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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.
Acknowledgements
The authors gratefully acknowledge Robert H. van Bremen and Reier Hoogendoorn for technical
assistance. This study was supported by the Netherlands Heart Foundation (2000T038 (DJD);
2000T042 (DM)).
Endothelin and coronary tone after myocardial infarction
14
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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