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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:1214 –1222, 1999
Vol. 288, No. 3
Printed in U.S.A.
Endogenous Endothelin-1 Depresses Left Ventricular Systolic
and Diastolic Performance in Congestive Heart Failure1
KATSUYA ONISHI, MICHIYA OHNO, WILLIAM C. LITTLE, and CHE-PING CHENG
Cardiology Section, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Accepted for publication October 20, 1998
This paper is available online at http://www.jpet.org
Endothelin-1 (ET-1) is a 21-amino-acid peptide, originally
isolated from endothelial cells (Yanagisawa et al., 1988), that
is also produced by the kidney and heart (Miller et al., 1989;
Luscher et al., 1991). It is a potent, arterial, and venous
constrictor (Kiowski et al., 1995), and it interacts with the
renin-angiotensin system (Miller et al., 1989). Plasma ET-1
levels are increased in both experimental (Cavero et al.,
1990; Margulies et al., 1990; Teerlink et al., 1994; Shimoyama et al., 1996) and clinical (McMurray et al., 1992;
Rodeheffer et al., 1992; Wei et al., 1994) congestive heart
failure (CHF), and endothelin receptors are increased in a rat
model of CHF (Sakai et al., 1996). Thus, ET-1 may play an
important role in the cardiac response to neurohormonal
activation in CHF.
Received for publication July 13, 1998.
1
This study was supported in part by grants from the National Institutes of
Health (HL45258 and HL53541) and the American Heart Association
(94006140) and the Alcohol Beverage Medical Research Foundation. Dr.
Cheng is an Established Investigator of the American Heart Association. This
work was presented in abstract form at the American Heart Association
Meeting in 1997.
end-systolic pressure (101 6 11 versus 93 6 8 mm Hg) and
total systemic resistance (0.084 6 0.022 versus 0.065 6 0.15
mm Hg/ml/min). The t (42 6 12 versus 38 6 10 ms), mean left
atrial P (22 6 5 versus 18 6 4 mm Hg) (p , .05), and minimum
LVP were also significantly decreased. After CHF, the slopes of
P-V relations, EES (3.4 6 0.4 versus 4.8 6 0.8 mm Hg/ml),
dE/dtmax (42.4 6 7.8 versus 50.0 6 7.8 mm Hg/s/ml), and
stroke work-end-diastolic V relation (58.1 6 3.3 versus 72.4 6
5.2 mm Hg) (p , .05) all increased after L-754,142, indicating
enhanced contractility. Before CHF, low levels of endogenous
ET-1 have little cardiac effect. However, after CHF, elevated
endogenous ET-1 produces arterial vasoconstriction, slows LV
relaxation, and depresses LV contractile performance. Thus,
elevated endogenous ET-1 may contribute to the functional
impairment in CHF in this canine model.
ET-1 exerts a positive inotropic action on normal myocardium (Ishikawa et al., 1988; Watanabe et al., 1989; Neubauer
et al., 1990). Thus, “upregulation of endothelin pathways
may be beneficial in providing short-term support for the
failing myocardium” (Goto et al., 1996). However, several
lines of evidence indicate that the effect of ET-1 on normal
myocardial contraction may be altered in a pathologic state
(Kohmoto et al., 1993; Thomas et al., 1996; Spinale et al.,
1997; Ito et al., 1997; Suzuki et al., 1998). We found that
angiotensin II has a positive inotropic effect in normal myocytes but depresses contraction in myocytes from dogs with
pacing-induced CHF (Cheng et al., 1996). The intracellular
signaling responsible for the inotropic effect of angiotensin II
and ET-1 in normal myocardium is similar (Fareh et al.,
1996; Touyz et al., 1996; Ito et al., 1997) and is altered by
myocardial hypertrophy in the rat (Ito et al., 1997). Thus, the
inotropic effect of ET-1 on normal myocardium may be altered in a similar fashion in CHF (Thomas et al., 1996;
Suzuki et al., 1998).
Previously, endogenous ET-1 has been reported variably to
ABBREVIATIONS: ET-1, endothelin-1; CHF, congestive heart failure; HR, heart rate; LA, left atrium; LV, left ventricular, left ventricle; P-V,
pressure-volume; PES, end-systolic pressure; PED, end-diastolic pressure; VES, left ventricular end-systolic volume; VED, left ventricular enddiastolic volume; SW, stroke work; TSR, total systemic resistance; dP/dt, derivatives of left ventricular pressure; EES, slope of PES, end-systolic
pressure-left ventricular end-systolic volume relation; dE/dtmax, slope of dP/dtmax-left ventricular end-diastolic volume relation; MSW, slope of
stroke work-left ventricular end-diastolic volume relation; EA, arterial elastance.
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ABSTRACT
Endothelin-1 (ET-1) is a positive inotrope in normal hearts;
however, the direct cardiac effects of endogenous ET-1 in
congestive heart failure (CHF) are unknown. We evaluated the
cardiac responses to endogenous ET-1 using an ETA and ETB
receptor blocker (L-754,142) in seven conscious dogs before
and after pacing-induced CHF. Before CHF, when the plasma
ET-1 was 7.3 6 1.7 fmol/ml, L-754,142 caused no significant
alterations in heart rate, left ventricular (LV) end-systolic pressure, total systemic resistance, and the time constant of LV
relaxation (t). LV contractile performance, measured by the
slopes of LV pressure (P)-volume (V) relation (EES), dP/dtmaxend-diastolic V relation (dE/dtmax), and stroke work-end-diastolic V relation, was also unaffected. After CHF, when the
plasma ET-1 was significantly increased to 14.1 6 3.0 fmol/ml
(p , .05), L-754,142 produced a significant decreases in LV
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Endogenous Endothelin-1 in CHF
Materials and Methods
Instrumentation
Seven healthy, adult, heartworm-negative mongrel dogs (weight,
25–36 kg) were instrumented using the technique that we described
previously (Cheng et al., 1990, 1996). Anesthesia was induced with
xylazine (2 mg/kg i.m.) and sodium thiopental (6 mg/kg i.v.) and
maintained with halothane (0.5–2.0%). They were intubated and
ventilated with oxygen-enriched room air to maintain arterial oxygen pressure greater than 100 mm Hg and pH between 7.38 and
7.42. The pericardium was opened through a left thoractomy. Micromanometer pressure transducers (Konisberg Instruments, Inc., Pasadena, CA) and polyvinyl catheters (1.1 mm I.D.) for transducer
calibration were inserted into the left ventricle (LV) through a LV
apical stab wound and into the left atrium (LA) through the LA
appendage. Three pairs of ultrasonic crystals (5 MHz) were implanted in the endocardium of the LV to measure the anterior-toposterior, septal-to-lateral, and base-to-apex (long-axis) dimensions
(Cheng et al., 1990). Hydraulic occluder cuffs were placed around the
inferior and superior venae cavae. A 540-mm sutureless myocardial
lead (model 4312; Cardiac Pacemakers, Inc., Minneapolis, MN) was
implanted within the myocardium of the right ventricle, and the lead
was attached to a unipolar multiprogrammable pacemakers (model
8329; Medtronics, Inc., Minneapolis, MN) positioned under the skin
of the chest. An ultrasonic transit-time flow probe (model 2R or 3R;
Transonic System Inc.) was placed around the proximal left anterior
descending coronary artery. All wires and tubing were exteriorized
through the posterior neck.
Data Collection
Studies were begun after full recovery from instrumentation (from
10 days to 2 weeks after surgery). The LV and LA catheters were
connected to pressure transducers (Statham P23Db; Gould, Cleveland, OH) calibrated with a mercury manometer. The signal from the
micromanometer was adjusted to match that of the catheter. The LA
micromanometer was adjusted to match LA and LV pressures at the
end of long periods of diastasis.
The analog signals were recorded on a 16-channel oscillograph
(Astro-Med, West Warwick, RI), digitized with an online analog-todigital converter (Data Translation Devices, Marlboro, MA) at 200
Hz, and stored on a magneto-optical disk memory system by use of a
486 computer system. Each data acquisition period lasted for 12 to
15 s, spanning several respiratory cycles. The derivatives of LV
pressure and volume were calculated using the five-point Lagrangian method (Cheng et al., 1990, 1993, 1996).
Experimental Protocol
To evaluate the potential functional role of endogenous ET-1 in the
progression of CHF, we used L-754,142, which is a potent mixed
ET-1 antagonist with more ETA selectivity (Williams et al., 1995a).
Studies before CHF
Effect of Exogenous ET-1 With and Without Pretreatment
of L-754,142. To determine the dosage for the ET-1 antagonist
L-754,142, we measured cardiovascular responses during ET-1 infusion with and without pretreatment of L-754,142. Data were initially
recorded with the animals lying quietly on their sides without medication to obtain baseline values. Three sets of variably loaded P-V
loops were generated by sudden transient occlusion of the cavae.
This caused a progressive fall in end-systolic pressure (PES)-LV
end-diastolic volume (VES) over a 12- to 15-s recording period. Immediately after the recording period, the caval occlusion was released, and hemodynamic parameters were allowed to restabilize.
After all parameters returned to their baseline levels, ET-1 (600
ng/kg i.v.) was administered. When the arterial pressure had
reached a stable level, steady-state and caval occlusion data at rest
were again collected. To assess the interactions of ET-1 with autonomic reflexes, the same protocol was repeated after the administration of metoprolol (0.5 mg/kg i.v.) and atropine (0.1 mg/kg i.v.).
On the following days, the adequacy of ET-1 blockade produced by
L-754,142 (3 mg/kg plus 3 mg/kg/h i.v.) was tested in the animals
both with and without autonomic blockade. First, L-754,142 was
administered, and the steady-state and caval occlusion data were
collected. Then, ET-1 (600 ng/kg i.v.) was infused. Data were acquired again.
Effect of Endogenous ET-1. On the next day, after the collection
of control steady-states and caval occlusion data, L-754,142 (3 mg/kg
i.v. followed by 3 mg/kg/h infusion) was administered. After 5 min,
when the arterial pressure had reached a stable level, steady-state
and caval occlusion data at rest were again collected.
Studies during Development of CHF. After the completion of
the baseline studies, the pacemaker rate was adjusted to 200 to 250
beats/min, using the external magnetic control unit. Three times per
week, the pacemaker rate was adjusted below the spontaneous rate.
The animal was allowed to equilibrate for 30 min, and then data
were collected. After each study, pacing rate was returned to 200 to
250 beats/min. After pacing for 4 to 5 weeks, when the LV enddiastolic pressure (PED) during nonpaced period had increased by
more than 15 mm Hg over the prepacing control level, CHF data
were obtained. This level of CHF was chosen because the animals
had begun to show clinical evidence of CHF (anorexia, mild ascites,
and pulmonary congestion).
Studies After Onset of CHF
Effect of Endogenous ET-1. Studies in dogs with CHF were
performed after the animal stabilized for at least 30 min after discontinuation of pacing. After recording of the baseline, steady-state,
and caval occlusion data, the same amount of L-754,142 as used in
the studies before CHF was administrated. At 5 min after drug
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produce positive (Sakai et al., 1996) or negative (Teerlink et
al., 1994; Shimoyama et al., 1996; Spinale et al., 1997) effects
on left ventricular (LV) contraction in animal models of CHF.
These inconsistent results may have resulted from the influence of ET-1-produced changes to loading conditions on conventional measures of LV contractile performance and the
variable effects of anesthesia. Thus, the inotropic effect of the
elevated endogenous ET-1 in CHF remains unclear.
The pacing-induced CHF model has been studied by many
investigators (Armstrong et al., 1986; Burchell et al., 1992;
Spinale et al., 1994), including those at our laboratory
(Cheng et al., 1993, 1996). The biochemical alterations are
similar to those reported for volume or pressure overload
(O’Brien et al., 1989). Chronic rapid pacing produces timedependent changes in LV and cardiomyocyte function, structure, hemodynamic compromise, and neurohormonal activation (including ET-1) that are very similar to the clinic
spectrum of CHF (Cohn, 1995). The National Institutes of
Health has identified this rapid pacing model as one of the
most promising for the elucidation of mechanisms that contribute to the initiation of the progression of CHF (Lenfant,
1994).
In the present study, we used pacing-induced CHF in dogs
to evaluate the hypothesis that endogenous ET-1 contributes
to a functional impairment of LV contraction and relaxation
in CHF independent of its effect on arterial load. To avoid the
potential confounding effects of ET-1-produced changes in
loading conditions on conventional measures of LV performance, we evaluated LV contractile performance in the pressure-volume (P-V) plane in conscious animals (Kass and
Maughan, 1988; Little et al., 1989; Cheng et al., 1996).
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Onishi et al.
Data Processing and Analysis
LV volume (VLV) was calculated as a modified general ellipsoid
using the following equation:
VLV5(p/6)DAPzDSLzDLA
where DAP is the anterior-to-posterior LV diameter, DSL is the septal-to-lateral LV diameter, and DLA is the long-axis LV diameter. We
previously demonstrated that this method gives a consistent measure of VLV despite changes in LV loading conditions, configurations,
and heart rate (Little et al., 1989; Cheng et al., 1993). To account for
respiratory changes in intrathoracic pressure, steady-state measurements were averaged over the 12- to 15-s recording period that
spanned multiple respiratory cycles. End-diastole was defined as the
relative minimum of LV pressure occurring after the A wave. Endsystole was defined as the top left corner of the LV P-V loop, identified using the iterative technique described by Kono et al. (1984). The
time of mitral valve opening was defined to be when LV pressure fell
below LA pressure. LV end-diastolic, end-systolic, and minimum
pressures and volumes were measured. LA pressure was measured
at the time of mitral valve opening (peak V wave) and at the peak of
the A wave. The mean LA pressure was determined.
The derivatives of LV pressure (dP/dt) were calculated using the
five-point Lagrangian method (Little et al., 1989; Cheng et al., 1993).
Stroke volume was calculated as VED minus VES. Cardiac output was
determined as stroke volume multiplied by heart rate. LV stroke
work (SW) was also calculated by point-by-point integration of the
LV P-V loop for each beat. The rate of LV relaxation was analyzed by
determining the time constant of the isovolumic fall of LV pressure.
LV pressure from the time of minimum dP/dt until mitral valve
opening was fit to an exponential equation:
P 5 PA exp~ 2t/T! 1 PB
where P is LV pressure, t is time, and PA, PB, and T are constants
determined by data. Although the fall in isovolumic pressure is not
exactly exponential, the time constant, derived from the exponential
approximation, provides an index of the rate of LV relaxation. The
effective arterial elastance, EA, was calculated as LV PES divided by
stroke volume. TSR was also calculated as PES divided by cardiac
output.
Only caval occlusions that produced a fall in LV PES of approxi-
mately 30 mm Hg were analyzed. Premature beats and the subsequent beat were excluded from analysis.
The LV PES-VES data during the fall of LV pressure, produced by
each caval occlusion, were fit using the least-squares method to:
PES5EES(VED2V0,ES)
where EES is the slope of linear PES-VES relation, representing the
LV end-systolic elastance, and V0,ES is the intercept with the volume
axis. The volume (V100,ES) associated with a PES of 100 mm Hg was
calculated as:
V100,ES5V0,ES1100/EES
The dP/dtmax-VED and SW-VED relations were quantified by fitting
the data from the same beats from each caval occlusion used to
evaluate the PES-VES relation to:
dP/dtmax5dE/dtmax(VED2V0,dP/dt)
and
SW 5 MSW(VED2V0,SW)
The position of the dP/dtmax-VED and SW-VED relations were calculated by determining the VED associated with a dP/dt of 1000 mm
Hg/s and an SW of 1000 mm Hg/ml:
V1000,dP/dt5V0,dP/dt11000/(dE/dtmax)
V1000,SW5V0,SW11000/MSW
The slopes, volume-axis intercepts, and positions in each of the
three relations for each condition were evaluated as the mean values
of the two or three caval occlusions performed under each condition
(Little et al., 1989).
The LV P-V area (PVA) was determined as the area under the
PES-VES relation and the systolic P-V trajectory and above the enddiastolic P-V relations curve. The mechanical efficiency of the heart
was calculated as SW/PVA. The coupling of the LV and arterial
system was quantified as EES/EA.
Postmortem Evaluation
At the conclusion of the studies, the animals were sacrificed by
lethal injections of sodium thiopental (100 mg/kg i.v.), and the heart
was examined to confirm the proper position of the instrumentation.
Statistical Analysis
Statistical comparisons were made with Student’s t test for paired
observations and ANOVA with the Bonferroni method of multiplepaired comparisons as appropriate. Significance was accepted when
p , .05. Data for steady-state and plasma ET-1 are expressed as
mean 6 S.D., and values for LV P-V relations are expressed as
mean 6 S.E.M.
Results
Effects of Exogenous ET-1 With and Without
Pretreatment of ET-1 Receptor Blocker Before CHF
As shown in Table 1, before CHF, with reflexes intact, the
infusion of ET-1 (600 ng/kg i.v.) produced significant increases in LV PES (103 6 2 versus 114 6 8 mm Hg, p , .05),
LV PED (11.6 6 2.7 versus 16.1 6 2.4 mm Hg, p , .05),
minimum LVP (1.2 6 1.1 versus 3.3 6 0.4 mm Hg, p , .05),
and TSR (0.063 6 0.023 versus 0.070 6 0.022 mm Hg/ml/min,
p , .05), indicating a vasoconstriction, and in EES (5.5 6 0.6
versus 6.8 6 0.7 mm Hg/ml, p , .05), dE/dtmax (80.6 6 8.4
versus 107.7 6 11.0 mm Hg/s/ml, p , .05), and MSW (71.5 6
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administration, when the arterial pressure had reached a stable
level, resting steady-state and caval occlusion data were again collected.
Effect of Nitroprusside. To assess the direct cardiac effect of
ET-1 blocker, independent of its effect on systolic load, we compared
the equal hypotension caused by nitroprusside and L-754,142. Nitroprusside (0.5–2.0 mg/kg/min) was administered to obtain a similar
decrease in LV PES. Data were collected in a similar way for the
L-754,142 study.
Plasma ET-1 Measurements. The plasma ET-1 concentrations
were measured before and after heart failure in five dogs. Before and
5 min after drug administration, 5 ml of blood was obtained from the
LA catheter, immediately placed into an EDTA tube on ice, and
centrifuged at 2500 rpm at 4°C. Plasma was separated and stored at
220°C until assay. Then, 1 ml of 20% acetic acid was added to the
1-ml plasma samples. The acidified samples were vortexed and centrifuged for 15 min at 2600g. Samples were applied to Si-C18 SepPak cartridges (500 mg C18 in a 3-ml syringe) that had been pretreated with 3 ml of metenolone, 3 ml of water, and 3 ml of 10% acetic
acid. Columns were washed with 3 ml of 10% acetic acid and 6 ml of
ethyl acetate. Columns were eluted with 3 ml of 80% methanol/20%
0.05 M ammonium bicarbonate. Eluted samples were dried overnight in a Savant Speed-Vac centrifugal evaporator. Dried samples
were assayed for immunoreactive ET-1 using an Amersham RIA kit
(RPA 545) (Wei et al., 1994).
Vol. 288
1999
Endogenous Endothelin-1 in CHF
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TABLE 1
Effects of exogenous ET-1 on the steady-state hemodynamics and P-V relations with and without L-754,142 before CHF
Control
Heart rate (beats/min)
Peak 1dP/dt (mm Hg/s)
Peak 2dP/dt (mm Hg/s)
LVPED (mm Hg)
LVPES (mm Hg)
Minimum LVP (mm Hg)
TSR (mm Hg/ml/min)
EES (mm Hg/ml)
V0,ES (ml)
dE/dtmax (mm Hg/s/ml)
V0, dP/dt (ml)
MSW (mm Hg)
V0,SW (ml)
L-754,142
Control
ET-1
Control
ET-1
114 6 6
2468 6 110
22162 6 49
11.6 6 2.7
103 6 2
1.2 6 1.1
0.063 6 0.023
5.5 6 0.6
7.4 6 2.0
80.6 6 8.4
5.3 6 1.0
71.5 6 4.7
22.2 6 3.2
110 6 3
2597 6 63
22252 6 175
16.1 6 2.4*
114 6 8*
3.3 6 0.4*
0.070 6 0.022*
6.8 6 0.7*
9.3 6 1.5*
107.7 6 11.0*
6.6 6 0.9*
81.1 6 5.6*
22.4 6 3.1*
114 6 7
2560 6 93
22283 6 58
10.6 6 2.8
106 6 4
0.7 6 0.6
0.063 6 0.017
5.4 6 0.3
10.4 6 1.4
72.6 6 14.9
4.1 6 1.3
80.4 6 6.1
26.2 6 4.7
112 6 7
2584 6 74
22252 6 84
10.2 6 2.3
104 6 8
0.6 6 0.8
0.064 6 0.018
5.4 6 0.3
10.1 6 1.3
75.7 6 13.3
5.6 6 2.6
80.3 6 6.4
27.5 6 4.9
LVP, LV pressure; V0,ES, intercept with volume axis; dE/dtmax, slope of dP/dtmax-VED relation; V0, dP/dt, intercept with volume axis; V0,SW, intercept with volume axis.
Values are mean 6 S.E. (n 5 4).
* P , .05, ET-1 versus corresponding control value.
Effect of Endogenous ET-1 Before CHF
Steady-State Measurements. Steady-state hemodynamic changes produced with L-754,142 in seven dogs before
CHF are summarized in Table 2 and displayed in Fig. 1.
Intravenous administration of L-754,142 caused no significant alternations in HR, LVPES (109 6 4 versus 107 6 6 mm
Hg), LVPED, left atrial pressure (LAP), 1dP/dtmax, TSR
(0.063 6 0.016 versus 0.059 6 0.013 mm Hg/ml/min), and SV.
Furthermore, there were no significant differences in t.
P-V Analysis. As shown in Table 3 and Fig. 2, in normal
dogs, L-754,142 produced no significant increases in the
slopes of the PES-VES relation (5.3 6 0.7 versus 5.3 6 0.8 mm
Fig. 1. An example of effect of L-754,142, an ET-1 blocker, on steady-state
LV P-V loops. Before CHF (left), administration of L-754,142 did not
significantly affect P-V loops. In contrast, after CHF (right), administration of L-754,142 produced a decrease in PES, PED, and minimum pressure.
Hg/ml), the dP/dtmax-VED relation (82.0 6 12.8 versus 82.3 6
11.4 mm Hg/s/ml), and the SW-VED relation (78.1 6 4.4
versus 81.2 6 7.5 mm Hg). There also were no significant
alterations in the positions of all three relations with rela-
TABLE 2
Effects of ET-1 blocker on steady-state hemodynamics and ET-1 plasma concentration before and after CHF
Before CHF
Heart rate (beats/min)
Peak 1dP/dt (mm Hg/s)
Peak 2dP/dt (mm Hg/s)
LVPED (mm Hg)
LVPES (mm Hg)
Minimum LVP (mm Hg)
Mean LAP (mm Hg)
VED (ml)
VES (ml)
Stroke volume (ml)
Ea (mm Hg/ml)
TSR (mm Hg ml/min)
t (ms)
CBF (ml/min)
Plasma ET-1 (fmol/ml)
After CHF
Control
ET-1 blocker
Control
ET-1 blocker
111 6 17
2604 6 295
22295 6 300
10.9 6 2.9
109 6 4
0.6 6 0.7
4.2 6 2.0
49.4 6 11.2
32.5 6 7.5
16.9 6 4.4
6.9 6 1.9
0.063 6 0.016
31.1 6 3.7
33.6 6 6.4
7.3 6 1.7
114 6 17
2549 6 282
22261 6 280
10.7 6 3.3
107 6 6
0.7 6 1.3
3.4 6 2.7
48.6 6 11.0
31.8 6 7.8
16.7 6 3.9
6.8 6 1.8
0.059 6 0.013
30.9 6 3.7
34.1 6 7.0
18.2 6 1.9**
118 6 16*
1550 6 433*
21631 6 325*
27.3 6 7.7*
101 6 11*
10.5 6 2.0*
22.0 6 5.3*
59.9 6 15.3*
48.6 6 12.3*
11.4 6 4.1*
9.6 6 2.9*
0.084 6 0.022*
41.9 6 12.1*
42.8 6 14.0*
14.1 6 3.0*
119 6 19
1757 6 289**
21780 6 228**
24.6 6 7.1**
95 6 10**
8.6 6 2.1**
17.7 6 4.2**
57.8 6 15.2**
44.7 6 11.5**
13.0 6 4.6**
7.9 6 2.3**
0.065 6 0.015**
37.7 6 10.4**
49.1 6 16.0**
26.1 6 3.4**
LVP, LV pressure; LAP, LA pressure; Ea, arterial elastance; t, time constant of LV relaxation; and CBF, coronary blood flow. Values are mean 6 S.D. (n 5 7).
* P , .05, control values after CHF versus corresponding control values before CHF.
** P , .05, ET-1 blocker versus corresponding control value.
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4.7 versus 81.1 6 5.6 mm Hg, p , .05), indicating an increase
in cardiac contractility (Table 1).
All of the ET-1-induced effects were completely blocked by
pretreatment with L-754,142 (3 mg/kg plus 3 mg/kg/h i.v.).
There were no significant differences in LV PES (106 6 4
versus 104 6 8 mm Hg) and TSR (0.063 6 0.017 versus
0.064 6 0.018 mm Hg/ml/min) and EES (5.4 6 0.3 versus
5.4 6 0.3 mm Hg/ml), dE/dtmax (72.6 6 14.9 versus 75.7 6
13.3 mm Hg/s/ml), and MSW (80.4 6 6.1 versus 80.3 6 6.4 mm
Hg). Similar observations were also obtained after autonomic
blockade.
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Onishi et al.
Vol. 288
TABLE 3
Effects of ET-1 blocker on the PES-VES, dP/dtmax-VED, and SW-VED relations before and after CHF
PES-VES Relation
EES
Before CHF
Control
ET-1 blocker
After CHF
Control
ET-1 blocker
5.3 6 0.7
5.3 6 0.8
3.4 6 0.4*
4.8 6 0.8**
V0,ES
9.0 6 2.6
8.8 6 3.0
15.7 6 0.6*
17.4 6 0.9**
dP/dtmax-VED Relation
V100,ES
dE/dtmax
V0,dP/dt
29.4 6 3.1
29.6 6 3.2
82.0 6 12.8
82.3 6 11.4
4.8 6 2.1
5.3 6 1.7
47.3 6 3.5*
41.2 6 3.9**
42.4 6 7.8*
50.0 6 7.8**
13.1 6 2.2*
15.5 6 2.0**
SW-VED Relation
V1000,dP/dt
MSW
V0,SW
V1000,SW
18.8 6 1.9
18.7 6 2.1
78.1 6 4.4
81.2 6 7.5
24.8 6 2.8
24.3 6 2.4
37.8 6 2.5
37.2 6 2.9
42.0 6 6.3*
38.8 6 5.0**
58.1 6 3.3*
72.4 6 5.2**
37.7 6 3.7*
38.2 6 3.4
55.2 6 3.6*
51.8 6 4.1**
V0,ES, intercept with volume axis; V100,ES, volume associated with a PES of 100 mm Hg; dE/dtmax, slope of dP/dtmax-VED relation; V0,dP/dt, intercept with volume axis;
V1000,dP/dt, volume associated with a dP/dt of 1000 mm Hg/s; V0,SW, intercept with volume axis, and V1000,SW, volume associated with SW of 1000 mm Hg ml. Values are
mean 6 S.E. (n 5 7).
* P , 0.05, control value after CHF versus corresponding normal control value before CHF.
** P , 0.05, ET-1 blocker versus corresponding control value.
tively unchanged V100,ES (29.4 6 3.1 versus 29.6 6 3.2 ml),
V1000,dP/dt (18.8 6 1.9 versus 18.7 6 2.1 ml), and V1000,SW
(37.8 6 2.5 versus 37.2 6 2.9 ml) before and after L-754,142.
This indicates L-754,142 has no effect on LV contractile performance in normal dogs.
Effects of Pacing-Induced CHF
After the development of CHF at rest, the mean PED increased from 10.9 6 2.9 to 27.3 6 7.7 mm Hg (p , .05) (Table
2). The minimum LVP (0.6 6 0.7 versus 10.5 6 2.0 mm Hg,
p , .05) and mean LAP (4.2 6 2.0 versus 22.0 6 5.3 mm Hg,
p , .05) also increased. The LV VES and VED increased,
whereas cardiac output was decreased due to the marked
reduction in stroke volume (16.9 6 4.4 versus 11.4 6 4.1 ml).
t increased (31.1 6 3.7 versus 41.9 6 12.1 ms, p , .05). LV
contractility was also significantly impaired as indicated by
the decreased slopes and rightward shifts of the P-V relations
(Table 3).
Effects of ET-1 Blocker in Dogs with CHF
Steady-State Measurements. Steady-state hemodynamic response produced by L-754,142 at rest after CHF is
summarized in Table 2 and displayed in Fig. 1. L-754,142
had no significant effect on HR. After CHF, L-754,142 produced a decrease in PES (101 6 11 versus 93 6 10 mm Hg,
p , .05) and EA (9.6 6 2.9 versus 7.9 6 2.3 mm Hg/ml, p ,
.05) and an increase in SV (11.4 6 4.1 versus 13.0 6 4.6 ml,
p , .05). TSR (0.084 6 0.022 versus 0.065 6 0.015 mm
Hg/ml/min, p , .05) decreased and CBF increased with
L-754,142. This indicated that L-754,142 caused marked arterial dilation of both the systemic and coronary arteries. In
addition, L-754,142 caused a marked improvement in LV
diastolic performance as indicated by a significant decrease
in t (41.9 6 12.1 versus 37.7 6 10.4 ms, p , .05), a decrease
in minimum LVP, and an increase in 2dP/dtmax. In addition,
with L-754,142, PED and mean LAP (22.0 6 5.3 versus 17.7 6
4.2 mm Hg, p , .05) also were significantly reduced.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Fig. 2. LV P-V loops produced by transient caval occlusion during control
and after administration of L-754,142
in a normal dog. LV end-systolic P-V
relations are indicated by lines. The
PES-VES (top), dP/dtmax-VED (bottom
left), and SW-VED (bottom right) relations were unchanged by administration of L-754,142.
1999
1219
TABLE 4
Effects of nitroprusside on the steady-state hemodynamics and P-V
relations after CHF
Heart rate (beats/min)
Peak 1dP/dt (mm Hg/s)
Peak 2dP/dt (mm Hg/s)
LVPED (mm Hg)
LVPES (mm Hg)
Minimum LVP (mm Hg)
TSR (mm Hg/ml/min)
CBF (ml/min)
EES (mm Hg ml)
V0,ES (ml)
dE/dtmax (mm Hg/s/ml)
V0, dP/dt (ml)
MSW (mm Hg)
V0,SW (ml)
Control
Nitroprusside
117 6 8
1629 6 116
21666 6 109
26.6 6 4.0
97.0 6 4.1
9.9 6 0.5
0.088 6 0.018
43.3 6 4.9
3.9 6 0.7
13.1 6 4.8
43.8 6 2.2
9.4 6 5.2
59.5 6 2.6
29.7 6 4.9
118 6 9
1741 6 84*
21712 6 114*
19.4 6 3.0*
85.1 6 4.9*
6.3 6 1.4
0.082 6 0.019*
49.2 6 4.9*
3.7 6 0.7
12.0 6 4.3
43.3 6 1.9
11.1 6 5.2
58.5 6 3.4
29.5 6 5.3
LVP, indicates LV pressure; CBF, coronary blood flow; V0,ES, intercept with
volume axis; dE/dtmax, slope of dP/dtmax-VED relation; V0, dP/dt, intercept with
volume axis; V0,SW, intercept with volume axis. Values are mean 6 S.E. (n 5 4).
* P , .05, ET-1 versus corresponding control value.
Discussion
We found in conscious dogs that the plasma ET-1 levels
approximately double after the induction of pacing-induced
CHF. Before CHF, ET-1 blockade has no direct effect on LV
contractility and relaxation. However, after pacing-induced
CHF, ET-1 blockade with L-754,142 improves LV contraction
and relaxation independent of its vasodilatory effect. These
results suggest that endogenous ET-1 may contribute to the
functional impairment of both systolic and diastolic performance in CHF.
Increased plasma ET-1 levels have been found in experimental (Cavero et al., 1990; Margulies et al., 1990; Teerlink
et al., 1994; Shimoyama et al., 1996) and clinical (McMurray
et al., 1992; Rodeheffer et al., 1992; Wei et al., 1994) CHF.
Fig. 3. LV P-V loops produced
by transient caval occlusion after development of pacing-induced heart failure in same
dog. L-754,142 produced leftward shifts of PES-VES (top),
dP/dtmax-VED (bottom left), and
SW-VED (bottom right) relations with increased slopes.
This indicates that L-754,142
improved LV contractile performance after CHF.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
P-V Analysis. The effect of L-754,142 on LV P-V relations
after CHF is summarized in Table 3. A typical example of the
effect of L-754,142 on variably loaded P-V relations from one
animal with CHF is shown in Fig. 3. After CHF, L-754,142
produced a markedly leftward shift of the PES-VES relation
with an increased slope (3.4 6 0.4 versus 4.8 6 0.8 mm
Hg/ml, p , .05). In addition, L-754,142 also increased the
slopes of the dP/dtmax-VED relation (42.4 6 7.8 versus 50.0 6
7.8 mm Hg/s/ml, p , .05) and the SW-VED relation (58.1 6 3.3
versus 72.4 6 5.2 mm Hg, p , .05). This result shows that
blocking endogenous ET-1 with L754,142 produces marked
augmentation of LV contractility in CHF.
Effect of Nitroprusside after CHF. As shown in Table 4,
nitroprusside produced similar decrease in LV PES (99 6 4.4
versus 90 6 4.5 mm Hg, p , .05) and a similar increase in
coronary blood flow (43.3 6 4.7 versus 49.2 6 4.9 ml/min, p ,
.05) as produced by L-754,142. However, nitroprusside produced no significant increases in the slopes of PES-VES (3.9 6
0.7 versus 3.7 6 0.7 mm Hg/ml), dP/dtmax-VED, and SW-VED
(59.5 6 2.6 versus 58.5 6 3.4 mm Hg) relations.
LV-Arterial Coupling and Work Efficiency of LV. We
evaluated LV-arterial coupling and the SW/PVA ratio in
normal dogs and dogs with CHF at rest. Data are summarized in Table 5. In normal dogs, L-754,142 had no significant
alternations in EES/EA, and SW/PVA. However, in dogs with
CHF, L-754,142 significantly increased the EES/EA ratio
(0.59 6 0.07 versus 0.36 6 0.03, p , .05). The SW/PVA ratio
was also significantly augmented (0.45 6 0.03 versus 0.37 6
0.03, p , .05).
Plasma ET-1 Activation. In five of the seven studied
dogs, the plasma ET-1 level was measured. The resting levels
of ET-1 were 7.3 6 1.7 fmol/ml before CHF and increased
2-fold to 14.1 6 3.0 fmol/ml (p , .05) after CHF.
Endogenous Endothelin-1 in CHF
1220
Onishi et al.
Vol. 288
TABLE 5
Effects of L-754,142 on LV EES/EA, SW, and work efficiency in dogs before and after CHF
Before CHF
EES/EA
SW (mm Hg ml)
SW/PVA
After CHF
Control
ET-1 blocker
Control
ET-1 blocker
0.78 6 0.03
1566 6 153
0.55 6 0.02
0.80 6 0.07
1544 6 111
0.57 6 0.03
0.36 6 0.03
985 6 182
0.37 6 0.03
0.59 6 0.07*
1074 6 199*
0.45 6 0.03*
EA, arterial elastance; SW, stroke work; PVA, LV pressure-volume area. Values are mean 6 S.E. (n 5 7).
* P , .05 versus control after CHF.
doubled. However, the local levels (in which myocardium
exposed) of ET-1 might be much higher than the plasma ET-1
concentrations. For example, Loffler et al. (1993) found that
the ET concentration in ventricle was roughly 4 orders of
magnitude higher than the plasma concentration in normal
rabbits (Loffler et al., 1993). Furthermore, in CHF, the myocardial ET system is up-regulated, producing much higher
cardiac ET-1 levels than in normal (Wei et al., 1994; Kiowski
et al., 1995; Sakai et al., 1996). Thus, it is likely that the
much elevated cardiac ET-1 levels are responsible for the
endogenous ET-1-induced cardiac depression in CHF. This
finding is in agreement with the studies performed in anesthetized dogs, in which Lerman et al. (1991) demonstrated
that a 2-fold increase in circulating levels of ET-1 was sufficient to locally reach a threshold that facilitate the appearance of coronary spasm. Similarly, Yang et al. (1990) concluded that subthreshold concentration of ET-1 could
facilitate the appearance of human vascular spasm. It has
also been shown that low ET-1 levels (0.1 nM) inhibited
substance pacing-induced dilation in middle cerebral canine
arteries.
The present observations of endogenous ET-1-depressed
LV contraction and relaxation in CHF are similar to our
previous observations with angiotensin II (Cheng et al.,
1996), that the inotropic effect is reversed in CHF, but the
magnitude of the angiotensin II-induced cardiac depression
was higher. A similar reversal of the inotropic effect of ET-1
has been observed in immature myocytes (Kohmoto et al.,
1993), in hypertrophy (Ito et al., 1997), and in isoproterenolinduced CHF (Suzuki et al., 1998). It is possible that there
would be an additive effect of ET-1 and angiotensin II type 1
receptor blockade.
Our observations are also consistent with several studies
showing that acute ET-1 receptor blockade improves LV myocardial function (Kiowski et al., 1995; Cheng et al., 1996;
Shimoyama et al., 1996) and survival in CHF (Spinale et al.,
1997). The current findings of the beneficial cardiac effect
with ETA receptor blockade also are compatible with the
recent observations in a study of the chronic actions of ETA
receptor blockade (Spinale et al., 1997). Our results differ
from the observations of Li and Rouleau (1996) and Sakai et
al. (1996). In an anesthetized left coronary artery ligated rat
model of CHF, Sakai and colleagues reported that blocking
the ETA receptor with BQ123 induced a negative inotropic
action. Similarly, using isolated papillary muscle from normal dogs and from dogs with CHF induced by pacing, Li and
Rouleau found that ET-1 caused a positive inotropic response. These inconsistencies may have resulted from the
influence of ET-1- or ET-1 receptor blocker-produced changes
in loading conditions on conventional measures of LV performance, variable effect of anesthesia, and different levels of
CHF.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Consistent with these findings, we observed that plasma
ET-1 levels increased about 2-fold in pacing-induced CHF.
Both norepinephrine and angiotensin II (which are elevated
in CHF) increase the expression of prepro-ET-1 mRNA in
cultured endothelial cells (Masaki et al., 1991). Thus, these
neurohormones may contribute to the increased ET-1 in
CHF. In addition, shear stress and stretch stimulate endothelial cell production in ET-1 (Emori et al., 1991) and may
be an increased conversion of big ET to ET-1 in CHF (Teerlink et al., 1994).
Although ET-1 increases systemic vascular resistance
(Miller et al., 1989; Luscher et al., 1991; Kiowski et al., 1995)
and coronary resistance, we found that blocking the effect of
the low endogenous level of ET-1 in normal animals had no
detectable effect on systolic pressure, systemic arterial resistance, and coronary flow or resistance. This is consistent with
previous observations (Shimoyama et al., 1996). However,
after CHF, ET-1 blockade decreased PES and TSR and increased coronary flow. These data indicate that endogenous
ET-1 does not normally play a substantial role in the regulation of resting arterial blood pressure. However, after CHF,
the elevated ET-1 levels contribute to both systemic and
coronary vasoconstriction.
To avoid the potentially confounding effects of the influence of ET-1 on loading conditions on conventional measures
of LV performance, we evaluated LV contractile performance
in the P-V plane (Kass and Maughan, 1988; Little et al.,
1989). We found that before CHF, exogenous ET-1 infusion
caused an increase in the slope of LV P-V relations, which is
consistent with previous studies of normal myocardium (Watanabe et al., 1989; Neubauer et al., 1990). However, ET-1
blockade had no effect on LV P-V relations, suggesting that
the low levels of ET-1 had no direct cardiac effects in normal
subjects. In contrast, after CHF, ET-1 blockade produced a
significant improvement in LV contractile performance, as
indicated by the increased slopes and leftward shift of the LV
P-V relations. These effects are independent on the alterations of loading conditions and coronary blood flow because
equally hypotensive doses of nitroprusside produced a similar increase in coronary blood flow, but there was no change
in the LV P-V relations. Thus, it appears that the beneficial
effect of blocking ET-1 receptors is due to a removal of the
direct inhibition by ET-1 of LV contraction and relaxation.
This view is supported by the study of the effect of chronic
ETA receptor blockade in the rabbit with pacing-induced
CHF (Spinale et al., 1997). They found that chronic rapid
ventricular pacing, plus concomitant ETA receptor blockade
(without marked change in blood pressure), significantly improved LV and cardiomyocyte functional performance, normalized myocyte inotropic responses to calcium and b-adrenergic stimulation, and improved survival.
In the present study, after CHF, the plasma ET-1 was
1999
1221
In conclusion, the present study demonstrates that the
plasma levels of ET-1 are increased in CHF and that ET-1
receptor blockade in a model of CHF has direct beneficial
effects on LV contraction and relaxation. Thus, endogenous
ET-1, despite its positive inotropic action in normal myocardium, may contribute to the impairment of LV contraction
and relaxation in CHF.
Acknowledgments
We are grateful to Merck and Company for supplies of L-754,142.
We gratefully acknowledge the computer programming of Ping Tan,
the technical assistance of Mack Williams, and the secretarial assistance of Carol S. Corum.
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