Effects of Captopril and Omapatrilat on Early Post-Myocardial Infarction Survival and Cardiac Hemodynamics in Rats: Interaction with Cardiac Cytokine Expression

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Effects of captopril and omapatrilat on early post-myocardial infarction survival and cardiac hemodynamics in rats: interaction with cardiac cytokine expression

Charles Blais, Jr., Nathalie Lapointe, Jean-Lucien Rouleau, Robert Clément, Dimcho R. Bachvarov, and Albert Adam

Abstract: The purpose of this study was to evaluate and compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (10 and 40 mg·kg–1·day–1) with those of the selective ACE inhibitor captopril (160 mg·kg–1·day–1) on survival, cardiac hemodynamics, and cytokine mRNA expression in left ventricular (LV) tissues 4 days after myocardial infarction (MI) –1 –1 in rats. The effects of the co-administration of both B1 and B2 kinin receptor antagonists (2.5 mg·kg ·day each) with and without omapatrilat were also evaluated to assess the role of bradykinin (BK) during this post-MI period. Both omapatrilat and captopril treatments improve early (4 days) post-MI survival when started 4 h post-MI. The use of kinin receptor antagonists had no significant effect on survival in untreated MI rats and omapatrilat-treated MI rats. This improvement in survival with omapatrilat and captopril is accompanied by a reduced LV end-diastolic pressure (LVEDP) and pulmonary congestion. The use of kinin receptor antagonists had little effect on cardiac hemodynamics α β or morphologic measurements. Acute MI significantly increased the expression of cardiac cytokines (TNF- ,TGF- 1, and IL-10). Captopril significantly attenuated this activation, while omapatrilat had variable effects: sometimes increasing but generally not changing activation depending on the cytokine measured and the dose of omapatrilat used. The α β co-administration of both kinin receptor antagonists attenuates the increase in expression of cardiac TNF- and TGF- 1 after omapatrilat treatment. Taken together, these results would suggest that despite very marked differences in the way these drugs modified the expression of cardiac cytokines, both omapatrilat and captopril improved early (4 days) post- MI survival and cardiac function to a similar extent. Key words: ACE inhibitor, cytokines, kinins, myocardial infarction, vasopeptidase inhibitor.

Reséumé : La présente étude a eu pour but d’évaluer et de comparer les effets de l’inhibition58 simultanée Blais et de al. l’enzyme de conversion de l’angiotensine (ECA) et de l’endopeptidase neutre 24.11 (EPN) par l’inhibiteur de la vasopeptidase, omapatrilat (10 et 14 mg·kg–1·jour–1), avec ceux de l’inhibiteur sélectif de l’ECA, captopril (160 mg·kg–1·jour–1), sur la survie, l’hémodynamique cardiaque et l’expression de l’ARNm des cytokines dans les tissus ventriculaires gauches (VG), 4 jours après un infarctus du myocarde (IM) chez des rats. On a aussi évalué les effets de la co-administration –1 –1 des antagonistes des récepteurs B1 et B2 des kinines (2,5 mg·kg ·jour chacun), avec et sans omapatrilat, afin d’évaluer le rôle de la bradykinine (BK) durant cette période post-IM. Tant le traitement à l’omapatrilat que le traitement au captopril ont amélioré rapidement (4 jours) la survie post-IM lorsqu’ils ont été amorcés 4 heures après l’IM. L’emploi des antagonistes des récepteurs des kinines n’a pas eu d’effet significatif sur la survie chez les rats non traités et chez les rats IM traités à l’omapatrilat. Cette amélioration de la survie par l’emploi de l’omapatrilat et du captopril est accompagnée d’une réduction de la pression télédiastolique VG et de la congestion pulmonaire. L’emploi des antagonistes des récepteurs des kinines a eu peu d’effet sur l’hémodynamique cardiaque ou les mesures $ morphologiques. L’IM aigu augmente de façon significative l’expression des cytokines cardiaques (TNF- ,TGF- 1 et IL-10). Le captopril atténue significativement cette activation, alors que l’omapatrilat a des effets variables, parfois augmentant, mais généralement ne modifiant pas l’activation, selon la cytokine mesurée et la dose d’omapatrilat

Received 29 May 2001. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 31 January 2002. C. Blais, Jr., and A. Adam.1 Faculté de Pharmacie, Université de Montréal, 2900, boul. Édouard-Montpetit, C.P. 6128, Succursale Centre-ville, Montréal, QC H3C 3J7, Canada. N. Lapointe and J.-L. Rouleau. Division of Cardiology, University Health Network, Toronto General Hospital, Toronto, ON M5G 2C4, Canada. R. Clément. Institut de Cardiologie de Montréal, Montréal, QC H1T 1C8, Canada. D.R. Bachvarov. Centre Hospitalier Universitaire de Québec, Centre de Recherche du Pavillon l’Hôtel-Dieu de Québec, Québec, QC G1R 2J6, Canada. 1Corresponding author (e-mail: [email protected]).

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utilisée. La co-administration des deux antagonistes des récepteurs des kinines atténue l’augmentation de l’expression $ de la TNF- et de la TGF- 1 cardiaques après le traitement à l’omapatrilat. Ensemble ces résultats donneraient à penser que, malgré des différences marquées dans la façon dont ils modifient l’expression des cytokines cardiaques, l’omapatrilat et le captopril améliorent la survie post-IM (4 jours) et la fonction cardiaque avec la même efficacité.

Mots clés : inhibiteur de l’ECA, cytokines, kinines, infarctus du myocarde, inhibiteur de la vasopeptidase.

[Traduit par Rédaction]

that cytokines may be important modulators in the post-MI Introduction process. Angiotensin-converting enzyme (ACE, EC 3.4.25.1) inhi- Neutral endopeptidase 24.11 (NEP, EC 3.4.24.11) is an- bition has been shown to improve the survival of patients other important enzyme involved in the degradation of when started early (<24 h) after myocardial infarction (MI), kinins and is the major enzyme inactivating natriuretic pep- particularly in the presence of left ventricular dysfunction tides (ANP, BNP, and CNP) (Erdös and Skidgel 1989). In- (Brown and Vaughan 1998; Yusuf et al. 2000). In MI pa- hibition of NEP potentiates the vasodilator, diuretic, and tients, the majority of the benefit is noted during the natriuretic effects of these two peptidergic systems and has first week post-MI. Since the renin–angiotensin system is beneficial effects in animal models of heart failure (Sey- activated in the setting of an MI, it is attractive to postulate mour et al. 1993; Rademaker et al. 1996; Willenbrock et al. that the beneficial effects of ACE inhibitors are only attribut- 1996). It may thus be postulated that the simultaneous inhi- able to a decreased synthesis of angiotensin II, a potent bition of ACE and NEP may be superior to ACE inhibition vasoconstrictive and salt-retentive peptide that stimulates alone post-MI. A new class of drugs has been developed, myocardial hypertrophy and plays a role in growth of the the vasopeptidase inhibitors, which are single molecules cardiac interstitium (Brown and Vaughan 1998). However, that simultaneously inhibit both ACE and NEP with similar ACE, also called kininase II, acts as a potent kinin-degrading nanomolar inhibitory constants. These vasopeptidase inhib- enzyme in plasma and tissues. Thus, there is mounting evi- itors were shown to prevent the development of cardiac hy- dence that at least part of the beneficial effect of ACE inhib- pertrophy and reduce cardiac preload in rats with MI itors are the result of the inhibition of kinin metabolism (Bralet et al. 1994; Marie et al. 1995). They display (Linz et al. 1995; Blais et al. 2000b). In acute MI, hemodynamic effects that are superior to those induced by bradykinin (BK) can exert beneficial effects by systemic and selective inhibition of ACE or NEP in cardiomyopathic coronary vasodilatation, thereby improving metabolic bal- hamsters (Trippodo et al. 1995) and, in the pacing-induced ance, lowering myocardial oxygen consumption, and poten- left ventricular (LV) dysfunction dog, they reduce the de- tially reducing infarct size (Linz et al. 1995). BK can also gree of LV dilation and improve isolated myocyte contrac- improve cardiac function through diuretic, natriuretic, anti- tile function, showing no apparent effect on LV-pump hypertrophic, anti-proliferative, anti-thrombotic, and fibrolytic function (Thomas et al. 1998). Omapatrilat is the clinically effects due to the release of prostacyclin, nitric oxide, and en- most advanced vasopeptidase inhibitor and is now being in- dothelium-derived hyperpolarizing factor (Levinsky 1979; vestigated in clinical trials for use in hypertension and con- Moncada et al. 1991; Linz et al. 1995; Blais et al. 2000b). gestive heart failure, and its use early post-MI is also being Thus, BK may help to counteract some of the detrimental ef- considered. fects of renin–angiotensin activation. In previous studies, we demonstrated that the vaso- Early post-MI is also characterized by activation of an in- peptidase inhibitor omapatrilat reduces the metabolism of flammatory process in which the pro-infammatory properties exogenous BK more than ACE or NEP inhibition in rat and of BK may play an important contributory role. As part of human hearts (Raut et al. 1999; Blais et al. 2000a). Be- this inflammatory process, there is increased expression of cause endogenous BK levels are cardioprotective in the several cytokines (Thompson et al. 1988; Herskowitz et al. acute MI setting, we hypothesized that omapatrilat could 1995; Ono et al. 1998). Indeed, increased plasma levels as have greater cardioprotective effects than ACE inhibitors in well as local myocardial production of several cytokines the early post-MI setting, and that this effect could be re- such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, lated to an increase in endogenous kinin cardiac levels. Al- β β IL-6, and transforming growth factor 1 (TGF- 1) have been ternatively, the extra protection of BK by simultaneous observed in patients early post-MI (Maury and Teppo 1989; ACE and NEP inhibition could excessively stimulate the Ikeda et al. 1992; Basaran et al. 1993). There is evidence post-MI inflammatory response and cardiac cytokine ex- that cytokines have been implicated in the pathogenesis of pression and lead to detrimental effects. myocardial dysfunction and cardiomyocyte death in MI and The purpose of this study was to evaluate and compare the congestive heart failure (CHF) (Pulkki 1997; Blum and effects of simultaneous ACE and NEP inhibition by the Miller 1998; Meldrum 1998). Moreover, several cytokines vasopeptidase inhibitor omapatrilat with those of the selec- have been reported to regulate cardiac myocyte growth, con- tive ACE inhibitor captopril on survival, cardiac hemo- tractile protein synthesis, fibroblast proliferation, and extra- dynamics, and cytokine mRNA expression in LV cardiac cellular matrix gene expression (Meldrum 1998; Sasayama tissues 4 days after MI in rats. The effects of the co- et al. 1999; Lijnen et al. 2000). These observations suggest administration of both B1 and B2 kinin receptor antagonists

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50 Can. J. Physiol. Pharmacol. Vol. 80, 2002

with and without omapatrilat were also evaluated to assess gation but prior to hemodynamic monitoring had morpho- the role of BK during this post-MI period. logic assessment for classification of MI size but were not used for other measurements except for survival. The survi- Materials and methods vors were randomized into different groups.

Drugs, peptides, and reagents Drug randomization The ACE inhibitor captopril (IC50 = 23 nM) and the At the time of the MI, rats were first divided to receive the vasopeptidase inhibitor omapatrilat, which acts by combined combination of both B2 (icatibant) and B1 (R-715) kinin re- –1 –1 inhibition of ACE (IC50 = 5 nM) and NEP (IC50 = 9 nM), ceptor antagonists (2.5 mg·kg ·day each) or not. B1 and were provided for research purposes by Bristol-Myers B2 antagonists were given together because, with inflamma- Squibb (Princeton, N.J.). The B2 kinin receptor antagonist tion such as may occur in this model, B1 receptors may be 3 5 7 8 icatibant (HOE 140, D-Arg-[Hyp ,Thi ,D-Tic ,Oic ]BK; expressed and exert effects similar to those of B2 receptors. Wirth et al. 1991) was a generous gift from Dr. K.J. Wirth Those randomized to receive the B2 and B1 receptor antago- (Hoechst AG, Frankfurt, Germany). The B1 kinin receptor nists received these agents dissolved in sterile saline via con- 7 8 9 antagonist R-715, AcLys-[D-βNal ,Ile ]des-Arg -BK (Gobeil tinuous infusion from a micro-osmotic pump (Alzet 1007D, et al. 1999), was obtained from Dr. D. Regoli (Université de Alza, Palo Alto, Calif.) that was inserted subcutaneously in Sherbrooke, Sherbrooke, Que.). Ketamine hydrochloride was the back of the animal during the surgery for the MI (Blais obtained from Rogar/STB (Montreal, Que.), xylazine was et al. 1997; Gobeil et al. 1999). Four hours after MI, rats from Bayer Canada (Etobicoke, Ont.), and buprenorphine HCl were again randomly divided into six groups according to was from Reckitt Colman Pharmaceuticals (Richmond, Va.). their therapeutic intervention. After randomization (4 h post- Halothane was manufactured by Halocarbon Laboratories MI), rats had an intraperitoneal injection of their assigned (Riveredge, N.J.). All other chemicals of analytic grade were medication. This procedure was repeated the next morning obtained from Fisher Scientific (Montreal, Que.). to assure adequate levels of medications post-MI. Thereafter rats received their medications with the food. The drugs Myocardial infarction were mixed with normal powdered chow, and total chow in- An MI was induced in 175 male Wistar rats (Charles take was monitored every day. A first group received River, St. Constant, Que.) weighing 200–250 g by ligation of intraperitoneal injections of saline followed by normal food the left anterior descending coronary artery (LAD) as de- (untreated group, n = 21). A second group received scribed earlier by Pfeffer et al. (1979). Animals were given intraperitoneal injections of 4 mg·kg–1 omapatrilat, and 5–7 days to adjust to their new environment. All of the ani- 40 mg·kg–1·day–1 in food thereafter (n = 20). A third group mal experiments followed the guidelines of the Canadian received intraperitoneal injections of 1 mg·kg–1 omapatrilat, Council on Animal Care and were approved by the Animal and 10 mg·kg–1·day–1 in food thereafter (n = 17). A fourth Care Ethics Committee of the Montreal Heart Institute group received intraperitoneal injections of 16 mg·kg–1 (Montreal, Que.). Animals were anesthetized with 3% captopril, and 160 mg·kg–1·day–1 in food thereafter (n = 27). halothane. During the surgery, they were artificially venti- A fifth group received both B2 and B1 kinin receptor antago- lated with humidified room air supplemented with oxygen, nists (icatibant and R-715, respectively) and intraperitoneal and the halothane concentration was gradually decreased to injections of 4 mg·kg–1 omapatrilat, and 40 mg·kg–1·day–1 in 1%. A Harvard rodent ventilator (Harvard Apparatus, South food thereafter (n = 31). A sixth group received both B2 and Natick, Mass.) set at 2 mL, 70 strokes/min, and a Fluotec 3 B1 kinin receptor antagonists (icatibant and R-715, respec- halothane vaporizer (Cyprane) were used for this procedure. tively) and intraperitoneal injections of normal saline solu- A left thoracotomy was performed via the fourth intercostal tion, and normal food thereafter (n = 15). The dosages of space, and the lungs were retracted to expose the heart. After captopril and omapatrilat were based on previous studies the pericardium was opened, the LAD was ligated ~2 mm (Pfeffer et al. 1985a, 1985b; Trippodo et al. 1999; d’Uscio et from its origin using a 4-0 silk (Ethicon, Johnson and John- al. 2001). son, U.S.A.); ligation was deemed successful when the anterior wall of the LV turned pale. The chest was closed with a Cardiac hemodynamic measurements 2-0 silk (Ethicon, Johnson and Johnson, U.S.A.) and the in- After 4 days of therapy, the rats were anesthetized with an cision was closed with a Mikron wound clip applicator (Clay intramuscular injection of a ketamine (50 mg·kg–1) and Addams, Parsipanny, N.J.) after the chest was gently pressed xylazine (10 mg·kg–1) mixture. The trachea was intubated by to expel air from the cavity to avoid a pneumothorax. Once a non-invasive method via the mouth and mechanically ven- awake, the rats were injected intramuscularly with tilated with room air supplemented with low-flow oxygen buprenorphine HCl 0.01 mg·kg–1 to reduce the pain during with a small-rodent ventilator (Harvard Apparatus). An elec- recovery. The sham ligation group underwent a similar pro- trocardiogram (ECG) was performed, and the left and right cedure except that the suture was not tightened around the ventricular pressures were measured by a Millar micro-tip coronary artery. The rats were housed for 4 days in clear catheter transducer (Millar Instruments, Houston, Tex.) with plastic cages in an air-conditioned room with a 12 h a pressure sensor at the tip. The catheter was inserted into photoperiod with free access to normal crush laboratory rat the jugular vein and carotid artery and advanced to the right chow and tap water. Of these 175 rats, 44 died 4 h post- and left ventricles, respectively. The maximum rate of pres- operation or during the 4 day follow-up period. Those dying sure rise (+dP/dt) and decline (–dP/dt) of both ventricles between 4 h and 24 h post-infarction were assumed to have were also measured. The ECG and pressures were recorded had a large MI. Those dying later than 24 h post-coronary li- on a Gould 2600S recorder (Gould, Cleveland, Ohio). Be-

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cause of equipment problems or death during the procedure, hibitor, 50 U of Moloney murine leukemia virus RT, and hemodynamic measurements could not be performed in 2.5 µM Random Hexamers (Perkin-Elmer, Roche Molecular some animals: 2 rats of group 1, 1 of group 2, 1 of group 3, Systems, Branchburg, N.J.). The RT reaction was performed 1 of group 4, 1 of group 5, and 1 of group 6. There was no at room temperature for 10 min followed by incubation at difference in cardiac hemodynamics between the untreated 42°C for 15 min. At the end of RT, the mixture was heated groups with or without the BK receptor antagonists, as well at 99°C for 5 min to inactivate the RT enzyme and denature as with both doses of omapatrilat, so these groups were the cDNA. combined. Each 100 µL PCR reaction contained 20 µL of RT mix, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, µ α β Cardiac morphologic assessment 0.4 M of each of the TNF- or TGF- 1 primers with Once the hemodynamic measurements were obtained, the 0.027 µM or 0.04 µM of the glyceraldehyde 3-phosphate rats were deeply anesthetized and killed by cardio- dehydrogenase (GAPDH) primers (needed for the amplifica- pulmonary excision. The heart was rapidly rinsed in saline tion of an internal standard), respectively, and 2.5 U of Taq solution, and dissected into right and left atria, right ventri- polymerase (AmpliTaq DNA Polymerase, Perkin-Elmer). cle, left ventricle, septum, and scar. The scarred area was The following oligonucleotides were utilized as PCR primers: pinned on a paper and its surface was determined by 5′-T ACT GAA CTT CGG GGT GAT TGG TCC-3′ and 5′- planimetry (Labtronics Inc., Guelph, Ont.). All portions of CAG CCT TGT CCC TTG AAG AGA ACC-3′ (Clonetech, the hearts as well as the lungs were then weighed individu- Palo Alto, Calif.) were used as sense and anti-sense primers, ally, frozen in liquid nitrogen, and stored at –80°C until be- respectively, for the amplification of a specific rat TNF-α ing used for biochemical investigations. fragment; 5′-C TTC AGC TCC ACA GAG AAG AAC TGC-3′ Animals were not classified according to MI size until the and 5′-CAC GAT CAT GTT GGA CAA CTG CTC C-3′ end of the study at which time they were separated into (Clonetech) were used as sense and anti-sense primers, respec- β sham to small MI or medium to large MI groups. Rats with a tively, for the amplification of a specific rat TGF- 1 fragment medium to large MI were defined as those having an LV scar and 5′-TG AAG GTC GGT GTC AAC GGA TTT GGC-3′, surface of ≥0.6 cm2. Those classified as having sham to and 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′ small MI had an LV scar ≤0.5 cm2. For more clarity, sham- (Clonetech) were used as sense and anti-sense primers, re- operated rats and those with a small MI are hereafter called spectively, for the amplification of a specific rat GAPDH “sham”, and those with a moderate to large MI are called fragment. The oligonucleotides were used according to the “MI”. Rats with an MI based on an LV scar surface conditions established by the manufacturer. The samples ≥0.6 cm2 had hemodynamic values similar to those with an were denatured initially for 4 min at 94°C and then submit- MI based on a ≥45% scar circumference (Nguyen et al. ted to 35 cycles of PCR for TNF-α and 27 cycles of PCR for β 1998). TGF- 1 (45 s at 94°C, 45 s at 60°C, and 2 min at 72°C) and a final 7-min elongation step at 72°C in a PTC-200 DNA Total RNA isolation Engine thermal cycler (MJ Research, Inc., Watertown, Total RNA was isolated using the TRIzol reagent (Gibco- Mass.). The number of cycles was chosen to keep the PCR- BRL), as described by the manufacturer. Briefly, ~100 mg of amplified DNA in the exponential phase of amplification. frozen non-infarcted LV tissue was ground in a mortar with One-fifth of each of the PCR reactions was loaded on a liquid nitrogen, and the resulting powder was resuspended in 1.8% agarose gel in 1× Tris-borate–EDTA buffer and 0.75 mL of TRIzol. The suspension was then homogenized 0.5 µg·mL–1 ethidium bromide and electrophoresed at 100 V using a Polytron homogenizer (Brinkmann Instrument, for approximately 1.5 h. The 1-kb DNA ladder (Gibco-BRL) Rexdale, Ont.) and incubated for 5 min at room temperature. was used to determine the molecular weight of each PCR The homogenate was extracted with 0.2 mL of chloroform, fragment. The resulting photographs were scanned with a and upon centrifugation (12 000 × g, 15 min, 4°C) the aque- ScanJet 5100C scanner (Hewlett-Packard, Palo Alto, Calif.) ous phase was mixed with 0.5 mL of isopropyl alcohol. The and analyzed with 1D Main densitometry software (AAB resulting pellet was washed with 1 mL of 75% ethanol and Software, Dayton, Ohio). finally resuspended in 50 µL of RNase-free sterile water. All total RNA samples were kept at –80°C until use. RT-PCR analysis of the IL-10 mRNA expression The IL-10 mRNA expression was evaluated in the probes α β RT-PCR analysis of the TNF- and TGF- 1 mRNA studied by a duplex RT-PCR approach (Dukas et al. 1993) us- expression ing the Ready-to-Go RT-PCR beads (Amersham Pharmacia The RT-PCR experiments were based on a method pub- Biotech) as indicated by the manufacturer. Each RT-PCR re- lished by Dukas et al. (1993). Briefly, 1 µg of total RNA was action (50 µL final volume) contained 4 µg of total RNA, treated for 15 min at room temperature in 20 mM Tris-HCl 20 ng of oligo dT13 primer, 100 ng each of the IL-10 prim- (pH 8.4), 2 mM MgCl2, 50 mM KCl, and1UofDNase I ers, and 10 ng each of the GAPDH primers (needed for the amplification grade (Gibco-BRL) to remove traces of genomic amplification of an internal standard, see above). The DNA. The DNase I was then inactivated by adding 1 µLof oligonucleotides utilized as PCR primers for IL-10 were 25 mM EDTA (pH 8.0) solution to the reaction mixture and purchased from BioSource International (Camarillo, Calif.) heating it for 10 min at 65°C. and used according to the conditions established by the man- DNase I-treated RNA (0.15 µg) was reverse transcribed in ufacturer. The samples were incubated initially for 30 min at a20µL reaction mix containing 10 mM Tris-HCl (pH 8.3), 42°C, followed by a denaturation step for 4 min at 95°C, and 50 mM KCl, 5 mM MgCl2, 1 mM dNTP, 20 U of RNase in- were then submitted to 30 cycles of PCR (1 min at 94°C,

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52 Can. J. Physiol. Pharmacol. Vol. 80, 2002

1 min at 60°C, 2 min at 72°C) with a 10 min final elonga- Fig. 1. Effect of omapatrilat, captopril, and omapatrilat plus BK tion step at 72°C. Twenty-five microlitres of each RT-PCR antagonists on survival of rats with large myocardial infarction reaction were run on a 1% agarose gel in 1× TBE buffer and (MI). Drug treatments were started 4 h after coronary artery liga- were then transferred to Hybond-N nylon membranes tion. Both omapatrilat (P = 0.002) and captopril (P = 0.016) (Amersham Pharmacia Biotech). The membranes were pre- treatments improved survival. hybridized for1hat65°C in a buffer containing 6× SSC, 5× Denhardt’s solution, 0.5% SDS, 100 µg·mL–1 salmon sperm DNA; then 106 cpm/mL of IL-10 random-primed 32P- labelled probe was added and hybridization was carried for 8–16 h at 65°C. The membranes were repeatedly washed (final wash 0.1× SSC – 0.1% SDS at 65°C) and exposed to Biomax MS (Kodak) autoradiographic films. The membranes were then stripped in boiling 0.1% SDS and rehybridized with a rat GAPDH probe used as an internal standard. The resulting autoradiograms were scanned with a ScanJet 6 (Hewlett-Packard) and analyzed with the 1D Main densitometry software (AAB Software).

Statistical analysis All data are expressed as means ± SE for n values. Statis- tical significance of differences was calculated using one- way analysis of variance and, when indicated, a post hoc Tukey’s test for multiple comparisons. Only probability values of P < 0.05 were accepted as statistically significant. Kaplan–Meier survival curves over the follow-up period were constructed and analyzed by the generalized savage (Mantel–Cox) test.

Results

Survival The sham-operated controls all survived until the end of all resulted in a further decrease in LVSP and a normaliza- the study. Of the untreated rats, 9 were sham and 28 had an tion of LVEDP. MI, of which 16 died and 12 survived (43% survival). Of the untreated rats that received B1 and B2 receptor antagonists Morphologic parameters (BK antagonists), 6 were sham and 19 had an MI, of which By definition, scar surface as well as the scar weight to 10 died and 9 survived (47% survival). Since survival rates body weight (SW/BW) ratio were significantly increased in were the same in the two untreated groups, they were con- hearts with an MI as compared with sham-operated rats. The sidered together (45% survival). The rats with an MI that re- SW/BW ratios were similar in all MI groups. The MI as ceived either of the doses of omapatrilat had a similar well as the various treatment regimens resulted in no other survival (82% and 83%) and thus these two groups were significant changes in total cardiac or individual cardiac considered together. Of the rats receiving omapatrilat, 18 chamber weights. There was, however, an increase in lung were sham and 23 had an MI, of which 4 died and 19 sur- weight to body weight (LW/BW) ratio in the untreated MI vived (83% survival, P < 0.05 versus untreated MI). Of the rats as compared with their sham counterparts, suggesting rats receiving omapatrilat and the BK antagonists, 11 were greater pulmonary congestion. This increase was not present sham and 30 had an MI, of which 10 died and 20 survived in the various MI treatment groups, which is consistent with (67% survival, P = 0.06 versus untreated MI). Of the rats re- a decrease in pulmonary congestion. This normalization was ceiving captopril, 13 were sham and 18 had an MI, of which particularly evident in the omapatrilat group without the BK 4 died and 14 survived (78% survival, P < 0.05 versus un- antagonist and in the captopril group. treated MI). Duplex RT-PCR analysis of cytokine mRNA expression Hemodynamic measurements in MI hearts α β In sham rats, the various treatment regimens resulted in no The TNF- , TGF- 1, and IL-10 mRNA expression levels change in any of the measured hemodynamic parameters, in non-infarcted LV tissues from MI hearts were determined except for the omapatrilat + BK antagonist group that had a using a semiquantitative RT-PCR assay. For these cytokines, slight rise in left ventricular end-diastolic pressure (LVEDP). a baseline mRNA expression was detected in LV tissues. As compared with untreated sham rats, the untreated MI rats had a decrease in LV systolic pressure (LVSP) and in maximum TNF-α rate of LV pressure rise (+dP/dt) and decline (–dP/dt), and The TNF-α mRNA expression was significantly increased an increase in LVEDP but no change in right-sided by 66% (P < 0.01) in the untreated MI group compared with hemodynamic parameters. The various treatment regimens the sham-operated rats. In both MI groups treated with

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omapatrilat (10 and 40 mg·kg–1·day–1), the TNF-α mRNA expression was not significantly different from that of the untreated MI group. The TNF-α mRNA expression in MI RV–dP/dt (mmHg/s) captopril-treated rats was not significantly increased in comparison with the sham-operated rats, but was also not different from that of the untreated MI group. However, a significant difference was observed between MI groups treated with 40 mg·kg–1·day–1 omapatrilat- and captopril-

RV+dP/dt (mmHg/s) treated rats (P < 0.01). The co-administration of both kinin receptor antagonists to the untreated MI and 40 mg·kg–1·day–1 omapatrilat-treated rats decreased the TNF-α mRNA expression, as compared with untreated MI (P < 0.05) and omapatrilat (P < 0.001) treated groups. RVEDP (mmHg)

β TGF- 1 β In the untreated MI rats, the TGF- 1 mRNA expression was significantly increased by 61% (P < 0.05) when com- RVSP (mmHg) pared with the sham-operated rats. In both MI groups treated –1 –1 β with omapatrilat 10 mg·kg ·day and captopril, the TGF- 1 eft ventricular rate of pressure development; LV–dP/dt, left ventricular mRNA expression was not significantly different from the untreated MI group, but was also not different from the β sham-operated group. However, the TGF- 1 mRNA expression was significantly increased in the MI group treated with LV–dP/dt (mmHg/s) 2750±151†2747±150†† 31±22594±64††2437±106† 25±1 31±1 4±1 27±1 3±1 4±1 1160±56 4±1 1070±47 742±21† 1180±115 1091±48 742±27† 746±72 719±42 –1 –1 ar rate of pressure development; RV–dP/dt, right ventricular rate of pressure decline. 40 mg·kg ·day omapatrilat, as compared with the sham- operated rats (P < 0.001), the untreated MI group (P < 0.01), –1 –1

† † †† † the MI 10 mg·kg ·day omapatrilat-treated rats (P < 0.001), and the MI captopril-treated group (P < 0.001). The co-administration of both icatibant and R-715 to the un- β LV+dP/dt (mmHg/s) treated MI rats did not modify the TGF- 1 mRNA expres- β sion, but decreased significantly the TGF- 1 mRNA expression in the 40 mg·kg–1·day–1 omapatrilat treated rats (P < 0.05), as compared with their respective untreated MI and omapatrilat-treated groups. LVEDP (mmHg) 13±1* 3918±186 IL-10

† The IL-10 mRNA expression was increased by 113% in the untreated MI group, by 121% in the MI 10 mg·kg–1·day–1 omapatrilat treated group, and by –1 –1 LVSP (mmHg) 1 2 3% in the MI 40 mg·kg ·day omapatrilat-treated rats. However, these increased expression values did not reach statistical significance (P = 0.094, P = 0.059, and P = 0.054, respectively) despite a significant ANOVA (F[6,35] = 3.028, P < 0.05). In the MI captopril-treated rats, the IL-10 mRNA expression was not significantly different from both Heart rate (beats/min) sham-operated and untreated MI groups. The co-administration of both kinin receptor antagonists with MI vehicle treated and 40 mg·kg–1·day–1 omapatrilat-treated rats had no effect on the IL-10 mRNA expression, as compared with the un-

) treated MI and the omapatrilat-treated groups, respectively. 2 Scar surface (cm Discussion 15 0.17±0.0511 259±9 0.16±0.0621 1.01±0.05† 258±8 109±720 267±6 11±2 0.98±0.04† 101±7 261±10 102±4 5223±341 13±2 3542±292 19±2† 82±2** 4864±433 31±5 4314±146 3286±305 4±1 28±1 1258±166 5±1 1063±171 1073±48 796±37 n This study demonstrated that the vasopeptidase inhibitor omapatrilat and the ACE inhibitor captopril both improved early (4 days) post-MI survival when started 4 h post-MI. The use of BK antagonists had no significant effect on survival in untreated MI rats and MI omapatrilat-treated rats

Cardiac hemodynamics 4 days postinfarction. (not tested with captopril). This improvement in survival Values are given as mean ± SE. HR, heart rate; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LV+dP/dt, l < 0.01 vs. untreated ± BK antagonists.

< 0.01 vs. respective Sham. with omapatrilat and captopril is accompanied by a reduced < 0.05 vs. untreated ± BK antagonists. < 0.05 vs. respective Sham. P P P P

antagonists antagonists antagonists antagonists LVEDP and pulmonary congestion. The use of BK antago- Note: † †† * ** Table 1. Sham Untreated ± BK OmapatrilatCaptoprilOmapatrilat + BK MI 18Untreated ± BK 13 0.17±0.05OmapatrilatCaptopril 0.12±0.05Omapatrilat + BK 267±8 19 267±8 14 0.97±0.06† 93±3 1.02±0.07† 103±7 267±5 10±1 260±8 11±2 86±3** 5136±237 5133±198 86±4* 13±1* 3707±192 3533±168 13±3* 4118±228 32±4 31±4 4079±122 3±1 4±1 1364±140 1321±184 1047±136 945±130 rate of pressure decline; RVSP, right ventricular systolic pressure; RVEDP, right ventricular end-diastolic pressure; RV+dP/dt, right ventricul nists had little effect on cardiac hemodynamics or morpho-

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54 Can. J. Physiol. Pharmacol. Vol. 80, 2002

Table 2. Morphologic parameters of heart at 4 days postinfarction. LVW/BW SW/BW RVW/BW AW/BW LW/BW n (g·kg–1) (g·kg–1) (g·kg–1) (g·kg–1) (g·kg–1) Sham Untreated ± BK antagonists 15 1.91±0.11 0.06±0.02 0.54±0.02 0.30±0.03 5.33±0.24 Omapatrilat 18 2.16±0.06 0.06±0.02 0.60±0.02 0.34±0.03 5.40±0.19 Captopril 13 2.02±0.05 0.03±0.02 0.57±0.02 0.27±0.02 5.12±0.13 Omapatrilat + BK antagonists 11 1.69±0.09 0.06±0.02 0.57±0.03 0.25±0.01 4.91±0.16 MI Untreated ± BK antagonists 21 2.01±0.06 0.31±0.02†† 0.58±0.03 0.32±0.03 7.43±0.60† Omapatrilat 19 2.12±0.08 0.35±0.03†† 0.64±0.03 0.31±0.02 5.74±0.27* Captopril 14 2.16±0.12 0.31±0.02†† 0.64±0.02 0.30±0.03 5.98±0.43 Omapatrilat + BK antagonists 20 1.93±0.06 0.39±0.02†† 0.64±0.02 0.31±0.02 6.36±0.27†† Note: Values are given as mean ± SE. LVW, left ventricular weight; BW, body weight; SW, scar weight; RVW, right ventricular weight; AW, atria weight; LW, lung weight. *P < 0.05 vs. untreated ± BK antagonists. **P < 0.01 vs. untreated ± BK antagonists †P < 0.05 vs. respective Sham. ††P < 0.01 vs. respective Sham.

logic measurements. An acute MI significantly increases the Fig. 2. RT-PCR analysis of TNF-α mRNA expression in the left expression of cardiac cytokines. Captopril significantly at- ventricle 4 days after induction of myocardial infarction (MI) or tenuates this activation, while omapatrilat has variable ef- after sham operation (SHAM). Drug treatments (10 mg·kg–1·day–1 fects: sometimes increasing but generally not changing omapatrilat (OMA-10), 40 mg·kg–1·day–1 omapatrilat (OMA-40), activation, depending on the cytokine measured and the dose and 160 mg·kg–1·day–1 captopril (CAP-160)) were started4haf- of omapatrilat used. In nearly every case, the BK antagonists ter coronary artery ligation. B1 and B2 receptor antagonist attenuated the increase in expression of cardiac cytokines. (ANTA) treatments (2.5 mg·kg–1·day–1 each) were started during Taken together, these results would suggest that despite very the surgery for MI. Values are means ± SE of 8 animals in each marked differences in cardiac cytokine expression both group. Values (TNF-α to GAPDH ratio, derived from RT-PCR) captopril and omapatrilat improve early post-MI survival are arbitrary scanning units normalized to sham group 1. Signifi- and cardiac function to a similar extent. It also demonstrates cance is indicated by the following: a, P < 0.05 vs. SHAM that under normal and MI circumstances, BK generation group; b, P < 0.05 vs. MI group; c, P < 0.05 vs. MI+OMA-10 during this period does not affect survival, cardiac group; d, P < 0.05 vs. MI+OMA-40 group. hemodynamics, or gross morphology, but has significant stimulatory effects on cardiac cytokine expression. Clearly the end result of the interaction between BK, cardiac cytokine expression, and the effects of these drugs in this setting are complex and linked with the convergence of nu- merous factors. Recent studies have reported the temporal sequence of pro-inflammatory cytokine gene expression after permanent LAD occlusion (Herskowitz et al. 1995; Ono et al. 1998). In this study, we found a significant increase of TNF-α mRNA expression. The co-administration of icatibant and R-715 (BK antagonists) prevented the increase in TNF-α mRNA expression in untreated MI rats. Tiffany and Burch (1989) have reported that kinins stimulate TNF-α and IL-1 release from macrophages through B1 kinin receptors. Moreover, infiltrating inflammatory cells such as macrophages, neutro- phils, and fibroblasts are at their peak in the area of tissue injury 4 days post-MI (Sun et al. 1994). Similarly, several studies demonstrated that TNF-α is produced by cardiac myocytes (Giroir et al. 1992; Kapadia et al. 1995). The presence of functional B2 receptors on cardiomyocytes have also been characterized (Minshall et al. 1995). Moreover, both B1 and B2 kinin receptors are up-regulated in both infarcted and and non-infarcted areas (Tschöpe et al. 2000a, 2000b). It is thus possible that, after MI, endogenous kinins increase TNF-α mRNA expression and the release of the protein TNF-α from infiltrating inflammatory cells and cardio-

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β Fig. 3. RT-PCR analysis of TGF- 1 mRNA expression in the left Fig. 4. RT-PCR analysis of IL-10 mRNA expression in the left ventricle 4 days after induction of myocardial infarction (MI) or ventricle 4 days after induction of myocardial infarction (MI) or after sham operation (SHAM). Drug treatments (10 mg·kg–1·day–1 after sham operation (SHAM). Drug treatments (10 mg·kg–1·day–1 omapatrilat (OMA-10), 40 mg·kg–1·day–1 omapatrilat (OMA-40), omapatrilat (OMA-10), 40 mg·kg–1·day–1 omapatrilat (OMA-40), and 160 mg·kg–1·day–1 captopril (CAP-160)) were started4haf- and 160 mg·kg–1·day–1 captopril (CAP-160)) were started4haf- ter coronary artery ligation. B1 and B2 receptor antagonist ter coronary artery ligation. B1 and B2 receptor antagonist (ANTA) treatments (2.5 mg·kg–1·day–1 each) were started during (ANTA) treatments (2.5 mg·kg–1·day–1 each) were started during the surgery for MI. Values are means ± SE of 8 animals in each the surgery for MI. Values are means ± SE of 6 animals in each β group. Values (TGF- 1 to GAPDH ratio, derived from RT-PCR) group. Values (IL-10 to GAPDH ratio, derived from RT-PCR) are arbitrary scanning units normalized to sham group 1. Signifi- are arbitrary scanning units normalized to sham group 1. Signifi- cance is indicated by the following: a, P < 0.05 vs. SHAM cance is indicated by the following: a, P < 0.05 vs. SHAM group; b, P < 0.05 vs. MI group; c, P < 0.05 vs. MI+OMA-10 group. group; d, P < 0.05 vs. MI+OMA-40 group.

present study, MI induced an increase of the IL-10 mRNA myocytes of the heart. Consistent with our data for a role of levels in LV non-infarcted tissue. This increased IL-10 ex- BK in the post-MI increase of TNF-α mRNA, we addition- pression could be a protective mechanism against uncon- ally found that BK receptor antagonists resulted in a signifi- trolled activation of inflammatory cytokines. As with TGF- α β cant reduction in cardiac TNF- expression levels 4 days 1, the co-administration of both kinin receptor antagonists post-MI. had no significant effect on IL-10 expression. β TGF- 1 is a major cytokine that regulates extracellular Our data show no significant effect of BK on survival rate matrix protein formation in many cell types and tissues or on any of the hemodynamic or morphologic parameters β (Border and Noble 1994). TGF- 1 induces collagen mRNA measured. This raises the question about the importance of expression and extracellular matrix formation in vitro in cul- BK on early (4 h to 4 days) post-MI prognosis. Because BK tured rabbit or rat cardiac fibroblasts (Eghbali et al. 1991; also significantly modified the expression of inflammatory β Villarreal et al. 1996). Increased TGF- 1 mRNA expression cytokines, it also raises the question on the importance of has been demonstrated in the non-infarcted portion of the suppressing the expression of these cytokines early post-MI. ventricle after MI (Thompson et al. 1988; Casscells et al. Indeed, although inflammatory cytokines are cardiodepres- 1990; Hanatani et al. 1995). Our present data also indicate sive and can contribute to adverse ventricular remodeling β that an MI induced a significant increase of TGF- 1 mRNA when chronically activated, it is possible that transient acti- expression in the LV non-infarcted rat tissue 4 days post-MI. vation early post-MI may exert beneficial effects by stabiliz- However, as opposed to inflammatory cytokines, BK did not ing the necrotic scar and by stimulating post-MI healing appear to play a role in this activation. (Mann 1996; Sack et al. 2000). Nevertheless, in this study IL-10 inhibits the production of the pro-inflammatory we did not evaluate the effects of direct cytokine inhibition, cytokines TNF-α, IL-1β, IL-6, and IL-8 and up-regulates the and cannot rule out an important beneficial effect due to in- expression of the IL-1 receptor antagonist (Howard and hibition of inflammatory cytokines. Indeed, it is known that O’Garra 1992). Consequently, IL-10 could have beneficial BK exerts a wide range of effects, some of which could effects in acute and chronic inflammatory diseases. In the mask potential beneficial effects of cytokine suppression.

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56 Can. J. Physiol. Pharmacol. Vol. 80, 2002

Captopril has been shown to improve early post-MI sur- beneficial effects of these drugs on early post-MI survival vival in both the experimental and clinical setting (Pfeffer et and their interaction with cardiac cytokine expression are al. 1985b, 1992). Indeed, in large clinical studies, most of complex and the result of the convergence of multiple com- the benefits of ACE inhibitors were found to occur within peting mechanisms. the first few days post-MI (GISSI-3 1994; ISIS-4 1995). The results of this study confirm the beneficial effects of Acknowledgments captopril in this setting and indicate that they are accompanied by a reduction in LVEDP and pulmonary congestion. This study was supported by a Pharmaceutical Manufac- Thus, our data probably indicate that captopril may exert turers Association of Canada – Medical Research Council of some of its beneficial effects by reducing the cardiac expres- Canada grant to A. Adam and J.-L. Rouleau. C. Blais, Jr., sion of various cytokines. Interestingly, captopril reduced and N. Lapointe are the recipients of a scholarship from the cardiac cytokine expression despite increasing cardiac BK Fonds de la recherche en santé du Québec (FRSQ). levels, a surprising result considering the suppression of cardiac cytokine expression with the BK receptor antagonists in References our study. Moreover, a recent study showed that the TNF-α production by peripheral blood mononuclear cells in patients Basaran, Y., Basaran, M.M., Babacan, K.F., Ener, B., Okay, T., with CHF was markedly decreased by captopril (Zhao and Gok, H., and Ozdemir, M. 1993. Serum tumor necrosis factor Xie 2001), but other potent ACE inhibitors such as ramipril, levels in acute myocardial infarction and unstable angina perindopril, and lisinopril do not influence cytokine produc- pectoris. Angiology, 44: 332–337. Blais, C., Jr., Couture, R., Drapeau, G., Colman, R.W., and Adam, tion (Schindler et al. 1995). These findings suggest that A. 1997. Involvement of endogenous kinins in the pathogenesis other factors that suppress cytokine expression may be in- of peptidoglycan-induced arthritis in the Lewis rat. Arthritis volved in captopril but not omapatrilat administration. Rheum. 40: 1327–1333. To our knowledge, this is the first report demonstrating Blais, C., Jr., Fortin, D., Rouleau, J.L., Molinaro, G., and Adam, A. the effects of omapatrilat on early experimental post-MI sur- 2000a. Protective effect of omapatrilat, a vasopeptidase inhibi- vival, cardiac hemodynamics, morphology, and cardiac cyto- tor, on the metabolism of bradykinin in normal and failing hu- kine expression. Omapatrilat administration resulted in an man hearts. J. Pharmacol. Exp. Ther. 295: 621–626. improvement in survival, cardiac hemodynamics, and mor- Blais, C., Jr., Marceau, F., Rouleau, J.L., and Adam, A. 2000b. The phology similar to that of captopril. This occurred despite kallikrein-kininogen-kinin system: lessons from the quantifica- marked differences in the way these drugs modified the ex- tion of endogenous kinins. Peptides, 21: 1903–1940. pression of cardiac cytokines. Indeed, omapatrilat generally Blais, C., Jr., Lapointe, N., Rouleau, J.L., Clément, R., Gervais, N., resulted in no change or, if anything, in an increase of the Geadah, D., and Adam, A. 2001. Effects of the vasopeptidase cardiac cytokine expression, while captopril reduced the ex- inhibitor omapatrilat on cardiac endogenous kinins in rats with pression of the same cytokines. That omapatrilat treatment acute myocardial infarction. Peptides, 22: 953–962. leads to elevated expression of cardiac cytokines is not Blum, A., and Miller, H. 1998. Role of cytokines in heart failure. surprising considering the marked increase in cardiac BK Am. Heart J. 135: 181–186. Border, W.A., and Noble, N.A. 1994. Transforming growth factor β that it causes (Blais et al. 2001). BK is a powerful pro- in tissue fibrosis. New Engl. J. Med. 331: 1286–1292. inflammatory substance, which resulted in increased expres- Bralet, J., Marie, C., Mossiat, C., Lecomte, J.M., Gros, C., and sion of inflammatory cytokines in this study. Consistent with Schwartz, J.C. 1994. Effects of alatriopril, a mixed inhibitor of an important role of BK in the effects of omapatrilat on car- atriopeptidase and angiotensin I-converting enzyme, on cardiac diac cytokine expression, the co-administration of BK recep- hypertrophy and hormonal responses in rats with myocardial in- tor antagonists markedly attenuated the effects of omapatrilat farction. Comparison with captopril. J. Pharmacol. Exp. Ther. α β on cardiac cytokine expression (TNF- and TGF- 1). 270: 8–14. The similar beneficial effects of omapatrilat and captopril Brown, N.J., and Vaughan, D.E. 1998. Angiotensin-converting en- on survival and cardiac hemodynamics despite their con- zyme inhibitors. Circulation, 97: 1411–1420. trasting effects on cardiac cytokine expression suggests that Casscells, W., Bazoberry, F., Speir, E., Thompson, N., Flanders, the effects of cardiac cytokines in these settings are com- K., Kondaiah, P., Ferrans, V.J., Epstein, S.E., and Sporn, M. β plex. Indeed, as already discussed, the effects of cardiac 1990. Transforming growth factor- 1 in normal heart and in cytokines post-MI may be potentially beneficial or detrimen- myocardial infarction. Ann. N.Y. Acad. Sci. 593: 148–160. tal (Mann 1996; Sack et al. 2000). The release of cytokines Dukas, K., Sarfati, P., Vaysse, N., and Pradayrol, L. 1993. from the myocardium following acute–subacute hemo- Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. Anal. Biochem. 215: dynamics and (or) ischemia–reperfusion biomechanical 66–72. stress may activate signaling pathways that promote cardiac d’Uscio, L.V., Quaschning, T., Burnett, J.C., Jr., and Luscher, T.F. adaptation and (or) protection. However, a sustained and (or) 2001. Vasopeptidase inhibition prevents endothelial dysfunction excessive release of cytokines for prolonged periods may be of resistance arteries in salt-sensitive hypertension in compari- maladaptative: producing cardiac decompensation (Mann son with single ACE inhibition. Hypertension, 37: 28–33. 1996; Sack et al. 2000). It seems that the cytokine–cardiac Eghbali, M., Tomek, R., Sukhatme, V.P., Woods, C., and Bhambi, β interaction early post-MI is quite complicated since the co- B. 1991. Differential effects of transforming growth factor- 1 administration of BK receptor antagonists with omapatrilat and phorbol myristate acetate on cardiac fibroblasts. 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