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1Experimental Cardiology, Thoraxcenter, and 2Internal Medicine, Cardiovascular Research School Rotterdam, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Submitted 6 May 2004 ; accepted in final form 25 September 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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coronary microcirculation; coronary blood flow; myocardial oxygen balance; left ventricular dysfunction
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 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 (LCx) or a sham procedure, and studied 2 wk later while running on a treadmill. Because we paradoxically found a decreased vasoconstrictor influence of endogenous ET on the coronary vasculature in vivo, we further investigated whether the blunted vasoconstrictor influence was due to a reduced local release of ET or a reduced responsiveness of the coronary vasculature by infusion of exogenous ET in vivo. Again, the vasoconstriction was blunted after MI. To investigate whether the reduced responsiveness was due to factors secreted by cardiac myocytes, we also studied the responsiveness of isolated coronary arterioles to ET.
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METHODS |
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Surgical Procedures
Thirty-two swine (22 ± 1 kg at the time of surgery) were sedated (20 mg/kg ketamine + 1 mg/kg midazolam im), anesthetized with thiopental sodium (15 mg/kg iv), intubated, and ventilated with 1:2 O2-N2O to which 0.2–1.0% (vol/vol) isoflurane was added (3, 25). Anesthesia was maintained with midazolam (2 mg/kg + 1 mg·kg–1·h–1 iv) and fentanyl (10 µg·kg–1·h–1 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 measurement of mean aortic pressure and blood sampling for determination of PO2, PCO2, and pH (model ABL 505, Radiometer), O2 saturation, and hemoglobin concentration (model OSM2, Radiometer). A fluid-filled catheter and a high-fidelity Konigsberg pressure transducer were inserted into the LV via the apex. Fluid-filled catheters were also implanted into the left atrium for pressure measurements and into 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 (LAD) (18).
In all 32 swine, the proximal part of the LCx was exposed; in 16 of these 32 swine, the LCx was permanently occluded with a silk suture to produce an MI (7). Three MI swine died during surgery as a result of refractory fibrillation. Electrical wires and catheters were tunneled subcutaneously to the back, the chest was closed, and the animals were allowed to recover. The animals received analgesia with buprenorphine (0.3 mg im) for 2 days and antibiotic prophylaxis with amoxicillin (25 mg/kg iv) and gentamicin (5 mg/kg iv) for 5 days. Three MI swine died overnight after surgery.
Exercise Protocols
Studies were performed 
2 wk after surgery. After hemodynamic measurements (prone lying and standing), blood samples (prone 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 were collected during the last 60 s of each 3-min exercise stage, when hemodynamics had reached a steady state. After 90 min of rest, the mixed ETA-ETB antagonist tezosentan (a gift from Dr. Clozel, Actelion Pharmaceuticals, Allschwil, Switzerland) was administered to 14 normal and 8 MI swine at 3 mg/kg followed by an infusion of 6 mg·kg–1·h–1 iv (18), and the exercise protocol was repeated. Tezosentan has a pA2 of 9.5 for ETA receptors and a pA2 of 7.7 for ETB receptors, indicating only a 63-fold selectivity for ETA compared with ETB receptors (1, 26). On a different day, the ETA receptor antagonist EMD-122946 (a gift from Prof. Schelling, Merck Darmstadt, Darmstadt, Germany) was administered to 10 normal and 8 MI swine at 3 mg/kg iv (18), and the exercise protocol was repeated. EMD-122946 has a pA2 of 9.5 for ETA receptors and a pA2 of 6.0 for ETB receptors, indicating a 3,200-fold selectivity for ETA compared with ETB receptors (15). The dose of EMD-122946 administered in the present study does not block ETB receptors, inasmuch as we previously found that ET plasma concentrations are not influenced by this dose of EMD-122946, whereas tezosentan at the dose used in this study does increase plasma ET levels, indicating that the ETB receptor, which is responsible for ET clearance, is blocked by tezosentan, but not by EMD-122946 (18). We previously showed excellent reproducibility of the hemodynamic response in two consecutive bouts of exercise in normal and MI swine (3, 8).
Digital recording and offline analysis of hemodynamic data and computation of myocardial O2 consumption (M
O2) have been described in detail elsewhere (4, 25). Myocardial O2 delivery was computed as the product of LAD blood flow and arterial blood O2 content. MO2 in the region of myocardium perfused by the LAD was calculated as the product of coronary blood flow (CBF) and the difference in O2 content between arterial and coronary venous blood. Myocardial O2 extraction was computed as the ratio of MO2 to myocardial O2 delivery.
Determination of Plasma Levels of ET
In seven normal and six MI swine, arterial and coronary venous blood samples (5 ml) were collected at rest (prone 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 immunoreactivity were determined using a radioimmunoassay (Euro-Diagnostica, Malmö, Sweden) that has a cross-reactivity of 100% toward ET-1, 48% toward ET-2, and 109% toward ET-3. Because production of ET-2 and ET-3 appears to be absent in the cardiovascular system of the pig (11), the concentration measured with the radioimmunoassay most likely represents ET-1.
In Vivo Responsiveness of Coronary Vasculature to Exogenous ET-1
To study the responsiveness of the coronary vasculature to ET in vivo, ET-1 (Sigma) was infused (10, 20, and 40 pmol·kg–1·min–1 iv) in chronically instrumented normal (n = 3) and MI (n = 3) swine under awake resting conditions. Changes in coronary venous PO2 were used as the index of coronary vasoconstriction.
Responsiveness of Isolated Coronary Arterioles to ET-1
For the study of isolated coronary arterioles, eight additional swine were sedated and anesthetized as described above, and a thoracotomy was performed under sterile conditions. In these animals, the pericardium was opened and the LCx was exposed, and in three swine the LCx was permanently ligated (28). Then the pericardium was closed to minimize inflammation, the chest was surgically closed, and the animals were allowed to recover.
At 3 wk after induction of MI or sham operation, swine were sedated with ketamine (20 mg/kg im) + midazolam (1 mg/kg im), anesthetized with pentobarbital sodium (15–20 mg/kg iv), intubated, and ventilated with 1:2 O2-N2O (28). Anesthesia was maintained with pentobarbital sodium (15–20 mg·kg–1·h–1). The thorax was opened by midline sternotomy, and the heart was fibrillated and instantaneously excised. Single arterioles (70–200 µm passive diameter) were dissected from the LV anterior free wall, as previously described (13, 17, 19).
The heart was placed in ice-cold physiological saline solution of the following composition (in mM): 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS, 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 (20- to 80-µm OD, depending on the size of the vessel) connected to pressurized reservoirs filled with physiological saline solution buffered at pH 7.4 at 37°C with a pressure-myograph 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 charge-coupled device camera, and diameter of the arterioles was measured. The vessel was slowly warmed to 37°C and allowed to develop spontaneous tone. ET-1 (Sigma) was added in cumulative steps (5 min per concentration) to the vessels in concentrations ranging from 10 pM to 5 nM, and the response of the vessels was measured. Vascular diameters were normalized to the diameter with tone before administration of 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) ANOVA for repeated measures, as appropriate. When significant effects were detected, post hoc testing for the effects of exercise, drug treatment, and MI was performed using Scheffé's test. To test for the effects of MI and drug treatment (EMD-122946 or tezosentan) on the relation between MO2 and coronary venous PO2, O2 saturation, myocardial O2 extraction, or O2 content, regression analysis was performed using MI, drug treatment, and MO2 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 ET-1-induced vasoconstriction of the coronary vasculature in vivo and in vitro using ET dose and infarct as independent variables. Statistical significance was accepted when P 
0.05. Values are means ± SE.
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RESULTS |
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Effect of MI. In accordance with previous reports from our laboratory (7, 8), MI had negligible effects on mean aortic blood pressure at rest and during exercise (Table 1) but resulted in LV dysfunction, as evidenced by a downward shift of the relation between heart rate and maximum rate of rise in LV pressure (dP/dtmax) or LV systolic pressure as well as markedly elevated left atrial pressures (Fig. 1). Plasma levels of ET were elevated in MI compared with normal swine at rest and during exercise. However, there were no differences in plasma ET between arterial and coronary venous blood (Table 2).
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ETA receptor blockade. The ETA receptor antagonist EMD-122946 decreased mean aortic pressure in MI and normal swine, similar to the decrease in blood pressure induced by tezosentan (Table 3). Similarly, EMD-122946 had no significant effects on any of the other hemodynamic variables in either group of animals.
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Effect of MI.
In normal swine, the exercise-induced increases in MO2 were met by commensurate increases in CBF (Table 4) and, hence, in myocardial O2 delivery, so that myocardial O2 extraction, coronary venous O2 saturation, and PO2 were maintained constant (Fig. 1). Proximal LCx occlusion in swine results in MI of 20% of the LV myocardium. Despite this loss of viable myocardial tissue, the LV weight-to-body weight ratio was slightly higher in MI than in normal swine (3.8 ± 0.2 vs. 3.2 ± 0.1 g/kg, P < 0.05), reflecting significant hypertrophy of surviving myocardium. Resting CBF and MO2 in the remote surviving myocardium of the LAD perfused area were larger in MI than in normal swine, likely because of the higher heart rate in conjunction with the LV hypertrophy.
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O2. Consequently, coronary venous O2 saturation and PO2 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 with normal swine.
Mixed ETA-ETB receptor blockade.
In normal resting swine, tezosentan had no effect on arterial O2 levels but resulted in increases in CBF and hemoglobin (Table 4) and, hence, myocardial O2 delivery from 262 ± 24 to 325 ± 34 µmol O2/min (P < 0.05). Also, MO2 increased slightly after administration of tezosentan, likely as the result of the increased heart rate. However, Fig. 2 (top) shows that the tezosentan-induced vasodilation and the commensurate increased O2 delivery slightly exceeded the increase in MO2, allowing myocardial O2 extraction to decrease and, consequently, coronary venous O2 saturation and PO2 (and, hence, coronary venous O2 content) 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 (Fig. 2, bottom). Thus, despite the increased plasma levels of ET, the vasoconstrictor influence of ET in the coronary microcirculation was lost after MI.
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To assess whether the reduced effect of ET receptor blockade after MI was due to a decreased ET production or to a reduced coronary vascular responsiveness, ET-1 was infused and the vasoconstriction was measured. Administration of ET-1 caused dose-dependent coronary vasoconstriction, as evidenced by decreased coronary venous PO2 in normal and MI swine (Fig. 4). However, the coronary vasoconstriction was blunted in MI compared with normal swine, indicating reduced vascular responsiveness to ET in vivo.
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To assess whether 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 Fig. 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.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 relation between coronary venous O2 levels and MO2 (7, 27). Thus an increase in coronary resistance vessel tone will limit CBF and, hence, myocardial O2 supply at a given level of MO2, forcing the myocardium to increase its O2 extraction (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 MO2, 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) that is determined by the coronary resistance vessel tone.
Myocardial O2 Balance in Remodeled Myocardium
Myocardial dysfunction due to MI is caused by the loss of viable pump tissue and results in compensatory LV remodeling and neurohumoral activation. The remaining viable tissue hypertrophies and heart rate increases to compensate for the decreased stroke volume (8). These adaptations result in an increased MO2 and thus require additional CBF. However, CBF is impeded by insufficient growth of the coronary microvasculature in conjunction with increased extravascular compressive forces, as a result of the increased heart rate and LV filling pressures (7). The additional vasodilation that is required to meet the increased O2 demand of the myocardium and overcome the augmented impediment of CBF 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 remodeled 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 investigated in the present study whether the increased circulating levels of ET contributed to the perturbations of myocardial O2 balance.
Altered Contribution of ET to Coronary Vasomotor Control After MI
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 sham-operated swine and swine with an MI that showed an increase in the ET responsiveness in vessels from animals with an MI.
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). Because adenosine production by cardiac myocytes may be increased in the hypertrophied myocardium as a result of mild underperfusion during exercise, especially in the subendocardium (7), ET receptors may have been desensitized in MI, but not normal, swine. Dissection of the isolated coronary arterioles results in removal of the cardiac myocytes and, thereby, loss of their possibly adenosine-mediated modulation of the ET receptors.
The increase in plasma ET levels after MI in combination with the potent vasoconstrictor properties of ET, as well as the possible role of ET as a growth factor in myocardial remodeling, has prompted 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 although 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. (14) found that intracoronary administration of an ETA antagonist reduced 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. In contrast, in failing rat hearts, the ET protein levels were increased and ETA receptor antagonism resulted 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 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 a therapeutic value of these compounds (6, 20). In 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 an 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 toward vasodilation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-adrenergic control. Circ Res 82: 1312–1322, 1998.This article has been cited by other articles:
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I. Bak, I. Lekli, B. Juhasz, N. Nagy, E. Varga, J. Varadi, R. Gesztelyi, G. Szabo, L. Szendrei, I. Bacskay, et al. Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1329 - H1336. [Abstract] [Full Text] [PDF]
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), and coronary venous PO2 (cvPO2). Values are means ± SE. *P 
Effect of tezosentan wanes during exercise (P 
