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Articles in PresS. Am J Physiol Heart Circ Physiol (September 30, 2004). doi:10.1152/ajpheart.00429.2004 Contribution of Endothelin to Coronary Vasomotor Tone is Abolished after Myocardial Infarction 1 Daphne Merkus PhD, 1Birgit Houweling BSc, 2Anton H. van den Meiracker MD PhD, 2 Frans Boomsma PhD, and 1Dirk J. Duncker MD PhD 1 Experimental Cardiology, Thoraxcenter, and 2Internal Medicine, Cardiovascular Research School Rotterdam,Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Running title: Endothelin and coronary tone after myocardial infarction Word count: Title Page 96 Abstract 279 Text 3774 References 1035 Figure Legends 354 Corresponding author: Daphne Merkus Experimental Cardiology, Thoraxcenter Erasmus MC, University Medical Center Rotterdam Box 1738 3000DR Rotterdam phone:+31-10-4088025 fax:+31-10-4089494 email: [email protected] Copyright © 2004 by the American Physiological Society. Endothelin and coronary tone after myocardial infarction 2 ABSTRACT Merkus Daphne, Birgit Houweling, Anton H. van den Meiracker, Frans Boomsma, and Dirk J. Duncker. Contribution of Endothelin to Coronary Vasomotor Tone is Abolished after Myocardial Infarction. Am J Physiol Heart Circ Physiol 000:H0000-H0000, 0000. – Left ventricular dysfunction in swine with a recent myocardial infarction (MI) is associated with neurohumoral activation, including increased catecholamines and endothelin (ET). Although the increase in ET may serve to maintain blood pressure and hence perfusion of essential organs like the heart and brain, it could also compromise myocardial perfusion, by evoking coronary vasoconstriction. In the present study, we tested the hypothesis that endogenous ET contributes to the perturbations in myocardial O2 balance that occur during exercise in remodeled myocardium of swine with a recent MI. For this purpose, 23 chronically instrumented swine (8 with and 15 without MI) were studied at rest and while running on a treadmill at 1-4 km/h. Plasma ET levels increased after MI from 3.2±0.4 pM to 4.9±0.3 pM (P<0.05). In normal swine, blockade of ETA (EMD122946) or ETA/ETB (tezosentan) receptors resulted in an increase in coronary venous O2 tension i.e. in coronary vasodilation at rest that decreased during exercise. In contrast, neither ETA nor ETA/ETB blockade resulted in coronary vasodilation in swine with MI. Coronary vasoconstriction to intravenous ET-1 infusion in awake resting swine was blunted after MI. To investigate whether factors released by cardiac myocytes contributed to the decreased vascular responsiveness to ET, we performed ET-1 dose-response curves in isolated coronary arterioles (70-200 µm). Vasoconstriction to ET-1 in the isolated arterioles from MI swine was enhanced. In conclusion, the vasoconstrictor influence of endogenous as well as exogenous ET on the coronary circulation in vivo is reduced. Since the response of isolated coronary arterioles to ET is increased after MI, the reduced vasoconstrictor influence in vivo suggests modulation of ET receptor sensitivity by cardiac myocytes, which may serve to maintain adequate myocardial perfusion. coronary microcirculation; coronary blood flow; endothelin; myocardial oxygen balance; left ventricular dysfunction Endothelin and coronary tone after myocardial infarction 3 INTRODUCTION Congestive heart failure is the only major cardiovascular disorder of which the incidence has increased over the past decade, which is principally due to a reduction in mortality associated with acute myocardial infarction (MI). Consequently, MI is becoming an increasingly important risk factor for the development of congestive heart failure (2). The loss of viable myocardial tissue and the consequent left ventricular dysfunction results in neurohumoral activation which, in conjunction with altered mechanical loading conditions of the left ventricle, initiate the process of left ventricular remodeling (consisting of left ventricular hypertrophy and dilation). Although these adaptations are aimed at restoring cardiac pump performance, left ventricular remodeling has been shown to be an independent risk factor for later development of congestive heart failure (2). The mechanisms that contribute to the progression from mild left ventricular dysfunction to overt congestive heart failure are still incompletely understood, but could involve an impaired supply of O2 to the hypertrophied myocardium. This concept is supported by in vivo studies in rats (9, 10) and pigs (30) that have shown a reduction in coronary flow reserve of up to 35% in the surviving post-infarct LV myocardium, three to eight weeks after infarction. Furthermore, hemodynamic and neurohumoral abnormalities associated with LV dysfunction are exacerbated during exercise (7), which is accompanied by mild perturbations in the myocardial O2-balance as O2 supply fails to match the increased O2 demand (7). A distinct feature of swine with a recent MI, is an elevation of circulating endothelin (ET) levels at rest and during treadmill exercise (7). Although an increase in ET levels may, on the one hand, aid in maintaining central aortic blood pressure (and hence coronary perfusion pressure) it may, on the other hand, compromise myocardial O2 supply by increasing coronary vascular tone. The aim of the present study was therefore to test the hypothesis that the increased plasma levels of ET in swine with a recent MI exert an increased vasoconstrictor influence on the coronary circulation, thereby contributing to the perturbation of myocardial O2 supply. For this purpose, swine were chronically instrumented, subjected to permanent ligation of the left circumflex coronary artery or a sham procedure, and studied two weeks later while running on a treadmill. Since we paradoxically found a decreased vasoconstrictor influence of endogenous ET on the coronary vasculature in vivo, we further investigated if the blunted vasoconstrictor influence was due to a reduced local release of endothelin or to a reduced responsiveness of the coronary vasculature by infusing exogenous ET in vivo. Again, the vasoconstriction was blunted after MI. To investigate if the reduced responsiveness was due to Endothelin and coronary tone after myocardial infarction 4 factors secreted by cardiac myocytes, we also studied the responsiveness of isolated coronary arterioles to ET. METHODS Studies were performed in accordance with the “Guiding Principles in the Care and Use of Laboratory Animals” as approved by the Council of the American Physiological Society, and with approval of the Animal Care Committee of the Erasmus MC Rotterdam. Forty crossbred Landrace x Yorkshire swine of either sex (2-3 months old) entered the study. Surgical procedures Thirty-two swine (22±1 kg at the time of surgery) were sedated (20 mg/kg ketamine and 1 mg/kg midazolam, im), anesthetized (thiopental 15 mg/kg, iv), intubated and ventilated with a mixture of O2 and N2O (1:2) to which 0.2-1.0% (v/v) isoflurane was added (3, 25). Anesthesia was maintained with midazolam (2 mg/kg + 1 mg/kg/h, iv;) and fentanyl (10 µg/kg/h, iv). Swine were instrumented under sterile conditions as previously described (3, 25). Briefly, a thoracotomy was performed in the fourth left intercostal space. Subsequently, a polyvinylchloride catheter was inserted into the aortic arch, for the measurement of mean aortic pressure and blood sampling for the determination of PO2, PCO2, pH (ABL 505, Radiometer), O2-saturation and hemoglobin concentration (OSM2, Radiometer). A fluid filled catheter and a high fidelity Konigsberg pressure transducer was inserted into the left ventricle (LV) via the apex. Fluid filled catheters were also implanted into the left atrium for pressure measurements and in the pulmonary artery for infusion of drugs. A small angiocatheter was inserted into the anterior interventricular vein for coronary venous blood sampling. Finally, a transit-time flow probe (Transonic Systems) was placed around the left anterior descending coronary artery (18). In all 32 swine the proximal part of the left coronary circumflex artery (LCx) was exposed, but only in 16 animals the LCx was permanently occluded with a silk suture to produce a MI (7). Three MI swine died during surgery due to recurrent fibrillation. Electrical wires and catheters were tunneled subcutaneously to the back, the chest was closed and animals were allowed to recover. Endothelin and coronary tone after myocardial infarction 5 Animals received analgesia (0.3 mg buprenorphine im, for 2 days) and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin iv, for 5 days). Three MI swine died overnight following surgery. Exercise protocols Studies were performed approximately 2 weeks after surgery. After hemodynamic measurements (lying and standing), blood samples (lying), and rectal temperature (standing) had been obtained, swine were subjected to a four-stage exercise protocol on a motor driven treadmill (1-4 km/h). Hemodynamic variables were continuously recorded and blood samples collected during the last 60 s of each 3 min exercise stage, at a time when hemodynamics had reached a steady state. After 90 minutes of rest, the mixed ETA/ETB antagonist tezosentan (a gift from Dr Clozel, Actelion Pharmaceuticals Ltd, Allschwil, Switzerland), was administered to 14 normal and 8 MI swine in a dose of 3 mg/kg followed by an infusion of 6 mg/kg/h iv (18) and the exercise protocol was repeated. Tezosentan has a pA2 of 9.5 for ETA and a pA2 of 7.7 for ETB receptors, indicating only a 63-fold selectivity for ETA compared to ETB receptors (1, 26). On a different day, the ETA receptor antagonist EMD122946 (a gift from Prof. Schelling, E. Merck Darmstadt, Darmstadt, Germany) was administered to 10 normal and 8 MI swine in a dose of 3 mg/kg iv (18) and the exercise protocol was repeated. EMD122946 has a pA2 of 9.5 for ETA and a pA2 of 6.0 for ETB receptors, indicating a 3200fold selectivity for ETA compared to ETB receptors (15). The dose of EMD122946 administered in the current study does not block ETB receptors as we have previously found that ET-plasma concentrations are not influenced by this dose of EMD122946, whereas tezosentan in the current dose does increase plasma ET levels, indicating that the ETB receptor, which is responsible for ETclearance is blocked by tezosentan, but not by EMD122946 (18). We have previously shown excellent reproducibility of the hemodynamic response in two consecutive bouts of exercise in both normal and MI swine (3, 7). Digital recording and off-line analysis of hemodynamic data and computation of myocardial VO2 have been described in detail elsewhere (4, 25). Myocardial O2 delivery (MDO2) was computed as the product of LAD coronary blood flow and arterial blood O2 content. Myocardial O2 consumption Endothelin and coronary tone after myocardial infarction 6 (MVO2) in the region of myocardium perfused by the left anterior descending coronary artery was calculated as the product of coronary blood flow and the difference in O2 content between arterial and coronary venous blood. Myocardial O2 extraction (MEO2) was computed as the ratio of MVO2 and MDO2. Determination of plasma levels of ET In 7 normal and 6 MI swine, arterial and coronary venous blood samples (5 ml) were collected at rest (lying) and at 2 and 4 km/h in the control exercise protocol and kept on ice until the end of the exercise trial. Then the blood samples were spun down and plasma was stored at -80°C. Plasma levels of ET-like immuno-reactivity were determined using a radio-immuno assay from Euro-Diagnostica (Malmö, Sweden), which has a cross reactivity of 100% toward ET-1, 48% toward ET-2 and 109% toward ET-3. Since production of ET-2 and ET-3 appears to be absent in the cardiovascular system of the pig (11), the concentrations measured with the radio-immuno assay most likely represents ET-1. In vivo responsiveness of the coronary vasculature to exogenous ET-1 To study the responsiveness of the coronary vasculature to ET in vivo, ET-1 (Sigma) was infused iv in doses of 10, 20 and 40 pmol/kg/min in chronically instrumented normal (n=3) and MI (n=3) swine under awake resting conditions. Changes in coronary venous PO2 were used as index of coronary vasoconstriction. Responsiveness of isolated coronary arterioles to ET-1 For the study of isolated coronary arterioles, 8 additional swine were sedated and anesthetized as described above, followed by a thoracotomy under sterile conditions. In these animals, the pericardium was opened, the LCx was exposed and in 3 swine the LCx was permanently ligated. Then the pericardium was closed, to minimize inflammation, followed by surgical closure of the chest and the animals were allowed to recover. Three weeks after induction of MI or sham operation, swine were sedated (20 mg/kg ketamine and 1 mg/kg midazolam, im), anesthetized (pentobarbital 15-20 mg/kg, iv), intubated and ventilated with a mixture (1:2) of O2 and N2O (28). Anesthesia was maintained with pentobarbital (15-20 Endothelin and coronary tone after myocardial infarction 7 mg/kg/h). The thorax was opened by midline-sternotomy, and the heart fibrillated and instantaneously excised. Single arterioles (70-200 µm passive diameter) were dissected from the left ventricular free wall, as previously described (13, 17, 19). The heart was placed in ice-cold physiological saline solution (PSS) of the following composition (in mM): NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02 and 3-N-morpholinopropanesulfonic acid (MOPS) 3.0, buffered to pH 7.4 at 4°C and filtered (dissection buffer). The heart was placed under a dissection microscope in a 4°C chamber and vessels were carefully dissected free from the surrounding myocardial tissue and placed in dissection buffer containing 1% bovine serum albumin (USB-Amersham). The vessels were cannulated on both ends with micropipettes (approximately 20-80 µm outer diameter, depending on the size of the vessel) connected to pressurized reservoirs filled with PSS buffered at pH 7.4 at 37°C, using a pressuremyograph system (Danish Myotechnology). Pressure in the reservoirs was set to obtain an intraluminal pressure of 60 mmHg. Vessels that failed to maintain pressure were excluded from analysis. Vessels were visualized on an inverted microscope. Images were digitized using a CCD camera and diameter of the arterioles was measured. The vessel was slowly warmed up to 37°C and allowed to develop spontaneous tone. ET-1 (Sigma) was added in cumulative steps (5 minutes per concentration) to the vessels in concentrations ranging from 10 pM to 5 nM and their response measured. Vascular diameters were normalized to the diameter with tone, prior to administration of the ET-1. Statistical analysis Statistical analysis of hemodynamic data and ET plasma levels was performed using three-way (MI, drug treatment and exercise) or two-way (MI and exercise) analysis of variance (ANOVA) for repeated measures, as appropriate. When significant effects were detected, post-hoc testing for the effects exercise, drug treatment and MI was performed using Scheffe's test. To test for the effects of MI and drug treatment (EMD122946 or tezosentan) on the relation between MVO2 and coronary venous O2 tension, saturation or content, regression analysis was performed using MI, drug treatment Endothelin and coronary tone after myocardial infarction 8 and MVO2 as well as their interaction as independent variables and assigning a dummy variable to each animal. Similarly, regression analysis was used to detect differences between normal and MI swine in the ET-1 induced vasoconstriction of the coronary vasculature in vivo and in vitro using ETdose and infarct as independent variables. Statistical significance was accepted when P≤ 0.05. Data are presented as mean±SEM. RESULTS Hemodynamics Effect of MI. In accordance with previous reports from our laboratory (7, 8), MI had no effect on mean aortic blood pressure either at rest or during exercise (Table 1) but resulted in LV dysfunction as evidenced by a rightward shift of the relation between heart rate and LVdP/dtmax or LV systolic pressure as well as the markedly elevated left atrial pressures (Figure 1). Plasma levels of ET were elevated in MI compared to normal swine both at rest and during exercise. However, there were no differences between plasma ET in arterial and coronary venous blood (Table 2). Mixed ETA/ETB receptor blockade. The mixed ETA/ETB receptor antagonist tezosentan decreased mean aortic pressure to a similar extent in MI and normal swine, which was accompanied by small increments in heart rate, particularly in normal animals (Table 1). Tezosentan had no significant effect on any of the other hemodynamic variables in either group of animals. ETA receptor blockade. The ETA receptor antagonist EMD122946 decreased mean aortic pressure in both MI and normal swine, which was similar to the decrease in blood pressure by tezosentan (Table 3). Similarly, EMD122946, had no significant effects on any of the other hemodynamic variables in either group of animals. Myocardial oxygen balance Effect of MI. In normal swine, the exercise-induced increases in MVO2 were met by commensurate increases in coronary blood flow (Table 4) and hence in MDO2, so that MEO2, and CVSO2 and CVPO2 were maintained constant (Figure 1). Proximal LCx occlusion in swine results in Endothelin and coronary tone after myocardial infarction 9 myocardial infarction of ~20% of the LV myocardium. Despite this loss of viable myocardial tissue, the LV weight to body weight ratio in MI swine (3.8±0.2 g/kg) was slightly higher than in normal swine (3.2±0.1 g/kg, P<0.05), reflecting significant hypertrophy of surviving myocardium. In MI swine, resting coronary blood flow and MVO2 in the remote surviving myocardium of the LAD perfused area were larger than in normal swine, likely due to the higher heart rate in conjunction with the LV hypertrophy. At rest, MEO2 in MI and normal swine were similar (Figure 1). In contrast, during exercise MEO2 increased in MI but not in normal swine (P<0.05), indicating that the increase in MDO2 during exercise did not completely match the increase in MVO2 (Table 4). Consequently, CVSO2 and CVPO2 decreased during exercise in MI swine (P<0.05 vs normal swine). These findings indicate that the exercise-induced coronary vasodilation was slightly blunted in MI compared to normal swine. Mixed ETA/ETB receptor blockade. In normal resting swine, tezosentan had no effect on arterial oxygen levels, but resulted in increases in coronary blood flow and Hb (Table 4), and hence MDO2 from 262±24 to 325±34 µmol O2/min (P<0.05). Also, MVO2 increased slightly after administration of tezosentan, likely as the result of the increased heart rate. However, inspection of Figure 2 (upper panels) shows that the tezosentan-induced vasodilation and the commensurate increased oxygen delivery slightly exceeded the increase in MVO2, allowing MEO2 to decrease and consequently CVSO2 and CVPO2 (and hence CVCONT O2) to increase. These changes reflect a direct coronary vasodilator effect of tezosentan independent of myocardial metabolic demand. This vasodilator effect waned gradually at higher levels of treadmill exercise. In contrast, mixed ETA/ETB receptor blockade did not affect myocardial O2 balance in MI swine (Figure 2, bottom panels). Thus, despite the increased plasma levels of ET, the vasoconstrictor influence of ET in the coronary microcirculation was lost after MI. ETA receptor blockade. The effects of EMD122946 on the myocardial O2 balance in normal swine were only slightly greater than the effects of tezosentan (Figure 3, top panels and Table 5), indicating that in the coronary resistance vessels of normal swine, ET exerts its vasomotor actions principally via ETA receptors. Similar to tezosentan, EMD122946 had no effect on myocardial O2 Endothelin and coronary tone after myocardial infarction 10 balance in MI swine (Figure 3, bottom panels), indicating that in coronary resistance vessels within remodeled myocardium of MI swine the ETA mediated vasoconstrictor influence is lost. Responsiveness of the coronary vasculature to exogenous ET-1 To assess if the reduced effect of ET-receptor blockade after myocardial infarction was due to a decreased ET-production or to a reduced coronary vascular responsiveness, ET-1 was infused and the vasoconstriction measured. Administration of ET-1 caused dose-dependent coronary vasoconstriction as evidenced by the decreased CVPO2 in both normal and MI swine (Figure 4). However, the coronary vasoconstriction was blunted in MI compared to normal swine indicating reduced vascular responsiveness to ET in vivo. Endothelin-1 sensitivity in isolated coronary arterioles To assess if the reduced vasoconstrictor influence of endogenous as well as exogenous ET in vivo was the result of factors secreted by cardiac myocytes that may have altered ET receptor sensitivity, the response of isolated coronary arterioles of swine with and without MI to ET was measured. As shown in Figure 4, the coronary arterioles isolated from remodeled myocardium of MI animals paradoxically demonstrated significantly greater vasoconstriction in response to ET than the arterioles from normal swine. DISCUSSION The main finding of the present study is that the vasoconstrictor influence of endogenous and exogenous ET on coronary resistance vessels, which is principally mediated via ETA receptors, is abolished after MI despite increased plasma ET-levels. The decreased coronary microvascular responsiveness to ET is likely due to factors secreted by cardiac myocytes that modify ET-receptor sensitivity, as the vasoconstrictor response of isolated coronary arterioles to ET is increased after MI. The implications of these findings are discussed below. Endothelin and coronary tone after myocardial infarction 11 Myocardial O2 balance and coronary resistance vessel tone Under basal resting conditions, the heart is characterized by a high level (80%) of myocardial O2 extraction (4, 5). Consequently, the ability of the coronary resistance vessels to dilate in response to increments in myocardial O2 demand is extremely important to maintain an adequate O2 supply. A sensitive way to study alterations in coronary vascular tone in relation to myocardial metabolism is the relationship between coronary venous O2 levels and myocardial O2 consumption (7, 27). Thus, an increase in coronary resistance vessel tone will limit CBF and hence myocardial O2 supply at a given level of myocardial O2 consumption, forcing the myocardium to increase its O2 extraction (in order to meet myocardial O2 demand), which results in a lower coronary venous O2 level. Conversely, a decrease in resistance vessel tone increases myocardial O2 supply at a given level of myocardial O2 consumption resulting in an increased coronary venous O2 level. The coronary venous O2 level thus represents an index of myocardial tissue oxygenation (i.e. the balance between myocardial O2 supply and O2 demand), which is determined by the coronary resistance vessel tone. Myocardial O2 balance in remodeled myocardium Myocardial dysfunction due to MI results in the loss of viable pump tissue and compensatory left ventricular remodeling and neurohumoral activation. The remaining viable tissue hypertrophies and heart rate increases to compensate for the decreased stroke volume (7). These adaptations result in an increased myocardial O2 consumption and thus require additional coronary blood flow. Yet, coronary blood flow is impeded by insufficient growth of the coronary microvasculature in conjunction with increased extravascular compressive forces, due to the increased heart rate and left ventricular filling pressures (7). The additional vasodilation that is required to meet the increased O2 demand of the myocardium and overcome the augmented impediment of coronary blood flow, results in a reduction in adenosine-recruitable flow reserve (9, 10, 30). Moreover, during exercise, when extravascular compressive forces increase further, the recruitment of vasodilator reserve in the remodelled heart is apparently not sufficient, thereby forcing the heart to increase its O2 extraction from the blood, and resulting in decreased coronary venous O2 levels (7). In view of the neurohumoral activation (7), we Endothelin and coronary tone after myocardial infarction 12 investigated in the present study whether the increased circulating levels of endothelin contributed to the perturbations of myocardial O2 balance. Altered contribution of endothelin to coronary vasomotor control after myocardial infarction Despite the increased plasma levels of endogenous ET, its vasoconstrictor influence on the coronary circulation was reduced. To determine whether this was the result of blunted receptor responsiveness or reduced local ET-production, we studied the vasoconstriction induced by exogenous ET in vivo. The coronary vasoconstrictor influence to exogenous ET-1 in vivo was also reduced, indicating a reduced coronary vascular responsiveness to ET. Paradoxically, a recent study showed that ischemic heart disease results in upregulation of ETA and ETB receptor mRNA in human coronary arteries (29). This is in accordance with our measurements in isolated coronary arterioles obtained from shamoperated swine and swine with a MI, which showed that the ET responsiveness in vessels from animals with a MI was actually increased. The discrepancy between the in vivo and the in vitro findings suggests that ET receptor sensitivity is modulated in vivo, and that this modulation is lost in vitro. A possible modulator of ET-receptors is adenosine, which has been shown to desensitize ET-receptors on the coronary vasculature (19). Since adenosine production by cardiac myocytes may be increased in the hypertrophied myocardium, due to mild underperfusion that occurs during exercise in especially the subendocardium (7), this may have resulted in desensitization of ET-receptors in MI, but not normal swine. The dissection of the isolated coronary arterioles results in removal of the cardiac myocytes and thereby in loss of their, possibly adenosine-mediated, modulation of the ET-receptors. The increased plasma ET-levels after MI in combination with the potent vasoconstrictor properties of ET, as well as its possible role as a growth factor in myocardial remodeling, have provoked both experimental and clinical studies using ET-receptor antagonists to improve outcome after MI. However, although plasma ET-levels are inversely correlated with myocardial function as well as survival after MI (23, 24) and while some studies suggest that especially ETA receptor antagonists improve cardiac function after MI (21, 23, 24), other studies are less unequivocal. For example, MacCarthy et al found that intracoronary administration of an ETA antagonist reduced Endothelin and coronary tone after myocardial infarction 13 myocardial contractility in normal but not failing human hearts, indicating that in this study, the cardiac effects of ET were reduced in the failing heart (14). In contrast, in failing rat hearts, the ET protein levels were increased and ETA receptor antagonism did result in decreased function in the failing but not the normal hearts (22). This is in accordance with increased ETA receptor expression in the failing human (31) as well as rat (12) heart. In the present study, we did not find any effects of the ET antagonists on myocardial function in either the normal heart or in the presence of myocardial dysfunction due to MI. These findings, in conjunction with the loss of vasoconstrictor influence of ET, may explain in part why clinical trials of ET-receptor antagonists in heart failure have failed to show therapeutic value of these compounds (6, 20). Conclusion In the normal heart, coronary vascular tone is regulated by a myriad of vasodilators and vasoconstrictors to ensure adequate myocardial perfusion (5, 16, 27). Previously, we showed that waning of a ETA- mediated vasoconstrictor influence aided in exercise-induced coronary vasodilation in normal swine (17, 18). The present study shows that when additional vasodilation is required in the hypertrophied myocardium after MI, withdrawal of the ET-mediated vasoconstrictor influence contributes to a shift in vasomotor tone towards vasodilation. 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Tune JD, Richmond KN, Gorman MW, and Feigl EO. Control of coronary blood flow during exercise. Exp Biol Med (Maywood) 227: 238-250, 2002. van Kats JP, Duncker DJ, Haitsma DB, Schuijt MP, Niebuur R, Stubenitsky R, Boomsma F, Schalekamp MA, Verdouw PD, and Danser AH. Angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs after myocardial infarction: role of tissue angiotensin II. Circulation 102: 1556-1563, 2000. Wackenfors A, Emilson M, Ingemansson R, Hortobagyi T, Szok D, Tajti J, Vecsei L, Edvinsson L, and Malmsjo M. Ischemic heart disease induce upregulation of endothelin receptor mRNA in human coronary arteries. Eur J Pharmacol 484: 103-109, 2004. Zhang J, Wilke N, Wang Y, Zhang Y, Wang C, Eijgelshoven MH, Cho YK, Murakami Y, Ugurbil K, Bache RJ, and From AH. Functional and bioenergetic consequences of postinfarction left ventricular remodeling in a new porcine model. MRI and 31 P-MRS study. Circulation 94: 1089-1100, 1996. Zolk O, Quattek J, Sitzler G, Schrader T, Nickenig G, Schnabel P, Shimada K, Takahashi M, and Bohm M. Expression of endothelin-1, endothelin-converting enzyme, and endothelin receptors in chronic heart failure. Circulation 99: 2118-2123, 1999. Endothelin and coronary tone after myocardial infarction 16 Legends Fig. 1. Top panels show the relations between heart rate and maximum rate of rise of LV pressure (LVdP/dtmax, left panel), left atrial pressure (middle panel) and LV systolic pressure (right panels). Shown are data points at rest (lying and standing) and during exercise (1-4 km/h) of normal (circles) and MI (triangles) swine. Bottom panels show the myocardial O2 balance, i.e. the relation between myocardial O2 consuption (MVO2) and myocardial O2 extraction (MEO2, left panels), coronary venous O2 saturation (CVSO2, middle panels) and coronary venous O2 tension (CVPO2 right panels). Data are means±SE; *P<0.05 MI versus normal swine. Fig. 2. Effect of mixed ETA/ETB receptor blockade with tezosentan (3 mg/kg followed by 6 mg/kg/h iv) on the relation between myocardial O2 consumption (MVO2) and myocardial O2 extraction (MEO2), coronary venous O2 saturation (CVSO2), coronary venous O2 tension (CVPO2), and coronary venous O2 content (CV Cont O2). Data are means±SE; *P<0.05 versus corresponding Control relation, †P<0.05 effect of tezosentan wanes during exercise. Fig. 3. Effect of ETA receptor blockade with EMD122946 (3 mg/kg iv) on the relation between myocardial O2 consumption (MVO2) and myocardial O2 extraction (MEO2), coronary venous O2 saturation (CVSO2), coronary venous O2 tension (CVPO2), and coronary venous O2 content (CV Cont O2). Data are means±SE; *P<0.05 versus corresponding Control relation, †P<0.05 effect of EMD122946 wanes during exercise. Fig. 4. Left panel: Increasing concentrations of ET-1 in vivo result in coronary vasoconstriction as evidenced by the changes in coronary venous O2 tension (∆CVPO2). These changes were more pronounced in normal as compared to MI swine, indicating that the coronary circulation of MI swine is less sensitive to ET. Right panel: Effects of increasing concentrations of ET-1 in vitro on the relative diameter of porcine coronary arterioles isolated from the subendocardium of the LV anterior wall of 5 normal swine Endothelin and coronary tone after myocardial infarction 17 (n=13, passive diameter 136±14 µm) and of the LV anterior wall of 3 swine with a myocardial infarction of the lateral LV wall (n=7, passive diameter 133±10 µm). Data are means±SE; *P<0.05 vasoconstriction induced by incrementing doses of endothelin; †P<0.05 effect of endothelin is significantly different in MI as compared to normal swine. Table 1. Hemodynamic effects of tezosentan in normal swine and swine with a recent myocardial infarction Treatment MI Exercise level (km/h) Rest Lying Standing 1 2 3 HR (bpm) Control Tezosentan Control Tezosentan + + AoP (mmHg) Control Tezosentan Control Tezosentan + + LVSP (mmHg) Control Tezosentan Control Tezosentan + + LVdP/dt max Control (mmHg/s) Tezosentan Control Tezosentan + + 2720 ± 110 3400 ± 210† 2590 ± 170 2540 ± 80 LAP (mmHg) + + 4.5 ± 0.8 4.5 ± 1.2 13.5 ± 2.3‡ 12.3 ± 2.2‡ Control Tezosentan Control Tezosentan 128 ± 4 149 ± 4† 148 ± 4‡ 159 ± 4 95 ± 3 89 ± 2† 90 ± 2 79 ± 2†‡ 103 ± 3 103 ± 2 99 ± 4 96 ± 2 4 148 ± 4* 162 ± 5*† 160 ± 5* 170 ± 4*† 169 184 187 191 ± ± ± ± 5* 6* 3*‡ 4* 185 ± 5* 197 ± 7*† 204 ± 4*‡ 205 ± 4* 201 ± 6* 214 ± 7*† 218 ± 7* 222 ± 8* 232 ± 7* 245 ± 8*† 245 ± 9* 241 ± 7* 88 ± 3* 80 ± 2*† 83 ± 2* 75 ± 2† 88 79 80 74 ± ± ± ± 2* 2*† 2*‡ 3† 88 ± 2* 77 ± 2*† 80 ± 2*‡ 76 ± 2 87 ± 2* 81 ± 2*† 81 ± 3* 75 ± 3 90 ± 2* 82 ± 1*† 82 ± 3* 77 ± 2† 105 102 103 102 ± ± ± ± 3 3 3 5 3380 ± 200* 3680 ± 270 2880 ± 210 2970 ± 180* 3690 3920 3210 3330 ± ± ± ± 220* 250* 200* 300* 4030 ± 270* 4170 ± 270* 3480 ± 240* 3570 ± 270* 4310 ± 290* 4620 ± 350*† 3900 ± 330* 3910 ± 360* 5100 ± 370* 5070 ± 360* 4490 ± 320* 4240 ± 320* 1.8 ± 1.4 0.4 ± 1.3* 12.0 ± 2.6‡ 11.1 ± 2.0‡ 3.8 3.4 14.0 13.2 ± ± ± ± 0.8 1.0 2.1‡ 2.1‡ 4.9 ± 0.8 4.2 ± 1.0 14.9 ± 1.9‡ 16.0 ± 2.2*‡ 6.1 ± 0.7* 6.5 ± 0.8* 17.0 ± 2.0*‡ 17.9 ± 2.1*‡ 8.0 ± 0.8* 8.5 ± 0.8* 18.7 ± 2.0*‡ 18.4 ± 2.1*‡ 101 ± 2 99 ± 3 100 ± 4 101 ± 3 107 ± 3 105 ± 3 104 ± 4 105 ± 3 109 ± 3 109 ± 4 109 ± 4* 107 ± 5 117 ± 4* 115 ± 5* 117 ± 5* 113 ± 6 Data are means ± SE; normal swine (-) n=14; MI swine (+) n=8; HR, heart rate; AoP, mean aortic pressure; LVSP, left ventricular peak systolic pressure; LVdP/dtmax, maximum rate of rise of left ventricular pressure; LAP, mean left atrial pressure. *P<0.05 vs Rest lying; †P<0.05 vs corresponding Control; ‡P<0.05 MI vs normal swine. Endothelin and coronary tone after myocardial infarction Table 2. Plasma ET-levels in 7 normal swine and 6 swine with a recent myocardial infarction MI Exercise level (km/h) Rest (Lying) 2 4 3.2 ± 0.4 3.0 ± 0.3 3.2 ± 0.3 ART ET CV ET 3.3 ± 0.5 3.0 ± 0.4 3.1 ± 0.4 ART ET CV ET ART ET, + 4.9 ± 0.3* 4.6 ± 0.6* 4.7 ± 0.5* + 4.9 ± 0.5* 4.9 ± 0.5* 5.1 ± 0.5* Arterial endothelin plasma concentration in pM; CV ET, Coronary venous endothelin plasma concentration in pM; * P<0.05 MI vs normal swine 19 Endothelin and coronary tone after myocardial infarction 20 Table 3. Hemodynamic effects of EMD122946 in normal swine and swine with a recent myocardial infarction Treatment MI Rest Exercise Level (km/h) Lying Standing 1 2 3 HR (bpm) Control EMD122946 Control EMD122946 + + 132 ± 6 133 ± 5 144 ± 5 152 ± 5‡ AoP (mmHg) Control EMD122946 Control EMD122946 + + 90 ± 2 82 ± 2† 95 ± 2 85 ± 2† LVSP (mmHg) Control EMD122946 Control EMD122946 + + 113 ± 4 110 ± 4 104 ± 4 95 ± 4 LVdP/dt max Control (mmHg/s) EMD122946 Control EMD122946 + + 3100 ± 250 3040 ± 300 2450 ± 220 2460 ± 240 LAP (mmHg) + + 3.8 ± 1.2 2.7 ± 1.9 9.9 ± 2.7‡ 10.3 ± 1.9‡ Control EMD122946 Control EMD122946 146 ± 7* 150 ± 5* 159 ± 5* 163 ± 5* 86 ± 3 73 ± 2*† 84 ± 2* 75 ± 2*† 115 ± 5 112 ± 6 98 ± 3 94 ± 4 168 ± 6* 173 ± 7* 190 ± 6*‡ 177 ± 6* 82 ± 2* 72 ± 1*† 81 ± 2* 74 ± 2*† 182 ± 6* 190 ± 8* 200 ± 7* 199 ± 5* 83 ± 2* 74 ± 2*† 81 ± 2* 73 ± 2*† 201 ± 8* 211 ± 8* 218 ± 8* 215 ± 4* 84 ± 2* 76 ± 2*† 83 ± 2* 75 ± 2*† 4 232 ± 8* 247 ± 8*† 239 ± 6* 241 ± 5* 87 ± 2 79 ± 1† 83 ± 3* 77 ± 2*† 114 ± 4 109 ± 5 101 ± 3 95 ± 4† 117 ± 4 113 ± 6 103 ± 3 95 ± 5† 122 ± 6 118 ± 8 107 ± 4 99 ± 5† 128 ± 6* 129 ± 6* 112 ± 6 103 ± 6† 3640 ± 420* 3590 ± 350* 2640 ± 280 2660 ± 260* 3740 ± 310* 3740 ± 390* 2920 ± 370 2800 ± 290* 3890 ± 270* 3980 ± 390* 3100 ± 360* 2890 ± 310* 4300 ± 400* 4320 ± 430* 3410 ± 440* 3170 ± 370* 5070 ± 400* 4930 ± 440* 3880 ± 610* 3560 ± 470* 2.8 ± 1.7 2.4 ± 1.7 8.4 ± 2.1‡ 6.7 ± 1.7* 4.2 ± 1.0 2.4 ± 1.5 11.0 ± 2.7‡ 9.1 ± 2.6‡ 5.2 ± 1.2 4.4 ± 1.8 12.4 ± 2.2‡ 12.3 ± 1.9‡ 7.6 ± 1.4* 6.6 ± 1.6* 14.8 ± 2.2‡ 14.5 ± 2.0*‡ 8.5 ± 1.4* 8.7 ± 1.8* 16.6 ± 1.9*‡ 16.9 ± 1.8*‡ Data are means ± SE; normal swine (-) n=10; MI swine (+) n=8; HR, heart rate; AoP, mean aortic pressure; LVSP, left ventricular peak systolic pressure; LVdP/dtmax, maximum rate of rise of left ventricular pressure; LAP, mean left atrial pressure. *P<0.05 vs Rest lying; †P<0.05 vs corresponding Control; ‡P<0.05 MI vs normal swine. Endothelin and coronary tone after myocardial infarction 21 Table 4. Effect of tezosentan on myocardial oxygen balance in normal swine and swine with a recent MI Treatment MI Rest Exercise Level (km/h) Lying 1 2 3 Hb (g%) ART SO2 (%) ART PO2 (mmHg) SO2 (%) CV CV PO2 (mmHg) CBF (ml/min) Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan Control Tezosentan + + + + + + + + + + + + 4 7.9 ± 0.2 8.5 ± 0.3† 7.5 ± 0.2 7.9 ± 0.3 8.3 ± 0.2* 8.6 ± 0.3 7.9 ± 0.4 8.2 ± 0.4* 8.4 ± 0.2* 8.6 ± 0.2 8.0 ± 0.4 8.1 ± 0.4 8.6 ± 0.2* 8.7 ± 0.2 8.3 ± 0.5* 8.0 ± 0.3 8.7 ± 0.2* 8.6 ± 0.2 8.4 ± 0.4* 8.1 ± 0.4 92 ± 1 92 ± 1 91 ± 1 90 ± 1‡ 92 ± 1 91 ± 1* 88 ± 1*‡ 89 ± 0‡ 92 ± 1 92 ± 1 88 ± 1*‡ 89 ± 1*‡ 93 ± 1 92 ± 1 90 ± 1‡ 89 ± 1†‡ 92 ± 1 92 ± 1 89 ± 1‡ 89 ± 1‡ 97 ± 2 100 ± 3 100 ± 3 91 ± 2†‡ 96 ± 3 96 ± 5 86 ± 4* 86 ± 3 95 ± 2 96 ± 2 84 ± 4*‡ 85 ± 3*‡ 95 ± 2 93 ± 3* 89 ± 4* 83 ± 3*†‡ 91 ± 3* 92 ± 3* 85 ± 3* 82 ± 3* 16.7 ± 1.0 20.3 ± 1.1† 16.7 ± 1.1 16.9 ± 1.2 17.4 ± 0.8 19.2 ± 1.0† 15.5 ± 0.6 16.8 ± 0.6 17.1 ± 0.7 18.6 ± 1.0 15.4 ± 0.6‡ 15.6 ± 0.9 17.5 ± 0.7 19.1 ± 1.0† 15.2 ± 0.4‡ 15.3 ± 0.7 18.0 ± 0.8 19.0 ± 0.9 14.4 ± 0.3‡ 13.8 ± 0.9 21.6 ± 1.1 24.0 ± 1.0† 22.9 ± 0.8 23.4 ± 0.9 21.6 ± 0.8 23.9 ± 1.0† 22.7 ± 0.8 23.4 ± 0.7 21.6 ± 0.7 22.8 ± 0.9 22.1 ± 0.9 22.6 ± 0.7 21.9 ± 0.7 23.3 ± 0.9† 22.0 ± 1.1 22.3 ± 0.9 22.4 ± 0.7 23.9 ± 0.9† 21.5 ± 1.0 21.4 ± 1.1 55 ± 6 62 ± 6† 76 ± 5‡ 76 ± 6 77 ± 7* 79 ± 9* 99 ± 6*‡ 108 ± 6*†‡ 86 ± 8* 87 ± 10* 107 ± 6* 115 ± 6*‡ 93 ± 8* 102 ± 11*† 117 ± 7* 126 ± 6* 112 ± 11* 121 ± 11*† 135 ± 7* 139 ± 9* Data are means ± SE; normal swine (-) n=14; MI swine (+) n=8; Hb, hemoglobin; ART SO2, arterial O2 saturation; ART PO2, arterial O2 tension; CV SO2, coronary venous O2 saturation; CV PO2, coronary venous O2 tension; CBF, coronary blood flow. *P<0.05 vs Rest lying; †P<0.05 vs corresponding Control; ‡P<0.05 MI vs normal swine. Endothelin and coronary tone after myocardial infarction 22 Table 5. Effect of EMD122946 on myocardial oxygen balance in normal swine and swine with a recent MI Treatment MI Rest Exercise level (km/h) Lying 1 2 3 4 Hb (g%) ART SO2 (%) ART PO2 (mmHg) SO2 (%) CV CV PO2 (mmHg) CBF (ml/min) Control EMD 122946 Control EMD 122946 + + 7.9 ± 0.3 8.0 ± 0.2 7.8 ± 0.5 8.0 ± 0.4 8.2 ± 0.3* 8.3 ± 0.2 7.9 ± 0.4 8.3 ± 0.3 8.5 ± 0.3* 8.4 ± 0.2* 8.3 ± 0.4 8.3 ± 0.3 8.5 ± 0.3* 8.5 ± 0.2* 8.2 ± 0.5 8.2 ± 0.3 8.7 ± 0.3* 8.4 ± 0.3 8.4 ± 0.5 8.4 ± 0.3* Control EMD 122946 Control EMD 122946 - 93 ± 1 92 ± 1 91 ± 1‡ 91 ± 1 93 ± 1 92 ± 0 91 ± 1 90 ± 1 92 ± 1 92 ± 1 91 ± 1 91 ± 1 93 ± 1 93 ± 1 92 ± 1* 91 ± 1 92 ± 1 92 ± 1 92 ± 1 91 ± 1 99 ± 2 91 ± 2*† 95 ± 4 95 ± 6 93 ± 3* 92 ± 2 94 ± 4 94 ± 5 93 ± 2* 95 ± 3 97 ± 3 95 ± 6 91 ± 2* 90 ± 2* 86 ± 11 89 ± 4 18.2 ± 1.5 22.5 ± 1.8† 17.6 ± 1.4 17.0 ± 1.4‡ 18.2 ± 1.0 20.8 ± 1.1† 17.0 ± 1.5 16.9 ± 1.4‡ 18.4 ± 1.1 20.6 ± 1.2 17.5 ± 1.4 16.6 ± 1.7‡ 18.3 ± 0.9 20.2 ± 1.1 16.4 ± 0.9‡ 16.1 ± 0.9‡ 18.6 ± 1.1 20.1 ± 1.3* 16.1 ± 1.2‡ 15.9 ± 1.7‡ 23.1 ± 1.3 25.7 ± 1.3† 23.2 ± 1.3 23.4 ± 1.5 22.7 ± 0.9 23.9 ± 0.9* 22.5 ± 1.0 22.6 ± 0.9 23.2 ± 0.8 24.0 ± 0.9* 22.3 ± 1.3 22.2 ± 1.0 22.5 ± 0.9 24.0 ± 0.8* 22.0 ± 0.9 21.4 ± 0.7‡ 22.8 ± 0.8 23.6 ± 0.9* 22.1 ± 0.6 22.3 ± 1.3 52 ± 4 57 ± 5 72 ± 4‡ 79 ± 7‡ 69 ± 5* 75 ± 7* 100 ± 6*‡ 100 ± 7*‡ 75 ± 6* 84 ± 8*† 105 ± 6*‡ 111 ± 9* 86 ± 8* 97 ± 11*† 115 ± 6*‡ 122 ± 8* 105 ± 10* 117 ± 12*† 130 ± 8* 141 ± 11* Control EMD 122946 Control EMD 122946 Control EMD 122946 Control EMD 122946 Control EMD 122946 Control EMD 122946 Control EMD 122946 Control EMD 122946 + + + + + + + + + + 103 ± 4 97 ± 2 100 ± 3 98 ± 4 Data are means ± SE; normal swine (-) n=10; MI swine (+) n=8; Hb, hemoglobin; ART SO2, arterial O2 saturation; ART PO2, arterial O2 tension; CV SO2, coronary venous O2 saturation; CV PO2, coronary venous O2 tension; CBF, coronary blood flow. *P<0.05 vs Rest lying; †P<0.05 vs corresponding Control; ‡P<0.05 MI vs normal swine. . Endothelin and coronary tone after myocardial infarction 4000 * 2000 LV Systolic Pressure (mmHg) Left Atrial Pressure (mmHg) 30 Normal MI * 20 10 0 0 100 150 200 250 300 140 120 * 100 80 100 Heart rate (bpm) 150 200 250 300 100 Heart rate (bpm) 90 80 70 20 15 * 10 100 300 500 MVO2 (µmol/min) 700 200 250 300 30 CVPO2 (mmHg) * 150 Heart rate (bpm) 25 CVSO2 (%) MEO2 (%) LV dP/dtmax (mmHg/s) 6000 23 25 * 20 15 100 300 500 MVO2 (µmol/min) 700 100 300 500 MVO2 (µmol/min) Figure 1 700 Endothelin and coronary tone after myocardial infarction † * 70 15 500 700 MI Swine 300 500 80 70 20 15 300 500 MVO2 (µmol/min) 700 20 0.6 100 300 500 700 300 500 MVO2 (µmol/min) 700 100 300 500 700 100 300 500 700 1.4 25 1.0 20 0.6 15 100 † * 1.0 30 10 100 * 700 25 Control Tezosentan 25 15 100 CVPO2 (mmHg) 300 CVSO2 (%) MEO2 (%) † * 10 100 90 20 CV Cont O2 (mM) 80 1.4 30 CVPO2 (mmHg) 25 Control Tezosentan CV Cont O2 (mM) Normal Swine CVSO2 (%) MEO2 (%) 90 24 100 300 500 700 MVO2 (µmol/min) MVO2 (µmol/min) Figure 2 Endothelin and coronary tone after myocardial infarction Normal Swine 25 Control EMD122946 70 500 700 300 500 70 15 10 100 300 500 MVO2 (µmol/min) 700 300 500 300 500 MVO2 (µmol/min) 700 100 300 500 700 100 300 500 700 1.4 25 20 15 100 1.0 700 30 20 † * 0.6 100 CVPO2 (mmHg) 80 * 20 700 25 Control EMD122946 † 25 15 100 MI Swine CV Cont O2 (mM) 15 10 300 CVSO2 (%) MEO2 (%) 90 20 CV Cont O2 (mM) † * CVPO2 (mmHg) * 80 100 1.4 30 † CVSO2 (%) MEO2 (%) 90 25 1.0 0.6 100 300 500 MVO2 (µmol/min) Figure 3 700 MVO2 (µmol/min) Endothelin and coronary tone after myocardial infarction 26 Ex-vivo 0 *† -10 * Relative diameter ∆CVPO2 (mmHg) In-vivo 1.0 * 0.6 Normal swine MI swine *† 0.2 -20 0 20 40 ET-1 (pmol/kg/min) 10-10 10-9 10-8 ET-1 (M) Figure 4