. 1991). Les hypothhes Cmises jusqu'k maintenant pour expliquer 11inefficacitt5des antagonistes du calcium de premihe gentration dam le traitement de l'insuffisance cardiaque sont l'amplification de la depression myocardique et/ou une stimulation neurohorrnonale (Packer 1989, Parameshwar et Poole-W ikon 1993). La dbensibil isat ion vasculaire mise en evidence dans notre projet de recherche constitue une hypothhe amayante et novatrice. Les antagonistes du calcium de deuxi5me gkneration semblent plus promettern dans le traitement de I'insufisance cardiaque. 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Appendice I TABLE 1-1 Mmimal comnaryjlbw hemme induced by mrn and clenikem in normal und failing hearts Normal hearts Failing hem Values are means f SEM (n = 5 per group). Data are expressed as percent increase from baseline. Data in parentheses are maximal flow values expressed in milliters per minute. Remerciements Au terme de ces travaux, je tiens ii remercier trks chaleureusement toutes les personnes qui m'ont aide clans la raisation de mon projet de doctorat. J'aimerais particulikrement remercier le Dr Louis Dumont, mon directeur de recherche, pour son devouement et sa participation sans reliche aux travaux, de mCme que pour son enthousiasme contagieux pour la recherche. Je voudrais kgalement hi exprimer toute ma reconnaissance pour la confiance qu'il m'a manifestee afin de me permettre de rhliser ce projet ainsi que Ie support et les encouragements incessants qu'il m'a prodigub. Je suis tigalement particulitrement redevable au Dr Gilbert Blaise. mon codirecteur, de ses judicieux conseils et des innombrables services qu'il m'a rendus. II me faut aussi souligner la precieuse collaboration du Dr Gdtan Jasmin et de M. Guy Lepage. Je tiens B remercier tous mes wll&guesde laboramire: Pierre Duguay, Chrystine Lacaille. Judith Pignac, Marc Vkromeau et Stiphanie Viau, qui m'ont apporte une aide inestimable et avec qui les longues heures d'exptrimentation Ctaient plus agrhbles. J'aimerais igalement remercier le Dr Richard Moisan pour son expertise en statistique et en inforrnatique et Elizabeth Pkrb pour son concours A la rhlisation de plusieurs figures de cet ouvrage. Je ne saurais passer sous silence le support financier que m'ont accord6 Nordic Merrel Dow Research et le Fonds pour la Formation de Chercheurs et 1'Aide A la Recherche (FCAR). Enfin, cette thiis= n'aurait pu &re men& terme sans l'amour et les encouragements de Sophie, ma wmpagne, pendant ces longs mois de travail.