European Journal of Pharmacology, 210 (1992) 85-90 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
85
EJP 52223
Protective effects of an a-tocopherol analogue against myocardial reperfusion injury in rats M a r g a r e t A. Petty, J. M a r t i n G r i s a r a n d W y b r e n D e J o n g Marion Merrell Dow Research Institute, B.P. 447R/9, 16 Rue d',4nkara, 67009 Strasbourg Cedex, France Received 25 March 1991, revised MS received 23 September 1991, accepted 15 October 1991
Free radicals may cause part of the irreversible injury which occurs during myocardial infarction and reperfusion. In the present study MDL 73404, an a-tocopherol analogue which is a free radical scavenger has been evaluated for its effects on infarct size in an anaesthetised rat model of coronary artery ligation (60 min) and reperfusion (30 min). Intravenous infusion of the compound began 10 min before occlusion until the end of reperfusion. MDL 73404 (0.3-3 mg/kg per h) reduced infarct size, although not in a dose-related manner. Infusion of MDL 73404 (3 mg/kg per h) commencing 30 min before reperfusion until the end of reperfusion also induced a significant reduction in infarct size. In the isolated rat heart (Langendorff technique) subjected to 30 min no-flow global ischaemia, pretreatment with MDL 73404 (0.03 and 0.1 mM) in the perfusion buffer and during 30 rain of reperfusion resulted in a significant increase in the maximal pressure development (+ dP/dt max) and relaxation ( - d P / d t max), left ventricular systolic pressure and heart rate during reperfusion, whereas left ventricular diastolic pressure was significantly reduced. In contrast, only one control heart out of five exhibited signs of recovery. Replacement, for 2 rain, with a cardioplegic solution before the 30 min period of ischaemia resulted in an increased heart rate and contractility during reperfusion compared to hearts that did not receive the cardioplegic solution. The presence of MDL 73404 (0.03 and 0.1 raM) in the perfusion fluid induced an additional increase in left ventricular systolic pressure to the pre-ischaemic levels. MDL 73404 may have potential for cardioprotective use in acute reperfusion of the myocardium following ischaemia. a-Tocopherol analogue; Free radical scavenger; Antioxidant; Reperfusion injury; Vitamin E; Myocardial ischaemia/reperfusion
1. Introduction
In patients with myocardial infarction, it has been shown that early reperfusion of the occluded coronary artery results in preserved left ventricular function and reduced mortality. However, there are also data to suggest that reperfusion, while terminating ischaemia can also cause further damage to jeopardized cells due to the deleterious effects of free radicals (Braunwald and Kloner, 1985). Implicated in reperfusion damage are oxygen-derived free radicals and active oxygen intermediates such as superoxyl radical, hydrogen peroxide and hydroxyl radicals. In fact, increased concentrations of oxygen-derived free radicals have been demonstrated in previously ischaemic, reperfused myocardium by electron-spin resonance spectroscopy (Arroyo et al., 1987) and electron paramagnetic resonance spectroscopy (Zweier et al., 1987; Bolli et al., 1989).
Correspondence to: M.A. Petty, Marion Merrell Dow Reseach Institute, B.P. 447 R/9, 16 Rue d'Ankara, 67009 Strasbourg Cedex, France. Tel. 33.88.41.45.00, fax 33.88.60.37.98.
Several scavengers of oxygen-derived free radicals have been found to effectively reduce myocardial infarct size and in some circumstances to preserve left ventricular function in animals subjected to coronary artery occlusion, followed by reperfusion. These include N-(2-mercaptopropionyl)glycine (Bolli et al., 1989), the tocopherol analogue Trolox in combination with vitamin C (Mickle et al., 1989), and superoxide dismutase, which was reported to be more effective on coronary venous retrograde infusion, due to increased accumulation in the ischaemic myocardium (Hatori et al., 1989). In the isolated rat heart, pretreatment with a-tocopherol (vitamin E) produces a high level of protection against oxidative injury (Guarnieri et al., 1988). However, the acute administration of a-tocopherol is much less effective in the in vivo situation of myocardial ischaemia and reperfusion presumably because of its slow incorporation into tissues due to its high lipophilicity (Klein et al., 1989). An a-tocopherol analogue ( M D L 73404) has been designed which is more hydrophilic in character is cardioselective, and retains the a-tocopherol antioxidant properties. M D L 74270 (6-acetyloxy-3,4-dihydro-
86 N,N,N,2,5,7,8-heptamethyl-2H-l-benzopyran-2-ethanaminium 4-methylbenzenesulfonate), the N,N,N-trimethylethanaminium analogue of o~-tocopherol was synthesized and demonstrated to be a free radical scavenger (Grisar et al., 1991). It was reported to reduce myocardial infarct size in a rat model of coronary artery occlusion followed by reperfusion at comparatively low doses (Petty et al., 1991). M D L 74270 is cardioselective resulting in heart tissue concentrations of total radioactivity 20 to 30-fold higher than in blood 1-6 h after intravenous (i.v.) administration (Dow et al., 1991). The high myocardial concentration was demonstrated to be M D L 73404 (3,4-dihydro-6-hydroxy-N,N,N,2,5,7,8,heptamethyl-2H-l-benzopyran-2ethanaminium 4-methylbenzenesulfonate; MW 463.64), which accumulates in heart tissue after hydrolysis of the O-acetate precursor M D L 74270 by endogenous esterases (Dow et al., 1991). Differential centrifugation of heart homogenates showed that radioactivity was equally distributed between the fraction containing nuclei and cell walls and the fraction containing microsomes and cytosol (Dow et al., 1991). M D L 73404 is a potent scavenger of hydroxyl, superoxyl and lipoperoxyl radicals in vitro (Bolkenius et al., 1991). Because of its hydrophilic character and antioxidant properties M D L 73404 has been examined in the present study in conditions of myocardial i n f a r c t i o n / r e p e r f u s i o n in the anaesthetised rat together with its effects in global ischaemia and reperfusion in isolated rat hearts.
2. Materials and methods
2.1. Animals Male normotensive S p r a g u e - D a w l e y rats (Charles River, France) were used. Animals were kept under controlled conditions with respect to temperature, humidity and light.
2.2. Myocardial infarction /reperfusion thetised rat
in the anaes-
S p r a g u e - D a w l e y rats weighing 225-275 g were anaesthetised with sodium pentobarbitone (60 m g / k g intraperitoneally (i.p.)) and were subjected to the inf a r c t i o n / r e p e r f u s i o n procedure as described previously (Petty et al., 1991). Briefly, a catheter was inserted into the femoral artery for the direct measurement of blood pressure and a jugular vein was cannulated for drug or saline infusion. The trachea was cannulated and artificial respiration was initiated with a frequency of 60 s t r o k e s / m i n and a tidal volume of 10 m l / k g . A left thoracotomy was performed in the fifth intercostal space and a ligature ( 2 / 0 , Ethicon, Neuilly, France) was placed around the left descending coronary artery between the pulmonary artery and the left
atrial appendage. The artery was occluded for a period of 1 h followed by 30 min of reperfusion. M D L 73404 dissolved in saline, was infused in doses ranging from 0.03 to 3.0 m g / k g per h, the infusion commencing 10 rain before ligation until the end of reperfusion. In a second group of investigations infusion of M D L 73404 (3.0 m g / k g per h) commenced 30 rain after the onset of occlusion until the end of reperfusion. At the end of the reperfusion period Evans Blue was injected into the jugular vein to denote the area at risk. Following a pentobarbitone overdose infarct-size was measured by means of triphenyl tetrazolium chloride (Petty et al., 1991).
2.3. Isolated heart preparation S p r a g u e - D a w l e y rats weighing 250-350 g were used. The rats were killed by decapitation and the hearts rapidly removed and immersed in a modified K r e b s Henseleit solution (composition in m m o l / l : NaCI 112; KC1 5; N a H C O 3 25; glucose 11; K H z P O 4 1.0; MgSO 4 1.2; CaC12 2.5). The extraneous fat and connective tissue were removed and the hearts were perfused with the K r e b s - H e n s e l e i t solution using a non-recirculating Langendorff technique with perfusate at 37°C, aerated with 95% 0 2 - 5 % CO 2. The solution was delivered to the aortic cannula at a constant pressure of 50 m m Hg. Myocardial contractions were monitored by means of a balloon, introduced into the left ventricle, attached to a Bentley Trantec 800 pressure transducer and recorded on a 7754 A Hewlett Packard recorder. Maximal pressure development ( + d P / d t max) and relaxation ( - d P / d t max), left ventricular pressure both systolic and diastolic together with heart rate were monitored. The volume of the ventricular balloon was always set to achieve maximum systolic and a consistant minimum diastolic pressure to avoid using different workloads and thus metabolic demands. Left ventricular diastolic pressure never attained zero in these experiments, due to the presence of the balloon. After an initial 10 min stabilisation period, the hearts were perfused for a further 20 min in the presence or absence of M D L 73404 in the K r e b s - H e n s e l e i t solution. They were then subjected to a 30 min period of ischaemia, achieved by closing the aortic cannula, followed by a 30 min reperfusion period in the presence or absence of M D L 73404. In a further series of experiments hearts, after the initial 10 min equilibration period followed by 20 min perfusion in the presence or absence of M D L 73404, were perfused for 2 rain with a cardioplegic solution (composition in m m o l / l : NaCI 98; KC1 16; procaine hydrochloride 1; CaC12 2; N a H C O 3 29; MgCI2.6H20 16) (Hearse, 1980), before being subjected to 30 m i n o t ischaemia followed by a 30 rain period of reperfusion in the presence or absence of M D L 73404 (Godfraind and Saleh, 1984).
87 TABLE 1
2.4. Statistics Results are expressed as the means _+ S.D. of 5 - 1 0 rats in each group in the in vivo study and 3 - 5 rat hearts in each group. Comparisons were carried out by means of analysis of variance ( A N O V A ) for in vivo study and A N O V A with repeated measures for the in vitro study, both being two-tailed.
The effects of increasing doses of MDL 734114 on infarct size and the area at risk. The left main descending coronary artery was occluded lk)r a 611 rain period followed by 311 rain reperfusion. Infusion (2.3 m l / h ) of MDL 73404 or saline (control) commenced 10 rain before occlusion and continued until the end of reperfusion. The results are expressed as the m e a n s + S . D . A particular treatmenl group was compared to its appropriate control by means of ANOVA. The number of rats in each group is written in parentheses.
3. Results
3.1. Effects of MDL 73404 on infarct size The effects of M D L 73404 in rats subjected to coronary artery occlusion for 60 rain followed by reperfusion for 30 rain have been evaluated (fig. 1). I.v. infusion of M D L 73404 beginning 10 min before occlusion of the coronary artery until the end of reperfusion reduced infarct size by 63, 48 and 67% when infused at 0.3, 1 and 3 m g / k g per h respectively. M D L 734[)4 reduced the infarct size, but not in a dose-dependent manner. The maximum reduction was attained with a dose of 0.3 m g / k g per h. A dose of 0.03 m g / k g per h, had no significant effect on infarct size, whereas treatment with doses higher than 0.3 m g / k g per h induced no significant greater decrease (table 1). The area at risk did not differ between control and treatment groups, indicating that any reduction in infarct size 100
-(D
80
Control(5) 0.03 m g / k g per h (5) Control(5) 11.1 m g / k g p e r h ( 5 ) Control(5) 11.3 m g / k g per h (5) Control (9) 1.11 m g / k g per h (11}) Control(6) 3mg/kgperh(6)
Infarcted area (rag)
Area at risk (mg)
21t9+ 61 197+ 54 267+ 2(3 221+ 30" 135+ 35 49+ 49 b 277+ 122 145_+ 117 ;' 170+ 53 56+- 24h
3111+ 300+ 329+ 315+ 311+ 294+415+ 338+ 294+ 272+
49 48 48 51 41 28 117 94 28 36
" P < 0.115, ~' P < 0.01.
could not be attributed to differences in the amount of tissue rendered ischaemic (table 11. Infusion of M D L 73404 (3 m g / k g per h) commencing 30 min before reperfusion until the end of reperfusion, in anaesthetised rats, also induced a significant (P < 0.01) decrease in infarct size compared to salinetreated controls. The infarct size was 57 _+ 16 mg after M D L 73404 infusion compared to 127 + 31 mg after saline. Infusion of M D L 73404 had no effect on blood pressure or heart rate, nor did it modify the blood pressure response to coronary artery occlusion.
3.2. Global ischaemia and reperfusion c-
-~
60
c~
~
4o
20
0,03
~
0,1
0,3
1
mg / kg per h
3
Saline MDL 734O4 Fig. 1. The effect of increasing doses of MDL 73404 and saline on infarct size expressed as a percentage of the area at risk. The left descending coronary artery was occluded for a 60 rain period followed by 30 rain reperfusion. Infusion (2.3 m l / h ) commenced 10 min before occlusion until the end of reperfusion. The results are expressed as the m e a n s + S . D , of 5-10 rats in each group and a particular treatment group was compared to its appropriate control by means of ANOVA, * P < 0.05, ** P < 0.01.
In isolated rat hearts M D L 73404 (0.03 and 0.1 raM) added to the perfusion buffer had no effect on left ventricular pressure, contractility and heart rate during the equilibration period (fig. 2). After a 30 min period of global ischaemia the presence of M D L 73404 significantly reduced left ventricular diastolic pressure and increased left ventricular systolic pressure in a dose-related manner (fig. 2). Similarly + d P / d t max, - d P / d t max and heart rate were also elevated (table 2). Only one control heart out of five exhibited signs of recovery during the 30 rain reperfusion period.
3.3. Global ischaemia and reperfusion after treatment with a cardioplegic solution R e p l a c e m e n t for 2 min with a cardioplegic solution before the 30 rain period of global ischaemia resulted in an increase in heart rate, left ventricular systolic
88 LEFT
VENTRICULAR
PRESSURE
( mm
Hg
)
150 120 O3
90
r',
~
30
10 Stab~lisat~on
20
0
10 20 Reperfus~on
30 mln
0
10 20 Reperluslon
30 mm
slop flow
100 80
~ n
60 40
C~
20
oF 0
43
4z]
,"
10 S~abdisation
2O
Fig. 2. The effects of increasing concentration of MDL 73404, • 0.1 mM, [] 0.03 mM and • control, in the perfusion buffer, during a 20 min equilibration period and reperfusion after 30 min of global ischaemia, on left ventricular systolic and diastolic pressures of isolated rat hearts. Results are expressed as the mean of 3-5 hearts in each group and were compared by means of A N O V A with repeated measures, * P < 0.05, • *P < 0.01. The S.D. have been omitted for the sake of clarity.
LEFT
VENTRICULAR
150
PRESSURE
( mm Hg )
I
120
~
9o
a_
6o
O9
:,5
3O 0
i 10 Stabilisation
20
0 stop flOW
i L 10 20 Reperfusion
30 mm
4O
~ if)
30
13_
2O .I
o C5
0
10 Stabilisabon
I
L
0
20
stop flow
i
i
10 20 Reperlusion
J
30 mln
Fig. 3. The effects of increasing concentrations of MDL 73404, • 0.1 mM, [] 0.03 mM and • control, in the perfusion buffer, during a 20 min equilibration period followed by 2 min perfusion with a cardioplegic solution, before being subjected to 30 min global ischaemia and 30 min reperfusion, on left ventricular systolic and diastolic pressures of isolated rat hearts. Results are expressed as the mean of 3-5 hearts in each group and were compared to control by means of A N O V A with repeated measures, *P < 0.05, **P < 0.01. The S.D. have been omitted for the sake of clarity.
89 TABLE 2 The effects of M D L 73404 on maximum pressure development + d P / d t max, relaxation - d P / d t max and heart rate at the end of the 30 rain reperfusion period following 30 min of hypoxia. Results are expressed as the means_+S.D, of 3-5 rat hearts in each group and were compared to control hearts by means of ANOVA .
Control 0.(/3 mM (1.1 mM
Heart rate (bpm)
+ d P / d t max (ram H g / s )
- d P / d t max (ram H g / s )
42 +- 95 243+_41 h 158+24 b
191 + 404 903+514 1444+ 48 ~1
167 +_372 625+375 ~ 1277_+ 96 i,
Aider a 2 rain treatment period with a cardioph'gie Control 249 +_62 1366 +_411 0.03 mM 18(/+2(/ 1735_+433 0.1 mM 161 +_34 1818_+ 158
sohaion: 1(183 + 353 1527+_432 1583+ 144
" P < 0./15, b p < 0.01.
pressure, maximal pressure development and relaxation during reperfusion compared to hearts that did not receive the cardioplegic solution. The presence of MDL 73404 (0.03 and 0.1 raM) in the reperfusion buffer resulted in an increase in left ventricular systolic pressure to the pre-ischaemic level (fig. 3). There was no further augmentation in heart rate or maximal pressure development and relaxation. Left ventricular diastolic pressure was not different compared to preischaemic values. 4. Discussion
In isolated rat hearts undergoing 25 min of ischaemia with varying periods of reperfusion a significant increase in lipid peroxidation (as indicated by the free radical-induced formation of conjugated diene free fatty acids) (Fridovich, 1978) was observed after 2 rain of reflow (Massey and Burton, 1988). The postulated production of free radicals during reperfusion is consistent with the findings of the present study in which the hydrophilic free radical scavenger M D L 73404 (Bolkenius et al., in press), in concentrations of 0.1 mM and less in the perfusion buffer, induced an increase in left ventricular systolic pressure, maximal pressure development and relaxation, and heart rate during reperfusion after a 30 rain period of global ischaemia. In contrast, only one control heart out of five exhibited signs of recovery during the 30 min reperfusion period. Similar findings have been reported by Massey and Burton (1989), who observed in hearts obtained from rats implanted with a s.c. timed-release vitamin E pellet 14 days before experimentation, a reduction in contractile dysfunction, after global ischaemia and reperfusion. This was associated with a 51% increase in myocardial vitamin E levels, and reduced formation of conjugated dienes as well as unesterified fatty acids, a decreased accumulation of tissue calcium and a reduced release of lactate dehydrogenase.
Weisel et al. (1989) have also reported a significant increase in coronary sinus phospholipid conjugated dienes and depletion of myocardial a-tocopherol during reperfusion after elective coronary artery bypass surgery despite cold blood cardioplegia. These authors suggested that the decrease in a-tocopherol concentrations during reperfusion may have resulted from free radical-mediated membrane lipid peroxidation. It was later demonstrated that pretreatment with vitamin E, for 14 days, before coronary artery bypass surgery reduced free radical mediated myocardial metabolic dysfunction (Yau et al., 1990). In the present study treatment with a cardioplegic solution for 2 min before 30 rain of global ischaemia resulted in a rapid reversal of the decreased contractility, heart rate, and left ventricular systolic pressure upon reperfusion. Incorporation of MDL 73404 into the reperfusion buffer further augmented the increase in contractility and left ventricular systolic pressure, without affecting heart rate. It is evident from the above findings that the damage induced by ischacmia and reperfusion is partially ameliorated by prior treatment with the cardioplegic solution, but the presence of MDL 73404 induces a still further improvement to pre-ischaemic levels. In the present study, an a-tocopherol analogue has been evaluated in a rat model of myocardial ischaemia and reperfusion. In this model, M D L 73404 reduced infarct size starting with a dose of 0.1 m g / k g per h. The maximum reduction occurred with a dose of 0.3 m g / k g per h, higher doses did not lead to enhanced salvage, equivalent to the findings obtained with MDL 74270 (Petty et al., 1990, 1991), the acetylated form of the free-radical scavenger. There appears to be no advantage, thus, to the use of the prodrug MDL 74270 and the active metabolite, MDL 73404, which also accumulates in heart tissue (Dow et al., in press), is to be preferred. Presumably the cardiac selectivity is the explanation why comparatively low doses of both MDL 73404 and MDL 74270 are required for myocardial salvage in conditions of reperfusion injury. It seems likely that the quaternary amine function is responsible for this cardiac selectivity, since the closely related tertiary amine analogue does not accumulate in heart tissue and a 30 times higher dose is required to produce a significant reduction in infarct size (Petty et al., 1991). Interestingly this cardioselectivity is not limited to quaternary ammonium compounds (Nature et al., 1975; Patterson e t a . , 1980), but is apparent with other quaternary substituted compounds, cations which may concentrate in myocytes due to the transmembrane potential gradient (Srivastava et al., 1990). The lipophylic cation, tetraphenylphosphonium enters myocytes by means of the sodium/calcium exchange (Reeves and Sutko, 1980), and similarly, perhaps, the cardioselective quaternary amine compounds are transported across the sarcolemma by this mechanism.
90
In patients with myocardial infarction it is proposed t o a d m i n i s t e r M D L 7 3 4 0 4 i.v. a t t h e s a m e t i m e o r e v e n b e f o r e a t h r o m b o l y t i c a g e n t s u c h as A P S A C ( a n i s o ylated plasminogen streptokinase activator complex), streptokinase itself or tissue plasminogen activator. Both thrombolytic agent and free radical scavenger ( M D L 7 3 4 0 4 ) m u s t b e a d m i n i s t e r e d as s o o n as p o s s i b l e a f t e r t h e o n s e t o f s y m p t o m s o f i n f a r c t i o n . H e n c e in t h e present infarction/ligation model further experiments have been carried out with MDL 73404 infusion comm e n c i n g 30 r a i n a f t e r o c c l u s i o n o f t h e a r t e r y , t o d e m o n s t r a t e t h a t t h e a - t o c o p h e r o l d e r i v a t i v e is still e f f e c t i v e a t r e d u c i n g i n f a r c t size. M D L 7 3 4 0 4 , 3 m g / k g p e r h, r e s u l t e d in a 5 5 % r e d u c t i o n in i n f a r c t size i n d i c a t i n g t h a t s u f f i c i e n t c o m p o u n d is d i f f u s i n g i n t o t h e area and/or entering the ischaemia area upon reperfusion; since the rat lacks a significant collateral circulat i o n ( M a x w e l l e t al., 1984). O n c e , in t h e i s c h a e m i c a r e a M D L 7 3 4 0 4 is a b l e t o s c a v e n g e o x y g e n - d e r i v e d f r e e radicals and to prevent any injury they cause. In c o n c l u s i o n , M D L 7 3 4 0 4 , a n a - t o c o p h e r o l a n a logue with free radical scavenging properties reduces m y o c a r d i a l i n f a r c t size in v i v o a n d i m p r o v e s c o n t r a c t i l ity u p o n r e p e r f u s i o n a f t e r g l o b a l i s c h a e m i a in i s o l a t e d r a t h e a r t s . It m a y h a v e t h e r a p e u t i c p o t e n t i a l f o r c a r d i o p r o t e c t i v e u s e in a c u t e r e p e r f u s i o n o f t h e m y o c a r d i u m as w e l l as in h e a r t t r a n s p l a n t a t i o n a n d b y p a s s s u r g e r y . The cardioselectivity of MDL 73404 provides a distinct advantage of this free radical scavenger for application in c o r o n a r y a r t e r y r e p e r f u s i o n .
Acknowledgements The authors wish to thank Mine. 1. Fornieles for her expert technical assistance and Mine. A.M. Bramonte for typing the manuscript.
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Wagner, K.D. Haegele and W. De Jong, 1991, A cardioselective hydrophilic, N,N,N-trimethylethanaminium ~-tocopherol analogue that reduces myocardial infarct size, J. Med. Chem. 34, 257. Guarnieri, C., C. Muscari, A. Fraticelli and C.M. Caldarera, 1988, Role of antioxidants in hypoxia-reoxygenation injury in the heart and in cardiac myocytes, in: Oxygen Radicals in the Pathophysiology of Heart Disease, ed. P.K. Singal (Kluwer Academic Publishers) p. 271. Hatori, N., A. Miyazaki, [I. Tadokovo, L. Ryden, J. Moll, R.E. Rajagopalan, M.C. Fishbein, S. Meerbaum, E. Corday and J.K. Drury, [989, Beneficial effects of coronary venous relroinfusion of superoxide dismutase and catalasc on reperfusion arrhythmias, myocardial function and infarct size in dogs, J. Cardiovasc. Pharmacol. 14, 396. Hearse, D.J., 1980, Cardioplegia: the protection of the myocardium during open heart surgery: a review, J. Physiol. (Paris) 76, 451. Klein, H.H., S. Pith, S. Lindert, K. Nebendahl, P. Niedmann and H. Kreuzer, 1989, Combined treatment with vitamins E and C in experimental myocardial infarction in pigs, Am. Heart J. 118, 667. Massey, K.D. and K.P. Burton, 1988, Myocardial ischemia and reperfusion induced membrane damage in isolated perfused rat hearts: attenuation by c~-tocopherol, Free Radical Res. Commun. 5, 199. Masscy, K.D. and K.P. Burton, 1989, a-Tocopherol anenuates myocardial membrane-related alterations resulting from ischaemia and reperfilsion, Am. J. Physiol. 256, 111192. Maxwell, M.P., D.J. Hearse and D.M. Yellon, 1984, The coronary collateral circulation during regional myocardial ischaemia: a comparative study in eight species, J. Mol. Cell. Cardiol. 16 (Suppl. 2), 47. Mickle, D.A.G., R,K. Ki, R.D. Weisel, P.L. Birnbaum, T.W. Tu, G, Jackowski, M.M. Madonik, G.W. Burton and K.U. lngold, 1989, Myocardial salvage with Trolox and ascorbic acid for an acute ew)lving infarction, Ann. Thorac. Surg. 47, 553. Namm, D.H., C.M. Wang, S. EI-Sayad, F.C. C,~)pp, and R.A. Maxwell, 1975, Effects of bretylium on rat cardiac muscle: the deetrophysiological effects and its uptake and binding in normal and immunosympathectomized rat hearts, J. Pharmacol. Exp. Ther. 193, 194. Patterson, E.. P. Stetson, and B,R. Lucchesi, 1980, Plasma and myocardial tissue concentrations of um-272 (N,N-dimethylpropranolol) after oral administration in dogs, J. Pharmacol. Exp. Ther. 214, 449. Petty, M.A., J.M. Grisar, J. Dow and W. De Jong, 1990, Effects of an ce-tocopherol analogue on myocardial ischaemia and reperfusion injury, in rats, Eur. J. Pharmacol. 179, 241. Petty, M.A., J. Dow. J.M. Grisar and W. De Jong, 1091, Effects of cardioselective c,-tocopherol analogue on reperfusion injury in rats induced by myocardial ischaemia, Eur. J. Pharmacol. 192, 383. Reeves, J.P. and J.L. Sutko, 1980, Sodium-calcium exchange activity generates a current in cardiac membrane vesicles, Science 208. 1461. Srivastava, P.C., F.F. Knapp, Jr., A.P. Callahan and B.M. Goldstein, 1990, Myocardial imaging agents: synthesis, characterization and evaluation of [(Z) and (Z,E)-(l-[SeBr]bromo-l-penten-5-yl]triphenylphosphonium cations, J. Labelled Comp. Radiopharmacol. 28, 1161. Weisel, R.D., D.A.G. Mickle, C.D. Finkle, L.C. Tumiati, M.M. Madonik, J. Ivanov, G.W. Burton and K.U. lngold, 1989, Myocardial free-radical injury after cardioplegia, Circulation 81) (Suppl. ll), Ill-14. Yau, T., R.D. Weisel, D.A.D. Mickle, J. lvanov, L.C. Tumiati, K.U. Ingold and G.W. Burton, 1990, Vitamin E improved myocardial protection, Circulation 82 (Suppl. IIl), II1-146. Zweier, J.L., J.T. Flaherty and M.L. Weisfeldt, 1987, Direct measurement of free radical generation following reperfusion ot ischaemic myocardium, Proc. Natl. Acad. Sci. 84, 1404.
85
EJP 52223
Protective effects of an a-tocopherol analogue against myocardial reperfusion injury in rats M a r g a r e t A. Petty, J. M a r t i n G r i s a r a n d W y b r e n D e J o n g Marion Merrell Dow Research Institute, B.P. 447R/9, 16 Rue d',4nkara, 67009 Strasbourg Cedex, France Received 25 March 1991, revised MS received 23 September 1991, accepted 15 October 1991
Free radicals may cause part of the irreversible injury which occurs during myocardial infarction and reperfusion. In the present study MDL 73404, an a-tocopherol analogue which is a free radical scavenger has been evaluated for its effects on infarct size in an anaesthetised rat model of coronary artery ligation (60 min) and reperfusion (30 min). Intravenous infusion of the compound began 10 min before occlusion until the end of reperfusion. MDL 73404 (0.3-3 mg/kg per h) reduced infarct size, although not in a dose-related manner. Infusion of MDL 73404 (3 mg/kg per h) commencing 30 min before reperfusion until the end of reperfusion also induced a significant reduction in infarct size. In the isolated rat heart (Langendorff technique) subjected to 30 min no-flow global ischaemia, pretreatment with MDL 73404 (0.03 and 0.1 mM) in the perfusion buffer and during 30 rain of reperfusion resulted in a significant increase in the maximal pressure development (+ dP/dt max) and relaxation ( - d P / d t max), left ventricular systolic pressure and heart rate during reperfusion, whereas left ventricular diastolic pressure was significantly reduced. In contrast, only one control heart out of five exhibited signs of recovery. Replacement, for 2 rain, with a cardioplegic solution before the 30 min period of ischaemia resulted in an increased heart rate and contractility during reperfusion compared to hearts that did not receive the cardioplegic solution. The presence of MDL 73404 (0.03 and 0.1 raM) in the perfusion fluid induced an additional increase in left ventricular systolic pressure to the pre-ischaemic levels. MDL 73404 may have potential for cardioprotective use in acute reperfusion of the myocardium following ischaemia. a-Tocopherol analogue; Free radical scavenger; Antioxidant; Reperfusion injury; Vitamin E; Myocardial ischaemia/reperfusion
1. Introduction
In patients with myocardial infarction, it has been shown that early reperfusion of the occluded coronary artery results in preserved left ventricular function and reduced mortality. However, there are also data to suggest that reperfusion, while terminating ischaemia can also cause further damage to jeopardized cells due to the deleterious effects of free radicals (Braunwald and Kloner, 1985). Implicated in reperfusion damage are oxygen-derived free radicals and active oxygen intermediates such as superoxyl radical, hydrogen peroxide and hydroxyl radicals. In fact, increased concentrations of oxygen-derived free radicals have been demonstrated in previously ischaemic, reperfused myocardium by electron-spin resonance spectroscopy (Arroyo et al., 1987) and electron paramagnetic resonance spectroscopy (Zweier et al., 1987; Bolli et al., 1989).
Correspondence to: M.A. Petty, Marion Merrell Dow Reseach Institute, B.P. 447 R/9, 16 Rue d'Ankara, 67009 Strasbourg Cedex, France. Tel. 33.88.41.45.00, fax 33.88.60.37.98.
Several scavengers of oxygen-derived free radicals have been found to effectively reduce myocardial infarct size and in some circumstances to preserve left ventricular function in animals subjected to coronary artery occlusion, followed by reperfusion. These include N-(2-mercaptopropionyl)glycine (Bolli et al., 1989), the tocopherol analogue Trolox in combination with vitamin C (Mickle et al., 1989), and superoxide dismutase, which was reported to be more effective on coronary venous retrograde infusion, due to increased accumulation in the ischaemic myocardium (Hatori et al., 1989). In the isolated rat heart, pretreatment with a-tocopherol (vitamin E) produces a high level of protection against oxidative injury (Guarnieri et al., 1988). However, the acute administration of a-tocopherol is much less effective in the in vivo situation of myocardial ischaemia and reperfusion presumably because of its slow incorporation into tissues due to its high lipophilicity (Klein et al., 1989). An a-tocopherol analogue ( M D L 73404) has been designed which is more hydrophilic in character is cardioselective, and retains the a-tocopherol antioxidant properties. M D L 74270 (6-acetyloxy-3,4-dihydro-
86 N,N,N,2,5,7,8-heptamethyl-2H-l-benzopyran-2-ethanaminium 4-methylbenzenesulfonate), the N,N,N-trimethylethanaminium analogue of o~-tocopherol was synthesized and demonstrated to be a free radical scavenger (Grisar et al., 1991). It was reported to reduce myocardial infarct size in a rat model of coronary artery occlusion followed by reperfusion at comparatively low doses (Petty et al., 1991). M D L 74270 is cardioselective resulting in heart tissue concentrations of total radioactivity 20 to 30-fold higher than in blood 1-6 h after intravenous (i.v.) administration (Dow et al., 1991). The high myocardial concentration was demonstrated to be M D L 73404 (3,4-dihydro-6-hydroxy-N,N,N,2,5,7,8,heptamethyl-2H-l-benzopyran-2ethanaminium 4-methylbenzenesulfonate; MW 463.64), which accumulates in heart tissue after hydrolysis of the O-acetate precursor M D L 74270 by endogenous esterases (Dow et al., 1991). Differential centrifugation of heart homogenates showed that radioactivity was equally distributed between the fraction containing nuclei and cell walls and the fraction containing microsomes and cytosol (Dow et al., 1991). M D L 73404 is a potent scavenger of hydroxyl, superoxyl and lipoperoxyl radicals in vitro (Bolkenius et al., 1991). Because of its hydrophilic character and antioxidant properties M D L 73404 has been examined in the present study in conditions of myocardial i n f a r c t i o n / r e p e r f u s i o n in the anaesthetised rat together with its effects in global ischaemia and reperfusion in isolated rat hearts.
2. Materials and methods
2.1. Animals Male normotensive S p r a g u e - D a w l e y rats (Charles River, France) were used. Animals were kept under controlled conditions with respect to temperature, humidity and light.
2.2. Myocardial infarction /reperfusion thetised rat
in the anaes-
S p r a g u e - D a w l e y rats weighing 225-275 g were anaesthetised with sodium pentobarbitone (60 m g / k g intraperitoneally (i.p.)) and were subjected to the inf a r c t i o n / r e p e r f u s i o n procedure as described previously (Petty et al., 1991). Briefly, a catheter was inserted into the femoral artery for the direct measurement of blood pressure and a jugular vein was cannulated for drug or saline infusion. The trachea was cannulated and artificial respiration was initiated with a frequency of 60 s t r o k e s / m i n and a tidal volume of 10 m l / k g . A left thoracotomy was performed in the fifth intercostal space and a ligature ( 2 / 0 , Ethicon, Neuilly, France) was placed around the left descending coronary artery between the pulmonary artery and the left
atrial appendage. The artery was occluded for a period of 1 h followed by 30 min of reperfusion. M D L 73404 dissolved in saline, was infused in doses ranging from 0.03 to 3.0 m g / k g per h, the infusion commencing 10 rain before ligation until the end of reperfusion. In a second group of investigations infusion of M D L 73404 (3.0 m g / k g per h) commenced 30 rain after the onset of occlusion until the end of reperfusion. At the end of the reperfusion period Evans Blue was injected into the jugular vein to denote the area at risk. Following a pentobarbitone overdose infarct-size was measured by means of triphenyl tetrazolium chloride (Petty et al., 1991).
2.3. Isolated heart preparation S p r a g u e - D a w l e y rats weighing 250-350 g were used. The rats were killed by decapitation and the hearts rapidly removed and immersed in a modified K r e b s Henseleit solution (composition in m m o l / l : NaCI 112; KC1 5; N a H C O 3 25; glucose 11; K H z P O 4 1.0; MgSO 4 1.2; CaC12 2.5). The extraneous fat and connective tissue were removed and the hearts were perfused with the K r e b s - H e n s e l e i t solution using a non-recirculating Langendorff technique with perfusate at 37°C, aerated with 95% 0 2 - 5 % CO 2. The solution was delivered to the aortic cannula at a constant pressure of 50 m m Hg. Myocardial contractions were monitored by means of a balloon, introduced into the left ventricle, attached to a Bentley Trantec 800 pressure transducer and recorded on a 7754 A Hewlett Packard recorder. Maximal pressure development ( + d P / d t max) and relaxation ( - d P / d t max), left ventricular pressure both systolic and diastolic together with heart rate were monitored. The volume of the ventricular balloon was always set to achieve maximum systolic and a consistant minimum diastolic pressure to avoid using different workloads and thus metabolic demands. Left ventricular diastolic pressure never attained zero in these experiments, due to the presence of the balloon. After an initial 10 min stabilisation period, the hearts were perfused for a further 20 min in the presence or absence of M D L 73404 in the K r e b s - H e n s e l e i t solution. They were then subjected to a 30 min period of ischaemia, achieved by closing the aortic cannula, followed by a 30 min reperfusion period in the presence or absence of M D L 73404. In a further series of experiments hearts, after the initial 10 min equilibration period followed by 20 min perfusion in the presence or absence of M D L 73404, were perfused for 2 rain with a cardioplegic solution (composition in m m o l / l : NaCI 98; KC1 16; procaine hydrochloride 1; CaC12 2; N a H C O 3 29; MgCI2.6H20 16) (Hearse, 1980), before being subjected to 30 m i n o t ischaemia followed by a 30 rain period of reperfusion in the presence or absence of M D L 73404 (Godfraind and Saleh, 1984).
87 TABLE 1
2.4. Statistics Results are expressed as the means _+ S.D. of 5 - 1 0 rats in each group in the in vivo study and 3 - 5 rat hearts in each group. Comparisons were carried out by means of analysis of variance ( A N O V A ) for in vivo study and A N O V A with repeated measures for the in vitro study, both being two-tailed.
The effects of increasing doses of MDL 734114 on infarct size and the area at risk. The left main descending coronary artery was occluded lk)r a 611 rain period followed by 311 rain reperfusion. Infusion (2.3 m l / h ) of MDL 73404 or saline (control) commenced 10 rain before occlusion and continued until the end of reperfusion. The results are expressed as the m e a n s + S . D . A particular treatmenl group was compared to its appropriate control by means of ANOVA. The number of rats in each group is written in parentheses.
3. Results
3.1. Effects of MDL 73404 on infarct size The effects of M D L 73404 in rats subjected to coronary artery occlusion for 60 rain followed by reperfusion for 30 rain have been evaluated (fig. 1). I.v. infusion of M D L 73404 beginning 10 min before occlusion of the coronary artery until the end of reperfusion reduced infarct size by 63, 48 and 67% when infused at 0.3, 1 and 3 m g / k g per h respectively. M D L 734[)4 reduced the infarct size, but not in a dose-dependent manner. The maximum reduction was attained with a dose of 0.3 m g / k g per h. A dose of 0.03 m g / k g per h, had no significant effect on infarct size, whereas treatment with doses higher than 0.3 m g / k g per h induced no significant greater decrease (table 1). The area at risk did not differ between control and treatment groups, indicating that any reduction in infarct size 100
-(D
80
Control(5) 0.03 m g / k g per h (5) Control(5) 11.1 m g / k g p e r h ( 5 ) Control(5) 11.3 m g / k g per h (5) Control (9) 1.11 m g / k g per h (11}) Control(6) 3mg/kgperh(6)
Infarcted area (rag)
Area at risk (mg)
21t9+ 61 197+ 54 267+ 2(3 221+ 30" 135+ 35 49+ 49 b 277+ 122 145_+ 117 ;' 170+ 53 56+- 24h
3111+ 300+ 329+ 315+ 311+ 294+415+ 338+ 294+ 272+
49 48 48 51 41 28 117 94 28 36
" P < 0.115, ~' P < 0.01.
could not be attributed to differences in the amount of tissue rendered ischaemic (table 11. Infusion of M D L 73404 (3 m g / k g per h) commencing 30 min before reperfusion until the end of reperfusion, in anaesthetised rats, also induced a significant (P < 0.01) decrease in infarct size compared to salinetreated controls. The infarct size was 57 _+ 16 mg after M D L 73404 infusion compared to 127 + 31 mg after saline. Infusion of M D L 73404 had no effect on blood pressure or heart rate, nor did it modify the blood pressure response to coronary artery occlusion.
3.2. Global ischaemia and reperfusion c-
-~
60
c~
~
4o
20
0,03
~
0,1
0,3
1
mg / kg per h
3
Saline MDL 734O4 Fig. 1. The effect of increasing doses of MDL 73404 and saline on infarct size expressed as a percentage of the area at risk. The left descending coronary artery was occluded for a 60 rain period followed by 30 rain reperfusion. Infusion (2.3 m l / h ) commenced 10 min before occlusion until the end of reperfusion. The results are expressed as the m e a n s + S . D , of 5-10 rats in each group and a particular treatment group was compared to its appropriate control by means of ANOVA, * P < 0.05, ** P < 0.01.
In isolated rat hearts M D L 73404 (0.03 and 0.1 raM) added to the perfusion buffer had no effect on left ventricular pressure, contractility and heart rate during the equilibration period (fig. 2). After a 30 min period of global ischaemia the presence of M D L 73404 significantly reduced left ventricular diastolic pressure and increased left ventricular systolic pressure in a dose-related manner (fig. 2). Similarly + d P / d t max, - d P / d t max and heart rate were also elevated (table 2). Only one control heart out of five exhibited signs of recovery during the 30 rain reperfusion period.
3.3. Global ischaemia and reperfusion after treatment with a cardioplegic solution R e p l a c e m e n t for 2 min with a cardioplegic solution before the 30 rain period of global ischaemia resulted in an increase in heart rate, left ventricular systolic
88 LEFT
VENTRICULAR
PRESSURE
( mm
Hg
)
150 120 O3
90
r',
~
30
10 Stab~lisat~on
20
0
10 20 Reperfus~on
30 mln
0
10 20 Reperluslon
30 mm
slop flow
100 80
~ n
60 40
C~
20
oF 0
43
4z]
,"
10 S~abdisation
2O
Fig. 2. The effects of increasing concentration of MDL 73404, • 0.1 mM, [] 0.03 mM and • control, in the perfusion buffer, during a 20 min equilibration period and reperfusion after 30 min of global ischaemia, on left ventricular systolic and diastolic pressures of isolated rat hearts. Results are expressed as the mean of 3-5 hearts in each group and were compared by means of A N O V A with repeated measures, * P < 0.05, • *P < 0.01. The S.D. have been omitted for the sake of clarity.
LEFT
VENTRICULAR
150
PRESSURE
( mm Hg )
I
120
~
9o
a_
6o
O9
:,5
3O 0
i 10 Stabilisation
20
0 stop flOW
i L 10 20 Reperfusion
30 mm
4O
~ if)
30
13_
2O .I
o C5
0
10 Stabilisabon
I
L
0
20
stop flow
i
i
10 20 Reperlusion
J
30 mln
Fig. 3. The effects of increasing concentrations of MDL 73404, • 0.1 mM, [] 0.03 mM and • control, in the perfusion buffer, during a 20 min equilibration period followed by 2 min perfusion with a cardioplegic solution, before being subjected to 30 min global ischaemia and 30 min reperfusion, on left ventricular systolic and diastolic pressures of isolated rat hearts. Results are expressed as the mean of 3-5 hearts in each group and were compared to control by means of A N O V A with repeated measures, *P < 0.05, **P < 0.01. The S.D. have been omitted for the sake of clarity.
89 TABLE 2 The effects of M D L 73404 on maximum pressure development + d P / d t max, relaxation - d P / d t max and heart rate at the end of the 30 rain reperfusion period following 30 min of hypoxia. Results are expressed as the means_+S.D, of 3-5 rat hearts in each group and were compared to control hearts by means of ANOVA .
Control 0.(/3 mM (1.1 mM
Heart rate (bpm)
+ d P / d t max (ram H g / s )
- d P / d t max (ram H g / s )
42 +- 95 243+_41 h 158+24 b
191 + 404 903+514 1444+ 48 ~1
167 +_372 625+375 ~ 1277_+ 96 i,
Aider a 2 rain treatment period with a cardioph'gie Control 249 +_62 1366 +_411 0.03 mM 18(/+2(/ 1735_+433 0.1 mM 161 +_34 1818_+ 158
sohaion: 1(183 + 353 1527+_432 1583+ 144
" P < 0./15, b p < 0.01.
pressure, maximal pressure development and relaxation during reperfusion compared to hearts that did not receive the cardioplegic solution. The presence of MDL 73404 (0.03 and 0.1 raM) in the reperfusion buffer resulted in an increase in left ventricular systolic pressure to the pre-ischaemic level (fig. 3). There was no further augmentation in heart rate or maximal pressure development and relaxation. Left ventricular diastolic pressure was not different compared to preischaemic values. 4. Discussion
In isolated rat hearts undergoing 25 min of ischaemia with varying periods of reperfusion a significant increase in lipid peroxidation (as indicated by the free radical-induced formation of conjugated diene free fatty acids) (Fridovich, 1978) was observed after 2 rain of reflow (Massey and Burton, 1988). The postulated production of free radicals during reperfusion is consistent with the findings of the present study in which the hydrophilic free radical scavenger M D L 73404 (Bolkenius et al., in press), in concentrations of 0.1 mM and less in the perfusion buffer, induced an increase in left ventricular systolic pressure, maximal pressure development and relaxation, and heart rate during reperfusion after a 30 rain period of global ischaemia. In contrast, only one control heart out of five exhibited signs of recovery during the 30 min reperfusion period. Similar findings have been reported by Massey and Burton (1989), who observed in hearts obtained from rats implanted with a s.c. timed-release vitamin E pellet 14 days before experimentation, a reduction in contractile dysfunction, after global ischaemia and reperfusion. This was associated with a 51% increase in myocardial vitamin E levels, and reduced formation of conjugated dienes as well as unesterified fatty acids, a decreased accumulation of tissue calcium and a reduced release of lactate dehydrogenase.
Weisel et al. (1989) have also reported a significant increase in coronary sinus phospholipid conjugated dienes and depletion of myocardial a-tocopherol during reperfusion after elective coronary artery bypass surgery despite cold blood cardioplegia. These authors suggested that the decrease in a-tocopherol concentrations during reperfusion may have resulted from free radical-mediated membrane lipid peroxidation. It was later demonstrated that pretreatment with vitamin E, for 14 days, before coronary artery bypass surgery reduced free radical mediated myocardial metabolic dysfunction (Yau et al., 1990). In the present study treatment with a cardioplegic solution for 2 min before 30 rain of global ischaemia resulted in a rapid reversal of the decreased contractility, heart rate, and left ventricular systolic pressure upon reperfusion. Incorporation of MDL 73404 into the reperfusion buffer further augmented the increase in contractility and left ventricular systolic pressure, without affecting heart rate. It is evident from the above findings that the damage induced by ischacmia and reperfusion is partially ameliorated by prior treatment with the cardioplegic solution, but the presence of MDL 73404 induces a still further improvement to pre-ischaemic levels. In the present study, an a-tocopherol analogue has been evaluated in a rat model of myocardial ischaemia and reperfusion. In this model, M D L 73404 reduced infarct size starting with a dose of 0.1 m g / k g per h. The maximum reduction occurred with a dose of 0.3 m g / k g per h, higher doses did not lead to enhanced salvage, equivalent to the findings obtained with MDL 74270 (Petty et al., 1990, 1991), the acetylated form of the free-radical scavenger. There appears to be no advantage, thus, to the use of the prodrug MDL 74270 and the active metabolite, MDL 73404, which also accumulates in heart tissue (Dow et al., in press), is to be preferred. Presumably the cardiac selectivity is the explanation why comparatively low doses of both MDL 73404 and MDL 74270 are required for myocardial salvage in conditions of reperfusion injury. It seems likely that the quaternary amine function is responsible for this cardiac selectivity, since the closely related tertiary amine analogue does not accumulate in heart tissue and a 30 times higher dose is required to produce a significant reduction in infarct size (Petty et al., 1991). Interestingly this cardioselectivity is not limited to quaternary ammonium compounds (Nature et al., 1975; Patterson e t a . , 1980), but is apparent with other quaternary substituted compounds, cations which may concentrate in myocytes due to the transmembrane potential gradient (Srivastava et al., 1990). The lipophylic cation, tetraphenylphosphonium enters myocytes by means of the sodium/calcium exchange (Reeves and Sutko, 1980), and similarly, perhaps, the cardioselective quaternary amine compounds are transported across the sarcolemma by this mechanism.
90
In patients with myocardial infarction it is proposed t o a d m i n i s t e r M D L 7 3 4 0 4 i.v. a t t h e s a m e t i m e o r e v e n b e f o r e a t h r o m b o l y t i c a g e n t s u c h as A P S A C ( a n i s o ylated plasminogen streptokinase activator complex), streptokinase itself or tissue plasminogen activator. Both thrombolytic agent and free radical scavenger ( M D L 7 3 4 0 4 ) m u s t b e a d m i n i s t e r e d as s o o n as p o s s i b l e a f t e r t h e o n s e t o f s y m p t o m s o f i n f a r c t i o n . H e n c e in t h e present infarction/ligation model further experiments have been carried out with MDL 73404 infusion comm e n c i n g 30 r a i n a f t e r o c c l u s i o n o f t h e a r t e r y , t o d e m o n s t r a t e t h a t t h e a - t o c o p h e r o l d e r i v a t i v e is still e f f e c t i v e a t r e d u c i n g i n f a r c t size. M D L 7 3 4 0 4 , 3 m g / k g p e r h, r e s u l t e d in a 5 5 % r e d u c t i o n in i n f a r c t size i n d i c a t i n g t h a t s u f f i c i e n t c o m p o u n d is d i f f u s i n g i n t o t h e area and/or entering the ischaemia area upon reperfusion; since the rat lacks a significant collateral circulat i o n ( M a x w e l l e t al., 1984). O n c e , in t h e i s c h a e m i c a r e a M D L 7 3 4 0 4 is a b l e t o s c a v e n g e o x y g e n - d e r i v e d f r e e radicals and to prevent any injury they cause. In c o n c l u s i o n , M D L 7 3 4 0 4 , a n a - t o c o p h e r o l a n a logue with free radical scavenging properties reduces m y o c a r d i a l i n f a r c t size in v i v o a n d i m p r o v e s c o n t r a c t i l ity u p o n r e p e r f u s i o n a f t e r g l o b a l i s c h a e m i a in i s o l a t e d r a t h e a r t s . It m a y h a v e t h e r a p e u t i c p o t e n t i a l f o r c a r d i o p r o t e c t i v e u s e in a c u t e r e p e r f u s i o n o f t h e m y o c a r d i u m as w e l l as in h e a r t t r a n s p l a n t a t i o n a n d b y p a s s s u r g e r y . The cardioselectivity of MDL 73404 provides a distinct advantage of this free radical scavenger for application in c o r o n a r y a r t e r y r e p e r f u s i o n .
Acknowledgements The authors wish to thank Mine. 1. Fornieles for her expert technical assistance and Mine. A.M. Bramonte for typing the manuscript.
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