J Mol Cell Cardiol 16, 43-52 (1984)

The Effects of Nafazatrom on Arrhythmias and Prostanoid Release During Coronary Artery Occlusion and Reperfusion

in Anaesthetized Greyhounds

S u s a n J. C o k e r a n d J a m e s R. Parra t t*

Department of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow, and Department of Surgery, University of Glasgow ( Western Infirmary),

Glasgow, UK

(Received 4 January 1983, accepted in revised form 25 May 1983)

S. J. COKER AND J. R. PARRATT. The Effects of Nafazatrom on Arrhythmias and Prostanoid Release During Coronary Artery Occlusion and Reperfusion in Anaesthetized Greyhounds. Journal of Molecular and Cellular Cardiology (1984) 16, 43-52. The effects of the antithrombotic drug nafazatrom (BAY g 6575) were investigated in chloralose-anaesthetized greyhounds subject to coronary artery occlusion and reperfusion. Pretreatrnent with nafazatrom 10 mg/kg p.o. did not significantly reduce the number of extra- systoles or the incidence of ventricular fibrillation (VF) during the first 30 min occlusion of the left anterior descending coronary artery. However, the incidence of VF resulting from release of a 40-min coronary artery occlusion was markedly reduced (from 88% in the controls to 14% in the nafazatrom group). Both thromboxane B, (TxB,) and 6-keto PGFI~ (breakdown products of TxA, and prostacyclin respec- tively) were released from the acutely ischaemic myocardium in control dogs. Nafazatrom did Dot alter the release of TxB2 but th~ concentrations of 6-keto PGFI~ were elevated in blood draining from both the ischaemic and normal regions of the myocardium. The pronounced anti-fibrillatory effect of nafazatrom during reperfusion of the ischaemic myocardium may be related to the ability of this drug to elevate prostacyclin concentrations in the coronary circulation.

KEY WORDS: Nafazatrom; Coronary artery occlusion; Reperfusion; Arrhythmias; Prostacyclin.

I n t r o d u c t i o n

We have reported previously that both th romboxane and prostacyclin are released from the acutely ischaemic myoca rd ium and that this release may be a major de t e rminan t of early ischaemic ar rhythmias [7], a higher incidence o fven t r i cu la r ectopic activity being associated with a shift in the balance of prostanoid release in favour of th romboxane [3]. We have also recently suggested that th romboxane may be involved in the serious ar rhythmias that occur dur ing reperfusion of the ischaemic myocardium. Thus, dazoxiben, a specific inhibi tor of th romboxane synthe- tase, markedly reduced the incidence of vent r icular fibril lation following the release of a 40 rain coronary artery occlusion [8].

Nafazat rom (BAY g 6575: 1-[2-(napthyl- oxy) ethyl] 3-methyl-2-pyrazolin-5-one) , an

* To whom requests for offprints should be addressed. 0022-2828/84/010043 4- 10 $03.00/0

an t i th rombot ic drug) has been reported to increase prostacyclin concentrat ions in vivo [21]. The present study was designed to examine the effects of p re t rea tment with nafaza t rom on ar rhythmias induced by coronary artery occlusion and reperfusion since increasing prostacyclin levels wi thin the myoca rd ium should, on the present evidence [5, 6], suppress ectopic activity.

M e t h o d s

Anaesthesia was induced in greyhounds (21 to 37 kg) of either sex by the in t ravenous inject ion of sodium thiopentone, 25 mg/kg. After endotracheal in tuba t ion , p a n c u r o n i u m 0.15 to 0 .20mg/kg was adminis tered and vent i la t ion, with oxygen con ta in ing 1% halothane, was ma in t a ined using a Palmer

�9 1984 Academic Press Inc. (London) Limited

44 S .J . Coker and J. R. Parratt

respiratory pump, 25 strokes/rain. The stroke volume was adjusted to give an arterial PCO 2 of approximately 40 mmHg. The descending aorta and vena cava were catheterized via the femoral vessels to enable pressure measure- ment and' blood sampling. After the insertion of these catheters, halothane was discon- tinued and anaesthesia maintained with chloralose (80 to 90 mg/kg i.v.). Catheters were then positioned within the heart under fluoroscopic control using a Siemens image intensifier. A catheter was placed in the coronary sinus via the left jugular vein for blood sampling, a No. 7 Cournand catheter was advanced down the left carotid artery and inserted into the left ventricle for pressure recording, and a Swan-Ganz catheter was placed in the pulmonary artery via the left jugular vein for pressure recording, blood sampling and for the measurement of cardiac output by thermodilution using a Devices cardiac output monitor.

After performing a left thoracotorny at the level of the sixth rib the pericardium was incised and the left anterior descending coronary artery (LAD) was dissected free from the surrounding tissue at a site approxi- mately 20 to 40 m m distal from the tip of the left atrial appendage. A ligature (Mersilk 4.0) was then passed under the LAD. A 6-inch 'Longdwel ' Teflon catheter (size 20G) was inserted into a major coronary vein running alongside the LAD and the tip advanced so that blood could be sampled from the area rendered ischaemic by occlusion of the LAD. Heparin (100 units/kg) was administered intravenously after completion of the surgical procedures.

Arterial blood pressure, pulmonary artery pressure and left ventricular pressure (LVP) were recorded via Elcomatic 751A trans- ducers on a Mingograf 82 ink-jet recorder along with a Lead I I electrocardiogram. A Siemens-Elema differentiating circuit was used to obtain LV dP/dt and LV end- diastolic pressure was measured from LVP at high gain. Mid-oesophageal temperature was monitored with an Ellab thermocouple. Blood gases (PO2, PCO2) and pH were measured with an IL213 analyzer, and 0 2 content was calculated by the method of Douglas et al. [9].

The plasma concentrations of TxB 2 and 6-keto PGFI~ (metabolites of TxA 2 and

prostacyclin, respectively) were measured using radioimmunoassay techniques [2]. The blood samples for prostanoid measurement were placed in tubes containing indomethacin to prevent ex vivo generation of prostanoids, and EDTA as anticoagulant. Plasma was stored at --20~ until assayed. Samples were acidified and the prostanoids extracted with ethyl acetate. Extracts were subject to radio- immunoassay using specific antibodies (Insti- rut Pasteur Production) and a dextran- coated charcoal separation procedure. The detection limits were 20 pg/ml for TxB 2 and 100 pg/ml for 6-keto PGFI~.

At the end of each experiment the heart was excised and a small volume of blue dye injected slowly into the LAD distal to the point of occlusion. The area outlined in this manner was then cut out and weighed; the 'occluded zone' was expressed as a percen- tage of the free left ventricular wall.

Statistics

All values have been expressed as the mean zk s.E.u, of n experiments. Changes within each group of dogs were assessed using a paired t test while differences between the groups were compared with an independent t test. The incidence of events was compared with a Chi-squared test. Results were considered to be statistically significant at P < 0.05.

Experimental protocol

Nafazatrom (10mg/kg) was administered orally 30 min to 1 h prior to the induction of anaesthesia. Approximately 1�89 h was required to prepare the animals for coronary artery occlusion and after completion of surgery at least 30 min were allowed to pass before the first control measurements were made. After obtaining stable control values for haemodynamics and blood gases, samples were taken for prostanoid analysis. The coronary artery was then occluded (3�89 to 4 h after drug administration) by tying a reversible knot in the ligature. Blood gases in the coronary sinus and local coronary vein were measured 7, 15 and 30 min after occlu- sion and samples for prostanoid analysis were taken at 2, 7, 15 and 30 min post-occlusion. After 40 min of occlusion the ligature was

N a f a z a t r o m Suppresses Reperfus ion-Induced VF 45

released and blood gases and prostanoids were m e a s u r e d 1, 5 a n d 15 m i n later .

R e s u l t s

The effects of nafazatrom on arrhythmias T h e a r r h y t h m i a s i n d u c e d by occ lus ion o f the left a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y w e r e q u a n t i f i e d by c o u n t i n g e a c h i n d i v i d u a l ec top ic b e a t (extrasystole) i n c l u d i n g those o c c u r r i n g as v e n t r i c u l a r t a c h y c a r d i a ( V T - - de f ined as six or m o r e consecu t ive ex t ra - systoles) d u r i n g the first 30 ra in occ lus ion in the survivors . T h e to ta l i n c i d e n c e o f ven t r i - cular fibrillation (VF) was ~ilso noted. Pretreatment of greyhounds with nafazatrom (10 m g / k g p.o.) d id no t s ign i f ican t ly a l t e r the to ta l n u m b e r o f extrasystoles t ha t o c c u r r e d d u r i n g the first 30 ra in c o r o n a r y a r t e ry occ lus ion (Fig. 1). I n the con t ro l dogs the m e a n v a l u e was 720 ~ 136 (S.E.M.) c o m - p a r e d wi th 450 • 202 in t he n a f a z a t r o m g roup . Closer e x a m i n a t i o n o f the a r r h y t h m i a d i s t r i bu t ion in i n d i v i d u a l an ima l s o f the n a f a z a t r o m g r o u p (see Fig. 2) reveals how- eve r t ha t some o f these dogs h a d ve ry few

arrhythmias (less than in any of the control dogs, Fig. 3) whereas others had more than 800 extrasystoles over the 30-min occlusion period which often included periods of rapid VT (e.g. 400 beats/min). This variation in arrhythmia distribution between dogs did not appear to be related to changes in local coronary venous blood gases (Table 1) or in p r o s t a n o i d release o r the size o f the o c c l u d e d

1000 I00 Occlusion Fteleose

0 g , ,

~ >

~o 50c 5o "6

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FIGURE 1. Arrhythmias and VF in control dogs and in those pretreated with nafazatrom 10 mg/kg p.o. * P < 0.01 compared with controls, Chi-squared test. [~], control; [], nafazatrom.

TABLE 1. Blood gases, pH and oxygen content in the local coronary vein draining the ischaemic region of the myocardium

PO~ PCO2 pH / O z content n (mmHg) (mmHg) (units) (ml/100 ml)

Controls

20 min pre-occlusion 9 32 • 5 min pre-occlusion 9 30 :~ 7 min post-occlusion 9 27 •

15 min post-occlusion 8 26 • 30 min post-occlusion 8 26 q=

Nafazatrom 10 mg/kg p.o.

20 min pre-occlusion 8 31 • 5 rain pre-occlusion 8 30 :~ 7 min post-occlusion 8 24

15 min post-occlusion 7 23 -i- 30 min post-occlusion 7 24 •

1 min post-reperfusion 7 49 -1- 5 min post-reperfusion 7 40 •

15 rain post-reperfusion 6 38

1 60 • 2 7.31 • 0.01 11.5 • 0.6 1 58 • 2 7.30 • 0.01 10.7 • 0.8 1 a 69 • 4 aaa 7.21 ~ 0.03 aa 8.1 ~_ 0.8 aa 1 a 72 :~ 3 aaa 7.19 ~- 0.03 aaa 7.4 • 0.7 an 1 aa 67 ~c 3 aaa 7.22 ~ 0.02 aa 8.2 ~ 0.8 aa

2 63 + 4 7.28 i 0.02 10.0 • 1.0 1 63 ~ 4 7.28 ~z 0.02 9.7 ~: 1.0 1 a 73 i 3 a 7.19 • 0.02 aa 6.1 ~ 1.8 aa 1 aa 75 • 4 a 7.17 i 0.04 aa 5.1 • 0.6 aa 2 a 73 • 3 7.19 • 0.03 aa 6.0 • 0.8 a 5 bb 66 i 6 7.18 ~ 0.05 15.7 -4- 2.2 bb 5 b 52 ~ 4 b 7.26 ~ 0.04 14.4 i 1.8 bb 5 bb 61 • 4 b 7.24 • 0.05 14.1 ~ 1.6 bb

Each value is the mean ~- S.E.M. a Indicates a significant difference from 5-min pre-occlusion value. b From 30-rain post-occluslon value. One symbol P < 0.05, two P < 0.001, three P < 0.01, paired t test.

46 S.J. Coker and J. R. Parratt

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[IJ,JllllJ

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FIGURE 2. Arrhythmia profiles in the nafazatrom group. Each panel represents one individual dog and illustrates the number of arrhythmias occurring in 1 min intervals during the first 30 min of coronary artery occlusion. The number at the upper right of each panel indicates the total number of extrasystoles that occurred in the 30 rain period.

zone. There was however some evidence that the nafazat rom-treated dogs with pronounced ventr icular ectopic activity had rather higher systemic arterial pressures than those with fewer arrhythmias. In the control dogs cardiac ou tpu t and stroke volume were de- creased and peripheral vascular resistance was increased 30 min . after coronary artery occlusion (Table 2) and rather similar haemodynamic changes were observed in the nafazatrom group except that occlusion

resulted in a decrease in arterial blood pres- sure.

The majori ty of dogs pretreated with nafazatrom survived reperfusion at the end of the 40 min ischaemic period. The one dog which fibrillated did so after 14 min of reper- fusion arrhythmias . This is in marked con- trast to the control group where eight out of nine animals died in VF, usually wi thin one minu te of restoration of flow. Further , the nafazat rom-treated dogs were haemodynami -

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48 S .J . Coker and J. R. Parratt

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FIGURE 3. Arrhythmia profiles in the control group which has been described previously [4]. Each panel represents one individual dog and illustrates the number of arrhythmias occurring in 1 rain intervals during the first 30 rain of coronary artery occlusion. The number at the upper right of each panel indicates the total number of extrasystoles that occurred in the 30 min period.

cal ly s table following reperfusion with good ca rd iac funct ion and norma[ systemic and p u l m o n a r y ar ter ia l pressures (Tab le 2). The occ luded zone was s imilar in bo th groups

0 9 ~ of the free left ven t r icu la r wall (35.7 ~ . /o in the controls and 37.2 ~ 1.4~ in the na faza t rom group) as was the m a g n i t u d e of the ST-segment depression after 30 min of i schaemia (0.38 ~ 0.7 m V and 0.31 • 0.09 m V respect ively) .

The effect of nafazatrom on plasma prostanoid concentrations

T h e p lasma concentra t ions of TxB~ and 6-keto P G F ~ in ar ter ia l , coronary sinus and

local coronary venous b lood (i.e. d ra in ing the i schaemic area) are shown in F igure 4. Dur ing coronary a r te ry occlusion both t h r o m b o x a n e and pros tacycl in (measured as 6-keto PGFI~ ) were released into b lood d ra in ing from the acu te ly ischaemic myo- ca rd ium. T h e concent ra t ions of TxB 2 in the aor ta , coronary sinus and local co ronary vein in the na faza t rom group were s imilar to those in the control g roup both before and dur ing coronary a r t e ry occlusion. However , with pros tacycl in a different s i tuat ion existed. A l though aor t ic concent ra t ions of 6-keto PGFI~ were not s ignif icant ly different from those in control dogs, coronary sinus and local

N a f a z a t r o m S u p p r e s s e s R e p e r f u s i o n - I n d u c e d V F 49

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Time (rain)

F I G U R E 4. P l a s m a pros tano id concen t ra t ions in the aor ta ( � 9 the co rona ry s inus ( I ) a n d the local co rona ry vein ( • ). * Signi f icant ly d i f ferent f rom 5 -m in pre-occlus ion va lue , ]- f rom 30 m i n post -occlus ion value , one symbo l P < 0.05, two P < 0.01, th ree P < 0.001, pa i red t test.

coronary venous concentrations were higher, There were significant increases in 6-keto PGFI~ in coronary sinus blood after coronary artery occlusion in the nafazatrom group whereas in the control group the changes were not statistically significant. The release of 6~ PGFI~ into local coronary venous blood was also greater in the dogs pretreated with nafazatrom. For example, at 7 min post-occlusion the concentration of 6-keto PGFI~ in the nafazatrom group (1271 • 148 pg/ml) was significantly higher than the corresponding value in the control group (718 -+- 178 pg/ml; P < 0.05, independent t test). Thus nafazatrom appears to have elevated prostacyclin concentrations during acute myocardial ischaemia.

Since all but one of the control dogs died in VF within one minute of release of the

coronary artery occlusion, very few samples were obtained for prostanoid analysis. In the nafazatrom group, however, local coronary venous concentrations of both TxB~ and 6-keto PGFI~ were significantly reduced one minute after reperfusion to values which were similar to the pre-occlusion concentra- tions (see Fig. 4). This suggests that arachi- donic acid derivatives which had accumu- lated during coronary occlusion were rapidly washed out of the ischaemic region within one minute of the occlusion being released.

D i s c u s s i o n

The results presented above are consistent with the idea that nafazatrom is a prosta- cyclin 'promoter ' . Although the arterial and coronary sinus concentrations of 6-keto

50 S .J . Coker and J. R. Parratt

PGFI= (a breakdown product of prostacyclin) were not significantly higher in the nafaza- trom group, the concentrations in local coronary venous blood were greater at certain times after coronary artery occlusion than those in the control group. Similarly, if these values are compared with correspond- ing values measured previously in other groups of dogs such as those receiving nife- dipine [4] or ZK36374 [5, 6], the concen- trations of 6-keto PGFa= in blood draining from the acutely ischaemic myocardium are significantly greater in dogs pretreated with nafazatrom. For example, 15 rnin after coronary artery occlusion the local coronary venous concentrations of 6-keto PGF1~ were 702 • 151 pg/ml in dogs which received nifedipine and 810-k l l 7 p g / m l in dogs which received ZK36374 (a drug which mimics the actions of prostacyclin) com- pared with 1477 • 154 pg/ml in the nafaza- trom-treated dogs. This suggests that during coronary artery occlusion-nafazatrom in- creases the concentrations of prostacyclin in blood draining from the ischaemic region of the myocardium.

There are a number of possible mechanisms which may explain this effect. Nafazatrorn was originally described as an antithrom- botic compound [20], although this activity could only be demonstrated in vivo, and Vermylen et al. [21] have reported that nafazatrom stimulates the release of prosta- cyclin from blood vessel walls. More recently, Maclntyre and Salzman [13] concluded that nafazatrom had no direct effects on platelets or on vascular tissue and that its antithrom- botic activity appeared to involve regulation of prostacyclin synthesis and metabolism, an effect mediated by factors, possibly nafazatrom metabolites, present in plasma after in vivo administration. Nafazatrom has been reported to inhibit 15-hydroxyprosta- glandin dehydrogenase, an enzyme which metabolises prostacyclin as well as PGE 2 and PGF2~ [22]. Substances such as 15-hydro- peroxyarachidonic acid, as well as other fatty acid peroxides, have been shown to inhibit the prostaeyclin synthetase enzyme [14, 19]. It has been suggested that nafazatrom may prevent such actions of naturally occurring peroxides of fatty acids, thus removing a 'brake' on the prostacyclin synthetase en-

zyme [10]. This would explain why the antithrombotic activity of nafazatrom is only apparent in vivo and would be consistent with the effects of nafazatrom on prostacyclin concentrations observed in this study.

The effects of nafazatrom on arrhythmias during coronary artery occlusion and reper- fusion are similar to those of prostacyclin itself. We have demonstrated that infusing prostacyclin directly into the coronary circu- lation in anaesthetized greyhounds reduced the number of extrasystoles during coronary occlusion by 50~ and prevented reperfusion- induced VF. Similar results were obtained in dogs which received intracoronary infusions of the prostacyclin analogue, ZK36374 [5, 6]. Similarly, preventing thromboxane synthesis with the specific inhibitor, dazoxi- ben, also markedly reduced the incidence of VF resulting from the release of a coronary artery occlusion [8]. Thus the present results obtained with nafazatrom provide further evidence in support of our hypothesis that thromboxane may be arrhythmogenic and prostacyclin antiarrhythmic during acute myocardial ischaemia, and particularly fol- lowing reperfusion.

There was no evidence in the present study to suggest that nafazatrom alters thrombox- ane concentrations. Nafazatrom has been reported to be a lipoxygenase inhibitor [1, 12] but it seems unlikely that, in the present experiments, nafazatrom had any actions that could be attributed to inhibition of lipoxygenase enzymes. If leukotrienes were released during myocardial ischaemia then inhibition of lipoxygenase would presumably result in diversion ofarachidonic acid to both prostacyclin and thromboxane. The lack of enhancement of thromboxane release sug- gests that there is probably no significant release of leukotrienes during our coronary artery occlusion experiments. Mullane and Moncada, however, have reported recently [15] that inhibition of lipoxygenase product formation with BW755C reduced the size of an infarct produced by 60 rain coronary occlusion followed by 5 h reperfusion in anaesthetized dogs. This might suggest that leukotrienes may be involved in the develop- ment of ischaemic damage. However, the drug was not given until 90 rain after coro- nary occlusion and it is possible that tile

Nafazatrom Suppresses Reperfusion-Induced VF 51

contribution of leukotrienes only becomes important in the later stages of myocardial ischaemia.

Prostacyclin has also been reported to have beneficial actions during acute myocardial ischaemia [16, 18] and to limit infarct size [17]. Thus it is possible that nafazatrom may have two separate actions on arachidonate metabolism which could be beneficial during myocardial ischaemia. The ability to increase prostacyclin concentrations may reduce ar- rhythmias during ischaemia, prevent VF on reperfusion and perhaps also limit the ulti- mate size of the infarct. A recent brief report by Fiedler [11] indicates that nafazatrom can reduce final infarct size and the number of arrhythmias resulting from experimental myocardial thrombosis in dogs.

Acknowledgements

We would like to thank Dr I . J . Zeitlin for his co-operation regarding the radioimmuno- assay of prostanoids and we are indebted to Professor I. McA. Ledingham for the continued use of laboratory facilities at the Western Infirmary, and to Ian Douglas, Dick Thomson, Alan Fleming and Margaret Macdonald for their expert technical assis- tance. Prostanoids used for assay standards were a gift from the Upjohn Co., Kalamazoo, Michigan, USA. Financial support was provided by Bayer AG, Wuppertal , F R G and the Scottish Hospital Endowments Research Trust.

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5 COKER, S.J . , PAgRATT, J. R. Different effects of intravenous and intracoronary prostacyclin and ZK36374 on cardiac arrhythmias in anaesthetized dogs (Abstract). Br J Pharmacol 78, 31 (1983).

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16 OOLETReE, M. L., LEFER, A. M., SMITH, J. B., NICOLAOU, K. C. Studies on the protective effect of prosta- cyclin in acute myocardial ischaemia. EurJ Pharmacol 56, 95-103 (1979).

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