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(Circulation. 1997;96:1953-1963.)
© 1997 American Heart Association, Inc.

Articles

Amelioration of Ischemia- and Reperfusion-Induced Myocardial Injury by 17ß-Estradiol

Role of Nitric Oxide and Calcium-Activated Potassium Channels

Koichi Node, MD; Masafumi Kitakaze, MD; Hiroaki Kosaka, MD; Tetsuo Minamino, MD; Hiroharu Funaya, MD; ; Masatsugu Hori, MD

From the First Department of Medicine and the First Department of Physiology (H.K.), Osaka University School of Medicine, Osaka, Japan.

Correspondence to Masafumi Kitakaze, MD, PhD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Japan. E-mail kitakaze{at}medone.med.osaka-u.ac.jp

	Abstract

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Background 17ß-Estradiol increases the production of nitric oxide (NO) and prostacyclin and opens Ca²⁺-activated K⁺ (K_Ca) channels. Whether these effects of 17ß-estradiol reduce infarct size and the incidence of ventricular arrhythmia was investigated in dogs subjected to myocardial ischemia and reperfusion.

Methods and Results Infarct size was measured in open-chest dogs after 90 minutes' occlusion of the left anterior descending coronary artery and a subsequent 6 hours of reperfusion. Infusion of 17ß-estradiol into the coronary artery was initiated 10 minutes before coronary occlusion and continued until after 1 hour of reperfusion, with the exception of the occlusion period. The difference in NO concentration between coronary venous and arterial blood 10 minutes after the onset of reperfusion was significantly greater in dogs treated with 17ß-estradiol (10 ng · kg^-1 · min^-1) than in control animals. Infarct size (13.1±3.0% versus 43.7±5.4% of the area at risk) and the incidence of ventricular arrhythmia during ischemia and reperfusion periods were significantly reduced in the 17ß-estradiol group. Both N^G-nitro-L-arginine methyl ester (an inhibitor of NO synthase) and iberiotoxin (a blocker of K_Ca channels) reduced both the infarct size-limiting effect (infarct size, 29.3±3.0% and 31.7±2.1%, respectively) and the antiarrhythmic effect of 17ß-estradiol; indomethacin (an inhibitor of cyclooxygenase) did not attenuate the beneficial effects of 17ß-estradiol.

Conclusions 17ß-Estradiol reduced both myocardial infarct size and the occurrence of ischemia- and reperfusion-induced ventricular arrhythmias, which appear to be mediated by NO and the opening of K_Ca channels in canine hearts.

Key Words: myocardial infarction • endothelium-derived factors • ischemia • reperfusion • hormones

	Introduction

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Estrogen replacement therapy reduces cardiovascular disease in postmenopausal women.¹ Such therapy may inhibit the growth of atherosclerotic plaques² and may reduce the occurrence of transient myocardial ischemia by modulating vasomotion in atherosclerotic coronary arteries.³ Estrogen increases the production of NO,⁴ and the biological effects of endogenous NO released during ischemia and reperfusion may result in the reduction of myocardial injury.⁵ These effects of NO include an increase in CBF during ischemia and reperfusion,⁶ inhibition of platelet aggregation,⁷ reduction of catecholamine-induced increases in myocardial contractility,⁸ inhibition of catecholamine release,⁹ reduction of Ca²⁺ overload in ischemic and reperfused myocardium,¹⁰ and inhibition of neutrophil activation.^{5 11} Thus, it is likely that enhancement of NO production during ischemia and reperfusion by administration of estrogen may reduce ischemia and reperfusion injury. Furthermore, 17ß-estradiol enhances the ability to open K_Ca channels,^{12 13} resulting in hyperpolarization of the cell membrane potential and attenuating Ca²⁺ overload during ischemia and reperfusion. Therefore, we investigated the effects of 17ß-estradiol on myocardial infarct size and the occurrence of ischemia- and reperfusion-induced ventricular arrhythmia.

	Methods

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Instrumentation
Mongrel dogs (weight, 15 to 23 kg) were anesthetized with intravenous sodium pentobarbital (30 mg · kg^-1), intubated with a cuffed endotracheal tube, and ventilated with room air mixed with oxygen (1.5 L · min^-1) with the use of a respirator. A left thoracotomy was performed through the fifth intercostal space, and the heart was suspended in a pericardial cradle. After the intravenous administration of heparin (500 U · kg^-1), a proximal portion of the LAD was cannulated and perfused with blood through an extracorporeal tube from the left carotid artery, and heparin (500 U · kg^-1) was administered intravenously every 3 hours throughout the protocol. The occluder was attached to the bypass tube via a carotid-to-LAD shunt and manually clamped to the zero level of CBF to complete the occlusion. An electromagnetic flow probe (FF-050T; Nihon Kohden) was attached to the bypass tube for measurement of CBF. We connected the pressure-resistant tube from the proximal portion of the cannula to a multichannel recorder (Rm-6000; Nihon Kohden) and measured coronary perfusion pressure (CPP). The femoral artery was cannulated to obtain a reference blood flow sample for determination of the absolute value of the regional myocardial blood flow. The left atrium was cannulated for microsphere injection. For blood sampling, a small-caliber (1 mm), short (70 mm) tube was inserted into the epicardial vein at the center of the perfused area, and the drained coronary venous blood was returned to the jugular vein. Arterial blood was sampled from the femoral artery. A miniature pressure transducer (model P-5; Konigsberg) was inserted into the cavity of the left ventricle through the apex, and we measured left ventricular pressure and dP/dt. A pair of ultrasonic crystals (5 MHz, 2 mm in diameter; Schuessler) were implanted in the left ventricular anterior wall in the endomyocardial segment in the center of the perfused area to measure segment length. We calculated FS from the equation FS=[(EDL-ESL)/EDL]x100%, where EDL and ESL are end-diastolic and end-systolic segment lengths (in mm), respectively.

We observed ventricular arrhythmias at baseline, during 90 minutes of ischemia, and during the first 10 minutes of reperfusion. Chart recorder speed was 25 mm/s for all timed intervals. VPBs were defined and quantified in accordance with the Lambeth convention,¹⁴ except that couplets and salvos were not analyzed separately but were included in the analysis as a single VPB, and VT was defined as a run of four or more premature beats from the same focus at a rate higher than the basal sinus rate. All studies conformed to the position of the NIH in its Guide for the Care and Use of Laboratory Animals, adopted in November 1984.

Experimental Protocols
Protocol 1: Effects of 17ß-Estradiol on Infarct Size and Ischemia- and Reperfusion-Induced Ventricular Arrhythmia
After 10 minutes of hemodynamic stability, an infusion of either 17ß-estradiol (1, 10, or 100 ng · kg^-1 · min^-1) (Research Biomedical Institute), 17-estradiol (a physiologically inactive stereoisomer of 17ß-estradiol; 10 ng · kg^-1 · min^-1) (Sigma Chemical Co), or vehicle (2.5% [vol/vol] ethanol) (control group) was initiated into the bypass tube at a rate of 0.1 mL · kg^-1 · min^-1 10 minutes before coronary occlusion and, with the exception of the occlusion period, continued until after 1 hour of reperfusion (control group, n=8; 17ß-estradiol groups: 1 ng · kg^-1 · min^-1, n=7; 10 ng · kg^-1 · min^-1, n=8; and 100 ng · kg^-1 · min^-1, n=8; and 17-estradiol group, n=7). The procedure using 17-estradiol instead of 17ß-estradiol was performed to examine the nonspecific action of 17ß-estradiol. In a previous study,¹⁵ an intracoronary continuous infusion of 17ß-estradiol at 75 ng · min^-1 · body^-1 potentiated the coronary microvascular vasodilatory response to acetylcholine in postmenopausal women. Considering the dose used in the study by Gilligan et al, we sampled the blood from the coronary vein of the LAD-perfused area at the end of infusion of three doses of 17ß-estradiol into the LAD for 20 minutes and measured each estrogen level. The dose of 10 ng · kg^-1 · min^-1 of 17ß-estradiol increased the plasma estrogen levels to physiological levels of estrogen (300 pg · mL^-1, typical midcycle premenopausal values¹⁶ ) from 24 pg · mL^-1, and 100 ng · kg^-1 · min^-1 of this agent increased the supraphysiological levels (528±37 pg · mL^-1). Furthermore 1 ng · kg^-1 · min^-1 of 17ß-estradiol increased these levels to a point between premenopausal and postmenopausal levels (97±17 pg · mL^-1).

After 10 minutes' infusion, the coronary artery was occluded for 90 minutes and then reperfused for 6 hours. Coronary arterial and venous blood were sampled before 90 minutes of coronary occlusion and after 10 minutes of reperfusion for measurement of lactate, norepinephrine, and nitrate plus nitrite and for the blood gas analysis. DVA(NO) reflects the amount of NO released from the myocardium. We did not measure the myocardial metabolic state during the complete coronary occlusion because there is no forward blood flow into the LAD area, and coronary venous blood may in part reflect the metabolic state of the left coronary artery–perfused area via collateral vessels. Hemodynamic parameters were measured before sustained ischemia, 80 minutes after the onset of ischemia, and 10 minutes and 3 hours after the onset of reperfusion.

To mimic the adjunctive therapy in acute myocardial infarction used in clinical settings, we also injected 17ß-estradiol intravenously for 10 seconds from the left carotid vein 10 minutes before the onset of reperfusion as a dose of 100 µg · kg^-1 dissolved in 1 mL of vehicle (2.5% [vol/vol] ethanol) (17ß-estradiol [reperfusion, IV] group, n=9).

Protocol 2: The Role of NO, Prostacyclin, and K_Ca Channels in the Effects of 17ß-Estradiol on Infarct Size and Ischemia- and Reperfusion-Induced Arrhythmia
The effects of 17ß-estradiol were examined in dogs pretreated with L-NAME (an inhibitor of NO synthase), indomethacin (an inhibitor of cyclooxygenase), or IBTX (a blocker of the K_Ca channel). An infusion of either L-NAME (10 µg · kg^-1 · min^-1) (Sigma), indomethacin (10 µg · kg^-1 · min^-1) (Sigma), or IBTX (1 µg · kg^-1 · min^-1) (Research Biomedical Institute) into the bypass tube was initiated 10 minutes before infusion of 17ß-estradiol (10 ng · kg^-1 · min^-1) or vehicle and 20 minutes before the onset of coronary occlusion; with the exception of the 90-minute occlusion period, all infusions were continued until after 1 hour of the 6-hour reperfusion period (the 17ß-estradiol+L-NAME group, n=8; the 17ß-estradiol+indomethacin group, n=7; the 17ß-estradiol+IBTX group, n=8; and the 17ß-estradiol+L-NAME+IBTX group, n=8). We also determined the effect of L-NAME, indomethacin, IBTX, and L-NAME+IBTX alone on infarct size (the L-NAME group, n=7; the indomethacin group, n=7; the IBTX group, n=7; and the L-NAME+IBTX group, n=7). Each agent was administered 20 minutes before the onset of coronary occlusion and, with the exception of the 90-minute occlusion period, continued until after 1 hour of reperfusion. We previously showed that this dose of L-NAME abolished the release of NO during ischemia.⁶ Indomethacin treatment prevented the coronary dilatory effect of arachidonic acid (600 µg IC), demonstrating inhibition of cyclooxygenase. This dose of IBTX caused maximal reduction of bradykinin (20 ng · kg^-1 · min^-1 IC)-induced coronary vasodilation.¹⁷ Hemodynamic parameters were measured, and blood was sampled at the same time as in protocol 1.

Protocol 3: Measurement of cGMP
In another 24 dogs, we tested whether 17ß-estradiol increases cGMP content of the coronary artery in the ischemic myocardium at baseline in the control, 17ß-estradiol (10 ng · kg^-1 · min^-1), and 17ß-estradiol+L-NAME groups (n=6). Before coronary occlusion or 10 minutes after reperfusion, we rapidly removed the epicardial LAD (ischemic region) with precooled stainless steel scissors and tongs. We rapidly stored samples in liquid nitrogen.

Protocol 4: Effects of 17ß-Estradiol on Infarct Size and Arrhythmia in Chemically Denervated Hearts
Estrogen may increase norepinephrine uptake into nerve terminals,¹⁸ which may reduce local norepinephrine concentrations and decrease coronary vascular tone via attenuation of -adrenoceptor activity. To clarify the role of norepinephrine in the effects of 17ß-estradiol, we administered the 17ß-estradiol (10 ng · kg^-1 · min^-1) as in protocol 1 to dogs that had undergone chemical denervation (the denervation group [n=7] and the 17ß-estradiol+denervation group [n=7]). Hemodynamic parameters were measured and blood was sampled at the same time as in protocol 1. Systemic chemical sympathectomy was performed by intravenous injection of 6-hydroxydopamine (50 mg · kg^-1), administered in three fractional doses (10, 20, and 20 mg · kg^-1) over a 24-hour period 5 days before the experiment. The deleterious side effects of 6-hydroxydopamine were prevented by prior injection of propranolol (1 mg · kg^-1). In another group, 5 dogs were killed after the denervation procedures, and myocardial tissue from the perfused area was sampled for the measurement of norepinephrine. In other innervated dogs (n=5), we measured norepinephrine content of the left ventricular myocardial tissues.

Chemical Analysis
MO₂ (in mL · 100 g^-1 · min^-1) was calculated as the product of CBF (mL · 100 g^-1 · min^-1) and the coronary arteriovenous blood oxygen difference (mL · dL^-1). Lactate was measured by enzymatic assay, and the LER (lactate extraction ratio) was calculated by dividing the coronary arteriovenous difference in lactate concentration by the arterial lactate concentration and multiplying by 100%. NO, cGMP, and norepinephrine levels⁶ were measured by use of methods described previously.

Criteria for Exclusion
To ensure that all of the animals included in the analysis of infarct size data were healthy and exposed to similar extents of ischemia, we adopted the following criteria for exclusion of unsatisfactory dogs: (1) subendocardial collateral flow >15 mL · 100 g^-1 · min^-1; (2) heart rate >170 bpm; or (3) more than two consecutive attempts required to correct ventricular fibrillation with low-energy DC pulses applied directly to the heart. We calculated survival percentage as Number of Dogs That Survived/Number of Assigned Dogsx100.

Measurement of Infarct Size
After 6 hours of reperfusion, the LAD was reoccluded and perfused with autologous blood, and Evans blue dye was injected into a systemic vein to determine the anatomic area at risk and the nonischemic area in the heart. The heart was then removed immediately and sliced into serial transverse sections 6 to 7 mm in thickness. The nonischemic area was identified by blue stain, and the ischemic region was incubated at 37°C for 20 to 30 minutes in sodium phosphate buffer (pH 7.4) containing 1% TTC (Sigma). TTC stained the noninfarcted myocardium brick red, indicating the presence of a formazan precipitate formed as a result of reduction of TTC by dehydrogenase enzymes present in viable tissue. The extents of the area at risk and area of necrosis in each slice were then quantified by planimetry, corrected for the weight of the tissue slice, and summed for each heart. Infarct size was expressed as a percentage of infarct zone against the area at risk.

Measurement of Regional Myocardial Blood Flow
Regional myocardial blood flow was determined by the microsphere technique.¹⁹ Nonradioactive microspheres (Sekisui Plastic) made of inert plastic and labeled with bromine or zirconium (mean diameter, 15 µm; specific gravity, 1.34 and 1.36, respectively) were suspended in isotonic saline with 0.01% Tween 80 to prevent aggregation. The microspheres were sonicated for 5 minutes and then agitated with a vortex mix for 5 minutes immediately before injection of 1 mL of the suspension (2x10⁶ to 4x10⁶ microspheres) into the left atrium, followed by several warm (37°C) saline flushes (5 mL). Microspheres were administered 45 minutes after the onset of coronary occlusion. Just before (15 seconds) microsphere administration, a reference blood sample was withdrawn from the femoral artery at a constant rate of 8 mL · min^-1 for 2 minutes. We previously confirmed the homogeneity of injected microspheres as follows: (1) by microscopic examination, microspheres before the injection were isolated from each other and were homogenous; (2) when we used the different microspheres simultaneously, the endocardial flow values were identical; and (3) when we divided myocardial tissue into several parts and measured the endocardial flow of each part, the variation of data was within 6%. The x-ray fluorescence of the stable heavy elements was measured with a wavelength dispersive spectrometer (model PW 1480; Phillips), the specifications of which have been described in detail.¹⁹ Myocardial blood flow (mL · 100 g^-1 · min^-1) was calculated from tissue counts multiplied by reference flow and divided by reference counts. We measured the endocardial blood flow of the inner half of the left ventricular wall.

Statistical Analysis
Data are expressed as mean±SE. Statistical significance was assessed by ANOVA followed by Bonferroni test except that the effect of collateral blood flow on infarct size was analyzed by ANCOVA, with regional collateral flow in the inner half of the left ventricle wall as the covariant, and differences in the incidence of VT and ventricular fibrillation as well as in survival among groups were assessed with the Fisher-Irwin test (with Yates' correction factor). A value of P<.05 was considered statistically significant.

	Results

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Mortality and Exclusions
We excluded 5 dogs from data analysis because subendocardial collateral blood flow was >15 mL · 100 g^-1 · min^-1. No dogs were excluded because of a heart rate >170 bpm. At least one episode of ventricular fibrillation occurred in 29 dogs; ventricular fibrillation that matched the exclusion criterion occurred in 24 of these animals during the 90 minutes of ischemia or during reperfusion (Table 1).

View this table: [in this window] [in a new window]   Table 1. Number of Dogs Assigned to and Excluded From Each Group for Measurement of Infarct Size

Effects of 17ß-Estradiol on Infarct Size
Systolic/diastolic blood pressure (142/90 mm Hg) and heart rate (139 bpm) remained stable throughout the study. The denervated dogs exhibited significantly lower heart rates (114 bpm) throughout the study. CPP, CBF, FS, pH of coronary arterial and venous blood, norepinephrine concentrations in coronary arterial and venous blood, LER, and MO₂ did not differ significantly among the innervated dogs immediately before the onset of 90 minutes of ischemia. MO₂ in the denervated dogs was significantly lower than that in the innervated dogs (Table 2). Although no significant differences were observed in FS 80 minutes after the onset of coronary occlusion, FS in the 17ß-estradiol group was lower than in the control group (4.2±1.1% versus 7.9±1.8%; P<.01) after 10 minutes of reperfusion, and FS in the 17ß-estradiol group increased more than in the control group 3 hours after the onset of reperfusion. These effects were not apparent in the 17ß-estradiol+L-NAME group. Eighty minutes after the onset of complete coronary occlusion, CPP in the 17ß-estradiol group was lower than in the control group or the 17ß-estradiol+L-NAME group (33.2±1.4 versus 38.1±1.9 and 39.1±1.8 mm Hg, respectively; P<.05). Both VA(NO) (Fig 1A) and the cGMP content of the LAD (Fig 1B) were increased after 10 minutes of reperfusion relative to the baseline values in the control group; these increases were more marked in dogs treated with 17ß-estradiol but were not apparent in dogs administered both 17ß-estradiol and L-NAME, and both LER and pH of coronary venous blood in the 17ß-estradiol group were higher than in the control or 17ß-estradiol+L-NAME or 17ß-estradiol+IBTX groups, whereas the plasma norepinephrine level did not differ among the innervated groups 10 minutes after the onset of reperfusion.

View this table: [in this window] [in a new window]   Table 2. Coronary Hemodynamic and Myocardial Metabolic Parameters Immediately Before Prolonged Ischemia

View larger version (0K): [in this window] [in a new window]   Figure 1. A, VA(NO) after 10 minutes of reperfusion in control, 17ß-estradiol (10 ng · kg-1 · min-1), and 17ß-estradiol+L-NAME groups compared with the baseline value. *P<.01 vs baseline, **P<.01 vs control group. B, cGMP levels in the LAD at baseline or after 10 minutes of reperfusion in control, 17ß-estradiol (10 ng · kg-1 · min-1), and 17ß-estradiol+L-NAME groups compared with the baseline value. *P<.01 vs baseline, **P<.01 vs control group.

The area at risk (34%) and collateral blood flow (8.1 mL · 100 g^-1 · min^-1) were similar among all groups. 17ß-Estradiol at 1 ng · kg^-1 · min^-1 significantly reduced infarct size, which was more marked at a dose of 10 ng · kg^-1 · min^-1; the extent of infarct size reduction obtained with 17ß-estradiol at 100 ng · kg^-1 · min^-1 was similar to that apparent at 10 ng · kg^-1 · min^-1 (Fig 2). A bolus intravenous injection of 17ß-estradiol (100 µg · kg^-1) 10 minutes before reperfusion also reduced infarct size, but the effect was only 50% of that obtained by pretreatment with 17ß-estradiol at 10 ng · kg^-1 · min^-1. Norepinephrine contents of the denervated and innervated myocardium of the perfused area were 11±3 and 366±28 pg/mg tissue (n=5; P<.01), respectively. Furthermore, 17ß-estradiol (10 ng · kg^-1 · min^-1) limited infarct size in innervated and denervated dogs to approximately the same extents. The infarct size-limiting effect of 17ß-estradiol was reduced by either L-NAME or IBTX and abolished by L-NAME+IBTX. Indomethacin had no effect on the infarct size-limiting action of 17ß-estradiol. Similar results were obtained by plotting infarct size normalized by risk area against the collateral blood flow to the inner half of the LAD-dependent endomyocardium during sustained ischemia (Fig 3). There was a negative correlation between infarct size and endocardial collateral blood flow during ischemia (r=-.81, P<.01).

View larger version (0K): [in this window] [in a new window]   Figure 2. Infarct size as a percentage of the area at risk in the various experimental groups. Data from individual animals and mean±SE are shown. *P<.01, **P<.001 vs control group; P<.01 vs denervation group.

View larger version (0K): [in this window] [in a new window]   Figure 3. Plots of infarct size expressed as a percentage of the area at risk and regional collateral blood flow during ischemia for the various experimental groups. There is a negative correlation between infarct size and endocardial collateral blood flow during ischemia (r=-.81, P<.01).

Effects of 17ß-Estradiol on Ischemia- and Reperfusion-Induced Ventricular Arrhythmia
Pretreatment of dogs with 17ß-estradiol (10 or 100 ng · kg^-1 · min^-1) significantly reduced the overall incidence of ventricular fibrillation and VT during the 90 minutes of LAD occlusion and the first 10 minutes of reperfusion and significantly increased the survival rate in both innervated and denervated dogs (Table 3). These effects of 17ß-estradiol were abolished by L-NAME and IBTX administered alone or together but were still apparent in dogs treated with indomethacin.

View this table: [in this window] [in a new window]   Table 3. Percentage Survival and Incidence of Ventricular Fibrillation and VT in Each Group

Dogs pretreated with 17ß-estradiol at 10 ng · kg^-1 · min^-1 showed significantly fewer VPBs (Fig 4) and decreased VT (Fig 5) during both ischemia and reperfusion periods. At a dose of 1 ng · kg^-1 · min^-1, 17ß-estradiol significantly reduced the number of VPBs during ischemia but not during reperfusion. The effects of 17ß-estradiol at 100 ng · kg^-1 · min^-1 were similar to those at 10 ng · kg^-1 · min^-1. Bolus intravenous injection of 17ß-estradiol 10 minutes before reperfusion reduced the occurrences of both VPBs and VT during reperfusion. 17ß-Estradiol reduced VPBs and VT in both innervated and denervated dogs. The effects of 17ß-estradiol on ischemia-induced VPBs and VT were attenuated by L-NAME alone or in combination with IBTX, and both reperfusion-induced VPBs and VT were reduced by L-NAME and IBTX administered alone or together. Indomethacin did not affect the antiarrhythmic action of 17ß-estradiol during either ischemia or reperfusion.

View larger version (0K): [in this window] [in a new window]   Figure 4. The total number of VPBs during ischemia (A) and the first 10 minutes of reperfusion for the various experimental groups (B). Data are mean±SE. *P<.01 vs control group; **P<.01 vs control group; #P<.01 vs denervation group.

View larger version (0K): [in this window] [in a new window]   Figure 5. The total number of episodes of VT during ischemia (A) and the first 10 minutes of reperfusion (B). Data are mean±SE. *P<.05 vs control group; **P<.01 vs control group; #P<.01 vs denervation group.

	Discussion

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We have shown that 17ß-estradiol reduces myocardial infarct size and the incidence of ischemia- and reperfusion-induced arrhythmia in dogs and that these cardioprotective effects are blocked by inhibition of NO synthase and antagonism of K_Ca channels. These results suggest that augmentation of endogenous NO release and the opening of K_Ca channels induced by 17ß-estradiol contribute to the alleviation of irreversible ischemia-reperfusion injury. However, we must carefully consider other possible mechanisms for the cardioprotective effects of 17ß-estradiol. First, because estrogen possesses antioxidant properties,²⁰ 17ß-estradiol may reduce reperfusion injury by attenuating the generation of oxygen-derived free radicals, which may contribute to the overall pathophysiology of ischemia and reperfusion. In the present study, L-NAME and IBTX abolished the cardioprotective effects of 17ß-estradiol, suggesting that an antioxidant action of 17ß-estradiol does not contribute to these effects directly. Second, because estrogen may increase the uptake of extraneuronal norepinephrine,¹⁸ the resulting decrease in local norepinephrine concentration may reduce catecholamine-induced myocardial injury. However, in the present study, plasma norepinephrine concentrations did not differ at baseline and 10 minutes after the onset of reperfusion among all innervated groups. Furthermore, estrogen reduced infarct size and the number of VPBs and episodes of VT in the chemically denervated hearts to the same extent as in the innervated hearts, suggesting that these effects are not mediated by a reduction in norepinephrine concentration.

Cellular Mechanisms by Which 17ß-Estradiol Increases NO Release and the Opening of K_Ca Channels
It is not clear how 17ß-estradiol increases the release of NO in the reperfused heart. It is possible that estrogen activates constitutive NO synthase or modulates the function of muscarinic receptors on endothelial or smooth muscle cells; estrogen increases both the concentration of inositol triphosphate and the release of Ca²⁺ in these cells. It is also possible that the antioxidant properties of estrogen result in a prolongation of the half-life of NO. Han et al²¹ showed that 17ß-estradiol does not affect the concentration of either cAMP or cGMP and that the NO synthase inhibitor, N^G-monomethyl-L-arginine, has no effect on 17ß-estradiol–induced relaxation in porcine coronary artery. In the present study, although 17ß-estradiol did not increase NO release under nonischemic conditions (data not shown), it significantly increased NO release during reperfusion after coronary hypoperfusion. The apparent discrepancy between the study of Han et al²¹ and the present study may be attributable to the differences in species (pig versus dog), experimental preparation (coronary artery strips versus whole hearts), or conditions (normoxia versus ischemia).

17ß-Estradiol increases the activity of the large- conductance K_Ca channels in isolated rabbit endothelial cells^{12 13} as well as K⁺ conductance in guinea pig coronary arteries and cardiomyocytes.²² White et al²³ showed that estrogen relaxes porcine coronary arteries by opening K_Ca channels through a cGMP-dependent mechanism. In the present study, however, IBTX and L-NAME appeared to act in an additive manner in reversing the infarct size-limiting effect of 17ß-estradiol. The hyperpolarization of vascular smooth muscle cells elicited by endothelium-derived hyperpolarizing factor is mediated by an increase in the K⁺ conductance of the cell membrane that results from activation of K⁺ channels.²⁴ Endothelium-dependent hyperpolarization and subsequent vascular relaxation are inhibited by a blocker of K_Ca channels in coronary arteries.²⁴ Therefore, the activation of K_Ca channels appears to play an important role in coronary vasodilation in the ischemic myocardium.¹⁷ Estrogen may open the K_Ca channels of smooth muscle via endothelium-derived hyperpolarizing factor released from the endothelium.

Mechanisms of Infarct Size-Limiting Effects Mediated by NO and the Opening of K_Ca Channels
Potentiation of NO release may be an effective pharmacological intervention to limit myocardial infarct size, given that administration of an NO donor markedly attenuates ischemia-reperfusion injury.^{5 11} Several possible mechanisms may underlie the beneficial effects of NO on infarct size. First, because NO regulates the membrane Ca²⁺ current of cardiac cells,²⁵ it may attenuate the severity of ischemia by reducing the cytosolic accumulation of Ca²⁺. Second, NO may also reduce MO₂⁸ as a result of a direct negative inotropic effect, as well as reduce the increase in ATP generation by stimulation of glycolysis²⁶ during early reperfusion. However, during the coronary occlusion, the negative inotropic effect of increased NO by 17ß-estradiol seemed to be masked by severe myocardial metabolic and contractile dysfunction. Third, in addition to the energy-sparing effects of NO on the myocardium during early reperfusion, cGMP-mediated coronary vasodilation may help to reduce myocardial ischemia. 17ß-Estradiol alone decreased CPP during complete coronary occlusion compared with either the control group or the 17ß-estradiol+L-NAME group, suggesting that increased NO relaxed coronary vessels distal to the occluded site and decreased perfusion pressure. Fourth, NO may reduce oxygen-derived free radical generation by decreasing lipolysis (thereby limiting the generation of radicals through lipid peroxidation). Furthermore, NO also inhibits platelet aggregation in the ischemic heart.⁷

The opening of K_Ca channels may hyperpolarize the cellular membrane and reduce Ca²⁺ overload during ischemia and reperfusion, similar to the protective effect of ATP-sensitive K⁺ channels,²⁷ as well as its coronary vasodilatory effect.¹⁷

Mechanisms of Antiarrhythmic Effects Mediated by NO and the Opening of K_Ca Channels
Estrogen has previously been shown to exert an antiarrhythmic action in the dog,²⁸ and the present results demonstrate that both NO and the opening of K_Ca channels contribute to this action. Increasing the concentration of cGMP in the myocardium reduces susceptibility to ventricular fibrillation and VPBs.^{29 30} Whereas the increases in cytosolic Ca²⁺ concentration can provoke oscillatory afterdepolarization of the cardiac cell membrane, which can trigger spontaneous and sustained extrasystoles,³¹ increases in myocardial cGMP may inhibit Ca²⁺ influx through L-type channels in the sarcolemma and reduce Ca²⁺ overload during ischemia and reperfusion. Therefore, cGMP may prevent afterdepolarization and the resulting arrhythmias by reducing the cytosolic Ca²⁺ concentration. In addition, cGMP reduces myocardial contractility and may improve the ratio between myocardial oxygen supply and demand, resulting in an energy-sparing effect during reperfusion. Furthermore, stimulation of cGMP-dependent cAMP phosphodiesterase should decrease myocardial cAMP concentrations³⁰ ; increased cAMP concentrations have been implicated in arrhythmogenesis in response to cardiac sympathetic nerve activation.

The precise mechanism by which the opening of K_Ca channels reduces only reperfusion-induced ventricular arrhythmias is not known. Whereas the opening of K_Ca channels may reduce the duration of action potential during coronary occlusion, resulting in an arrhythmogenic effect, the opening of K_Ca channels reduces Ca²⁺ overload in cardiomyocytes as a result of a hyperpolarized membrane potential or increased CBF, leading to a reduction in the total number of ventricular arrhythmias during the reperfusion period.

We used both male and female dogs in the present study. There was no statistically significant difference in the effect of 17ß-estradiol on ischemia-reperfusion injury between male and female dogs; however, we could not draw further conclusions concerning the sex difference because of insufficient number of data. All dogs (four male, four female) in the group treated with 17ß-estradiol at 10 ng · kg^-1 · min^-1 survived after the reperfusion and exhibited less severe myocardial necrosis and arrhythmias, regardless of sex. Sudhir et al³² showed that an estrogen receptor antagonist did not significantly inhibit estrogen-induced vasodilation in canine coronary arteries. Therefore, it is not clear whether the beneficial effects of estrogen are receptor mediated. Estrogen is likely to induce the direct and nongenomic effects through membrane receptors that differ from the classic intracellular estrogen receptor. Steroid responses mediated by the superfamily of intracellular receptors occur with a latency of 1 to 2 hours. Thus, the rapid time course of the beneficial action of 17ß-estradiol in the present study suggests that it is not mediated by the classic nuclear estrogen receptors.

	Selected Abbreviations and Acronyms

 

CBF = coronary blood flow CPP = coronary perfusion pressure FS = fractional shortening IBTX = iberiotoxin KCa channel = Ca2+-activated potassium channel LAD = left anterior descending coronary artery L-NAME = NG-nitro-L-arginine methyl ester MO2 = myocardial oxygen consumption NO = nitric oxide TTC = tetraphenyl tetrazolium chloride VA(NO) = difference in total nitrate plus nitrite concentration between coronary venous and arterial blood VPB = ventricular premature beat VT = ventricular tachycardia

	Acknowledgments

 
We thank Kayoko Yoshida, Yukiyo Nomura, and Makoto Hasegawa for technical assistance.

Received February 3, 1997; revision received April 14, 1997; accepted April 18, 1997.

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