inhibition of cox pathway in experimental myocardial infarction

Original Article

Inhibition of COX pathway in experimental myocardial infarction

Takayuki Saito a, Ian W. Rodger b, Fu Hu a, Richard Robinson a, Thao Huynh a, Adel Giaid a,*a The Montreal General Hospital and McGill University, Montreal, Que., Canada H3G 1A4

b Merck Frosst Inc., White Lane Station, NJ 08889, USA

Received 20 February 2004; received in revised form 29 March 2004; accepted 1 April 2004

Available online 25 May 2004

Abstract

Release of inflammatory mediators within the ischemic myocardium has long been thought to contribute to myocardial damage anddysfunction. Myocardial infarction (MI) and congestive heart failure (CHF) were induced in rats by ligating the left coronary artery. Animalswere treated with the selective cyclooxygenase-2 (COX-2) inhibitor-5,5-dimethyl-3-(3-fluorophenyl1)-4-(4-methyl-sulphonyl-2(5H)-fluranone (DFU), low-, high-dose acetyl salicylic acid (aspirin), or vehicle for 3 months. Strong immunoreactivity for COX-2 was detected inthe cardiomyocytes, vascular endothelial cells, and macrophages in the infarcted myocardium. Compared to the vehicle, treatment with DFUsignificantly reduced left ventricular end-diastolic pressure, central venous pressure, lung wet/dry ratio and infarct size, and improved cardiaccontractility (P < 0.05). In comparison, treatment with low or high doses of aspirin did not significantly impact any of these parameters. Thesefindings demonstrate that induction of myocardial COX-2 in rats with CHF secondary to MI contributes to the cardiac injury and dysfunctionassociated with this disease, and that therapy aimed at inhibiting this enzymatic pathway at the onset of the disease may be beneficial in thetreatment of MI and CHF.© 2004 Elsevier Ltd. All rights reserved.

Keywords: Cyclooxygenase; Acetyl salicylic acid (aspirin); Heart; Rat; Immunohistochemistry

1. Introduction

Myocardial infarction (MI) is caused by interruption ofcoronary blood flow to the myocardium which if persistedwould lead to chronic heart failure and death. There arenumerous inflammatory mediators produced by resident andinfiltrating inflammatory cells as well as cardiac myocytesthat are known to contribute to myocardial remodeling anddysfunction associated with MI and congestive heart failure(CHF). Of particular interest are prostanoids. The latter areproduced by cardiac myocytes and inflammatory cellsthrough activation of cyclooxygenase (COX) which catalyzearachidonic acid [1]. There are two known types of COX:COX-1 and COX-2 [2]. These two isoforms have differentphysiologic and pathologic roles. COX-1 is present in almostall tissues and cells, and plays an important role in themaintenance of vascular homeostasis and gastrointestinalintegrity [1,2]. COX-2 is expressed in several cell types suchas macrophages, endothelial cells, fibroblasts, and smooth

muscle cells, and is highly inducible by cytokines, growthfactors, hormones, and oncogenes [1,2]. The induction ofCOX-2, with resultant production of prostanoids, can con-tribute to inflammation, pain, parturition, and certain types ofcancer [3].

Acetyl salicylic acid (aspirin), which has long been usedto treat patients with MI is intended to block platelet-derivedthromboxane A2 (TXA2) formation and prevent thrombusformation, is a non-selective inhibitor of COX. Whether ornot chronic aspirin therapy has any effects on cardiac remod-eling remains to be fully elucidated. However, there is evi-dence to suggest that aspirin treatment reduces myocardialfibrosis in the non-ischemic myocardium of rats with acuteMI [4]. One of the well-established side effects of chronicaspirin therapy is cell toxicity in the stomach and kidneys,and the development of hemorrhagic stroke. Nowadays,there is a new generation of more selective COX-2 inhibitors.These molecules are well characterized in terms of theirsparing of gastric mucosa and kidney toxicity. However, theirefficacy in MI remains to be established. We have previouslydemonstrated the induction of COX-2 in the myocardium ofpatients with ischemic heart disease and CHF [5]. Recently,we have shown that selective inhibition of COX-2 in a rat

* Corresponding author. Tel.: +1-514-934-1934x43841;fax: +1-514-934-8448.

E-mail address: [email protected] (A. Giaid).

Journal of Molecular and Cellular Cardiology 37 (2004) 71–77

www.elsevier.com/locate/yjmcc

© 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.yjmcc.2004.04.002

model of MI improves myocardial dysfunction [6]. The pur-pose of the present study was to determine whether myocar-dial induction of COX-2 contribute to cardiac injury anddysfunction in the setting of chronic MI and CHF. We havetherefore investigated the effect of selective and non-selective inhibition of COX-2, using 5,5-dimethyl-3-(3-fluorophenyl1)-4-(4-methyl-sulphonyl-2(5H)-fluranone(DFU) and high- and low-dose aspirin on infarct size and thedevelopment of CHF in a rat model of left coronary arteryligation.

2. Materials and methods

2.1. Experimental MI

Lewis rats (240–270 g) were used for this study. Allanimal works were performed in accordance with the guide-lines and policies of the Canadian Council on Animal Care.Protocol has been approved by McGill University AnimalCare Committee. Left ventricular (LV) free-wall MI wasinduced as previously described [6]. In brief, each rat wasanesthetized with isoflurane, intubated with an 18-gaugeintravenous (i.v.) catheter and mechanically ventilated withroom air by use of a Harvard rodent ventilator (HarvardApparatus Co., South Natick, MA) at a rate of 60 cycles perminute and a tidal volume of 1 ml/100 g body weight (BW)with a positive end-expiratory pressure of 3 cm H2O. A leftthoracotomy was performed in the fourth intercostal space.After the pericardium was incised, the proximal portion ofthe left coronary artery was ligated with one suture of 5-0silk. Apart from the coronary artery ligation, sham-operatedrats underwent an identical procedure. Subsequently, thechest was closed in three layers of 3-0 Vicryl (Ethicon,Somerville, NJ) and the rats were allowed to recover.

2.2. Experimental groups

Rats were divided into five groups: (1) sham rats (shamgroup, n = 5); (2) MI rats treated with methylcellulose (ve-hicle) solution (vehicle group, n = 8); (3) MI rats treated withDFU (5 mg/kg/d) (DFU group, n = 9), DFU (Merck FrosstCanada Inc.), which is an orally active and highly selectiveCOX-2 inhibitor [7], was dissolved in 1% methylcellulosesolution. The dosage of DFU was determined according toour previously published data, where it was not only effectivebut also selective to COX-2 inhibition [6–8]. (4) MI ratstreated with low-dose aspirin (1 mg/kg/d) (low-dose acetylsalicylic acid (L-ASA) group, n = 9); and (5) MI rats treatedwith high-dose aspirin (25 mg/kg/d) (high-dose acetyl sali-cylic acid (H-ASA) group, n = 11), aspirin (Sigma, Ontario,Canada) was dissolved in water. Drugs or vehicle solutionwere administered to rats by gavage 30 min prior to ligationand continued for the ensuing 3 months.

2.3. Immunohistochemistry for COX-2

Multiple-step heart cryostat sections from MI and shamrats at 3, 7, and 14 d, and 1, 2, and 3 months after surgery,

were immunostained with antiserum to COX-2 [5]. The im-munohistochemical procedure used here is described in de-tail elsewhere [9]. COX-2 immunoreactivity was examinedin cardiomyocytes, endothelial cells, and endocardium fromboth the infarcted and non-infarcted zones and in macro-phages within the infarcted zone.

2.4. Echocardiographic measurements

Three months following MI or sham surgery, transthoracicechocardiographic measurements were performed under an-esthesia with 1.5% of isoflurane as mentioned above at theend of the 3 months treatment period. Echocardiograms wereperformed with an echocardiographic system equipped witha 7.5-MHz transducer (Hewlett Packard Sonos 5500). Atwo-dimensional short-axis view of the LV was obtained atthe level of papillary muscles to record M-mode tracings. LVend-diastolic diameter (LVEDD) and LV end-systolic diam-eter (LVESD) were measured according to the AmericanSociety of Echocardiography leading-edge method from atleast three consecutive cycles [10]. LV fractional shortening(LVFS) and ejection fraction (LVEF) were calculated.

2.5. Hemodynamic measurements

Few days after echocardiography, the same groups ofanimals were anesthetized and a fluid-filled catheter con-nected to a transducer was inserted into the right carotidartery. After measuring arterial blood pressure and heart rate(HR), the catheter was advanced into the LV and LV systolicpressure (LVSP), LV end-diastolic pressure (LVEDP), andfirst derivative of LV pressure (±dP/dt) were determined.Then the chest was opened and 18-gauge catheter was in-serted into the right atrium to measure central venous pres-sure (CVP). At the end of the experiment, the heart–lung blocwas removed. Heart was weighed prior to fixation for calcu-lation of heart/body weight (heart/BW) ratio. The left lungwas weighed and backed for 2 weeks at 37 °C for calculationof wet/dry ratio.

2.6. Infarct size

The 5 µm-thick histologic slides from middle paraffinsections of vehicle, DFU, L-ASA and H-ASA groups werestained with Masson trichrome. These slides were examinedby light microscope coupled to a computerized morphometrysystem (Image Pro Plus, Media Cybernetics, MD). Infarctsize was morphologically measured and calculated [8,11,12]as the ratio of scar average circumferences of the endocar-dium and the epicardium to LV average circumferences ofthe endocardium and the epicardium.

2.7. Statistical analysis

Data are presented as mean ± S.E. One-way ANOVAfollowed by Tukey HSD multiple comparisons were used for

72 T. Saito et al. / Journal of Molecular and Cellular Cardiology 37 (2004) 71–77

analyzing the differences among multiple groups. Significantdifferences among groups were defined by a value ofP < 0.05.

3. Results

3.1. Detection of COX-2 immunoreactivity

The myocardium of sham rats exhibited little COX-2immunoreactivity in cardiomyocytes or endothelial cells. Incontrast, there was strong expression of COX-2 in the inf-arcted myocardium mainly in the cardiomyocytes, macroph-ages, vascular endothelium, and endocardium (Fig. 1A,B).There was similar level of COX-2 expression in the cardi-omyocytes and endothelial cells of the non-infarcted myo-cardium (Fig. 1C). The strongest expression of COX-2 in all

cells was seen at 2 weeks after coronary artery ligation, andslightly decreased thereafter except in macrophages where itwas prominent throughout the study (Fig. 1D).

3.2. Echocardiography

LVEDD and LVESD were significantly increased, andLVFS and LVEF were significantly reduced in vehicle,L-ASA and H-ASA groups compared to sham group(Table 1, P < 0.05). There was no significant difference inLVEDD, LVESD, LVFS, and LVEF between DFU and shamgroup (Table 1).

3.3. Hemodynamic and cardiac function data

Three months after ligation of the left coronary artery,there was no significant difference among the five groups in

Fig. 1. COX-2 immunohistochemistry. COX-2 immunostaining in cryostat heart sections of MI (A–D,F) and sham (E) rats. Panel A shows COX-2immunoreactivity in endocardium (arrow), cardiomyocytes and inflammatory cells in infarcted myocardium of MI rats 2 weeks after ligation. Panel B is a highermagnification of the same heart in A showing strong COX-2 immunoreactivity in inflammatory cells. Panel C shows COX-2 immunostaining in thenon-infarcted area of MI rats. Panel D shows the presence of little COX-2 immunostaining in the infarcted myocardium 12 weeks after coronary artery ligation.Panel E shows the myocardium of sham-operated rat. Panel F represents negative control experiments of the infarcted myocardium. Arrows in D and F alsoindicate the endocardium. Magnifications: A,D,F (×200); B,C,E (×400).

73T. Saito et al. / Journal of Molecular and Cellular Cardiology 37 (2004) 71–77

HR, mean arterial pressure (MAP), LV end-systolic pressure(LVESP), body weight gain (BW gain), heart weight (heartwt) and the heart/body weight ratio (heart/BW) (Table 1).The latter two parameters were higher in the vehicle com-pared to the sham group, but did not reach statistical signifi-cance with the use of the rigorous Tukey test. Heart/BW ratioof the DFU-treated group was the lowest among the four MIgroups, and was almost identical to that of the sham group(Table 1). Lung wet/dry ratio (lung wet/dry) was significantlyincreased in the vehicle compared to the sham group(Table 1, P < 0.01). Treatment with DFU significantly re-duced lung wet/dry ratio when compared to the vehicle group(Table 1, P < 0.05), and it did not significantly differ fromthat of the sham group. Treatment with either dosages ofaspirin did not significantly differ from that of the vehicletreatment, and was significantly higher than that of the sham(Table 1, P < 0.01) and DFU (Table 1, P < 0.05) groups.LVEDP of the vehicle group was significantly higher thanthat of the sham group (Fig. 2A, P < 0.001). DFU treatmentresulted in a significant reduction in LVEDP compared withvehicle (Fig. 2A, P < 0.001), and it did not significantly differfrom that of the sham group (Fig. 2A). Treatment with low orhigh dose of aspirin did not significantly reduce LVEDPcompared with the vehicle, and it remained significantlyhigher than that of the sham control group (Fig. 2A,P < 0.002). CVP was significantly higher in the vehicle whencompared to the sham group (Fig. 2B; P < 0.05). In contrast,CVP of the DFU-treated group was significantly lower thanthat of the vehicle group (Fig. 2B, P < 0.05), and it did notsignificantly differ from that of the sham group (Fig. 2B).Treatment with low or high dose of aspirin did significantlyimprove CVP compared with the vehicle (Fig. 2B). Therewas a significant decrease in +dP/dt and –dP/dt in the vehiclegroup compared to the sham group (Fig. 2C,D, P < 0.01). Incontrast, there was a significant increase in +dP/dt and–dP/dt in the DFU-treated group compared to the vehicle-treated group (Fig. 2C,D, P < 0.05). There was no significantimprovement in either +dP/dt or –dP/dt with the either of theaspirin treatments compared to the vehicle treatment.

3.4. Infarct size

Treatment with the selective COX-2 inhibitor, DFU, sig-nificantly reduced infarct size when compared to animalstreated either with vehicle or low-dose aspirin (Fig. 3,P < 0.05). There was no significant difference in infarct sizebetween low-aspirin-, high-aspirin-, and vehicle-treatedgroups.

3.5. Mortality rate

There was no mortality in the sham group. In the vehiclegroup, two rats died on days 1 and 65 after surgery. In theDFU group, two rats died on days 5 and 12. In the L-ASAgroup, three rats died on days 1, 2, and 52. In the H-ASAgroup, three rats died on days 1, 2, and 84. There was nosignificant difference in mortality between any of the treat-ment groups and the vehicle group.

4. Discussion

The role of inflammatory mediators in the pathogenesis ofCHF has gained a great deal of attention during the pastdecade. Among the important mediators of CHF are theprostanoids. The latter exert wide range of actions on thecoronary circulation and the myocardium [1]. One obstaclethat has faced research into the role of each of the twoenzymes (COX-1 and COX-2), responsible for prostanoidsgeneration, in heart failure, is the availability of highly selec-tive inhibitors of these enzymes. The new generations of painkillers targeting COX-2 have made that possible. Here, wedemonstrate that the animals in the vehicle group showsignificantly higher lung wet/dry ratio, LVEDD, LVESD,LVEDP, and CVP; and significantly lower LVFS, LVEF, and+dP/dt when compared with those in the sham group, indi-cating the development of CHF in these animals. Long-termtreatment with the selective COX-2 inhibitor, DFU, signifi-

Table 1Physiologic parameters of study groups

Sham (n = 5) Vehicle (n = 6) DFU (n = 7) L-ASA (n = 6) H-ASA (n = 8)LVEDD (mm) 5.59 ± 0.11 9.23 ± 0.57 a 7.07 ± 0.17 8.96 ± 0.25 a 8.00 ± 0.70 b

LVESD (mm) 3.75 ± 0.18 8.17 ± 0.50 a 5.54 ± 0.17 7.78 ± 0.33 a 6.75 ± 0.75 b

LVFS (%) 32.83 ± 4.23 11.44 ± 0.69 a 21.58 ± 1.83 13.27 ± 1.49 a 17.30 ± 2.84 a

LVEF (%) 68.63 ± 5.43 30.49 ± 1.61 a 51.45 ± 3.45 34.48 ± 3.16 a 41.85 ± 5.86 b

HR (bpm) 317.40 ± 5.55 292.40 ± 27.41 305.33 ± 15.97 314.17 ± 9.98 326.29 ± 7.75MAP (mmHg) 103.80 ± 4.98 82.67 ± 8.69 101.06 ± 8.71 89.71 ± 9.81 101.18 ± 7.52LVSP (mmHg) 121.00 ± 4.49 90.00 ± 8.09 122.17 ± 8.07 96.40 ± 8.20 113.00 ± 8.97BW (g) 448.60 ± 9.49 434.33 ± 6.92 453.43 ± 9.15 467.33 ± 8.79 477.14 ± 9.97BW gain (g/d) 2.04 ± 0.16 1.89 ± 0.12 1.99 ± 0.10 2.12 ± 0.16 2.20 ± 0.05Heart wt (g) 1.11 ± 0.06 1.32 ± 0.14 1.19 ± 0.04 1.29 ± 0.06 1.27 ± 0.04Heart/BW (%) 0.25 ± 0.01 0.31 ± 0.04 0.26 ± 0.01 0.28 ± 0.01 0.27 ± 0.01Lung wet/dry 4.69 ± 0.07 5.15 ± 0.09 a,c 4.77 ± 0.06 5.12 ± 0.10 a,c 5.08 ± 0.07 a,c

Value is expressed as mean ± S.E.a P < 0.01 compared to sham group.b P < 0.05 compared to sham group.c P < 0.05 compared to DFU group.


cantly reduced lung wet/dry ratio, LVEDP, CVP and infarctsize, and improved cardiac contractility in chronic rat modelof left coronary artery ligation. In comparison, non-selectiveCOX-1/COX-2 using high or low dose of aspirin did notsignificantly improve these parameters. The present studydemonstrates that COX-2 plays an important role in thepathogenesis of the CHF secondary to MI, and suggests that

therapy aimed at inhibiting myocardial COX-2 may provebeneficial in altering the course of this debilitating disease.

Here, we selected to use aspirin as a non-selective inhibi-tor of COX due it its wide use as an anti-thrombotic agent inpatients with MI. Indeed, i.v. infusion of 50 mg aspirin over a60-min period significantly reduced platelet aggregation andreduced TXA2 release [13]. Moreover, an aspirin dosage of5 mg/kg/d has been shown to diminish the concentrations ofprostaglandin I2 (PGI2) and TXA2 in infracted heart muscle2 days after coronary artery ligation in rabbits [14]. However,in a canine model of left coronary artery ligation, aspirintreatment at a dose of 3 mg/kg/d i.v. for 72 h did not reduceinfarct size [15]. Moreover, in similar model to the one usedin our study, chronic treatment with a high-dose aspirin,25 mg/kg/d intraperitonially, did not affect heart dryweight/BW ratio, infarct size, wall thickness, LV diameter, orLV function [4]. In general, these findings are consistent withour observation that chronic aspirin therapy, although isbeneficial in preventing platelet aggregation, does not signifi-cantly improve cardiac remodeling and dysfunction in thisanimal model. A higher dosage of aspirin than the one used inthe present study may result in a better outcome, however it

Fig. 2. Hemodynamic measurements and cardiac function. LVEDP, CVP, and cardiac contractility (+dP/dt and –dP/dt) for all study groups are shown in PanelsA–D. Values are expressed as mean ± S.E.

Fig. 3. Infarct size of the LV. The effect of treatment with the selectiveCOX-2 inhibitor (DFU) and low- or high-dose aspirin on the infarct size isshown. Values are expressed as mean ± S.E.


would most likely be associated with higher risk of gas-trointestinal adverse effects.

There is evidence to suggest that inhibition of COX-2aggravates the ischemia/reperfusion injury in guinea pig per-fused hearts [16]. These findings imply that in the immediatecourse following coronary obstruction, certain prostanoidssuch as PGI2 may have cardioprotective role; however, ourstudy shows that long-term exposure of the ischemic myo-cardium to these mediators may have adverse effects oncardiomyocytes viability and function. Of note, in one groupof rats we administered similar dosage of DFU 1 week aftercoronary artery ligation, and observed a significant improve-ment in LVEDP (9.6 ± 0.4) and CVP (7.2 ± 0.9) comparableto our DFU group that received treatment prior to ligation,with no significant improvement in other parameters (datanot shown). These findings suggest that, at least in the settingof experimental MI, COX-2 has no beneficial role in the earlycourse of disease. In addition, there is evidence to suggestthat COX-2 contributes to endothelial generation of PGI2,and that selective COX-2 inhibition may be associated withincreased risk of cardiovascular events [17]. Evidently, in thepresent study, 3-month treatment with DFU did not signifi-cantly increase MAP. Second, the increased risk of cardio-vascular events seen in the gastrointestinal safety trial for theselective COX-2 inhibitor Vioxx, may result from aspirinwithdrawal rather than a direct effect of the enzyme inhibitor[18] as shown in the recently published clinical trials [19].Moreover, recent reports have shown that COX-2 contributesto the process of atherosclerosis and plaque instability inpatients [20,21] and in animal models [22] therefore targetedmyocardial inhibition of COX-2 in the setting of MI and CHFis warranted.

Although, the mechanism by which inhibition of COX-2improves cardiac function and reduces infarct size was notthe focus of the current study, one can speculate severalscenarios: First, there is several evidence to suggest thatmetabolites of COX affect the inotropic state of the myocar-dium via changes in Ca2+ cycling [23,24]. Second, the gen-eration and release of PGs is known to induce expression ofinflammatory and growth mediators. Thus, inhibiting theirformation via COX-2 would lead to a reduction in the extentof inflammation [25], thereby reducing release of other in-flammatory mediators known to cause exudation, injury, andtissue scaring. Similarly, inhibition of COX-2 leads to areduction in oxidant formation [26]. Oxidant injury has beenshown to induce negative effects on the function and struc-ture of the myocardium [27]. In addition, consequent in-creased formation of 8-iso-PGF-2a in endothelial cells mighthave modified coronary blood flow in vehicle-treated rats.This isporostane binds to TXA2 receptors on vascular smoothmuscle cells and act as a potent vasoconstrictor and mitogen[28], and may decrease renal plasma and glomerular filtra-tion rate [29].

In the present study, effects of DFU appear to be specificto the heart since we saw no effects on other organs (kidneysand lungs; unpublished data). The drug we used here, DFU,

has been used in a number of diseased animal models, and itsselectivity in inhibiting COX-2 over COX-1 has also beendetermined in several animals including rats [7,30,31]. Forexample, Riendeau et al. [7] have shown that DFU inhibitedthe arachidonic acid-dependent production of PGE2 inChinese hamster ovarian cells with at least a 1000-fold selec-tivity for COX-2 (IC50 = 41 ± 14 nM) over COX-1(IC50 > 50 µM).

In conclusion, we clearly demonstrate that selective inhi-bition of COX-2 significantly improved cardiac dysfunctionand reduced infarct size in a chronic model of MI and CHF.In comparison, non-selective COX inhibition using low andhigh doses of aspirin did not significantly impact cardiacfunction or structure. Therefore, the findings support an im-portant role for the COX-2 enzyme in myocardial injury anddysfunction associated with chronic MI. The pharmacologi-cal benefits of the new generation of selective COX-2 inhibi-tors or targeted myocardial COX-2 inhibition in chronic MIand CHF warrant further investigation.

Acknowledgements

This work was supported by the Canadian Institute forHealth Research and Heart and Stroke Foundation of Que-bec. Dr. Adel Giaid is supported by the Fonds de Recherchéen Santé du Quebec.

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inhibition of cox pathway in experimental myocardial infarction

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