Download Investigation of distal aortic compliance and

Clinical Science (1999) 96, 241–251 (Printed in Great Britain)
Investigation of distal aortic compliance and
vasodilator responsiveness in heart failure due
to proximal aortic stenosis in the guinea pig
Martyn P. KINGSBURY, Wenxin HUANG, Silvana GIULIATTI, Mark TURNER,
Ross HUNTER, Kim PARKER* and Desmond J. SHERIDAN
Academic Cardiology Unit, National Heart and Lung Institute, Imperial College of Science Technology and Medicine, St Mary’s
Hospital, Paddington, London W2 1NY, U.K., and *Physiological Flow Studies Group, Department of Biological and Medical
Systems, Imperial College of Science Technology and Medicine, Prince Consort Road, London SW7 2AZ, U.K.
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Hypotension and syncope are recognized features of chronic aortic stenosis. This study examined
vasomotor responses and dynamic compliance in isolated abdominal aortae after chronic
constriction of the ascending aorta. Guinea pigs underwent constriction of the ascending aorta
or sham operation. Sections of descending aorta were removed for studies of contractile
performance and compliance. Dynamic compliance was measured using a feedback-controlled
pulsatile pressure system at frequencies of 0.5, 1.5 and 2.5 Hz and mean pressures from 40 to
100 mmHg. Chronic (149p6 days) aortic constriction resulted in significant increases in organ
weight/body weight ratios for left ventricle (58 %), right ventricle (100 %) and lung (61 %). The
presence of heart failure was indicated by increased lung weights, left ventricular end-diastolic
pressure and systemic vascular resistance, reduced cardiac output and increased levels of plasma
atrial natriuretic peptide (166 %), adrenaline (i20), noradrenaline (106 %) and dopamine (i3).
Aortic rings showed similar constrictor responses to phenylephrine and angiotensin II, but
maximal vasodilator responses to acetylcholine and isoprenaline were significantly increased
(144 % and 48 % respectively). Dilator responses to sodium nitroprusside, forskolin and
cromokalim were unchanged. Compliance of all vessels decreased with increasing pulsatile
frequency and to a lesser extent with increased mean pressure, but were similar in aorticconstricted and control groups. Chronic constriction of the ascending aorta resulted in heart
failure and increased vasodilator responses to acetylcholine and isoprenaline in the distal aorta
while dynamic compliance was unchanged. We hypothesize that increased endotheliummediated vasodilatation may contribute to hypotension and syncope in patients with left
ventricular outflow obstruction.
INTRODUCTION
Heart failure is the inability of the heart to satisfy the
perfusion requirements of the body under ordinary
conditions despite an adequate venous return. It may
result from abnormalities of left ventricular systolic
function, diastolic function or both [1–3]. Left ventricular
obstruction due to aortic stenosis is another important
cause of heart failure and represents a pathological
increase in afterload. Despite some progress in the
treatment of heart failure, it is still a disabling disorder
with both high morbidity and high mortality [4,5]. One
of the principal haemodynamic characteristics of patients
with heart failure is the diminished response of cardiac
Key words : arteries, heart failure, vasoconstriction, vasodilatation.
Abbreviations : ANP, atrial natriuretic peptide ; L-NAME, NG-nitro-L-arginine methyl ester.
Correspondence : Professor D. Sheridan.
# 1999 The Biochemical Society and the Medical Research Society
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M. P. Kingsbury and others
output with physiological stress [5–7]. There are a
number of circulatory mechanisms, both central and
peripheral, which may be responsible [6,8].
Afterload is an important determinant of cardiac
output in patients with heart failure [9,10]. In patients
with low cardiac output due to heart failure, arterial
pressure is supported by an increase in systemic vascular
resistance [7,11–13]. Thus, abnormal vasoconstriction
both at rest and during exercise is frequently observed in
heart failure [11,14,15] and contributes to the low cardiac
output and elevated cardiac afterload. Systemic vascular
resistance is one component of afterload and represents
the resistance to steady flow. Arterial compliance, elegantly described as ‘ a pressure-flow buffer that stores a
portion of the mechanical energy discharged by the heart
during systole for delivery to the tissues during diastole ’
[16], is another important contributing factor to ventricular afterload. The pulsatile component of arterial
hydraulic load [3,11] is dependent on arterial compliance
and on the distribution of vascular resistance between the
proximal and peripheral circulation. Several conditions
are known to cause a reduction in arterial compliance in
man including hypertension [17], age [18,19] and atherosclerosis [20–22] and may therefore contribute to
increased ventricular afterload. Changes in any of the
components of ventricular afterload may have significant
effects on cardiac output. Understanding how these may
change in various forms of heart failure is therefore of
considerable importance.
Left ventricular outflow obstruction increases afterload and acts to disconnect the ejecting ventricle from the
normal sources of afterload described above. It also
masks the impact of left ventricular ejection on the
systemic circulation. We hypothesized that this may alter
distal arterial properties and this receives some support
from evidence that forearm vasodilatation occurs in
response to leg exercise in patients with aortic stenosis, in
contrast to the vasoconstriction seen in normal subjects
[23]. To test this we measured dynamic compliance and
vasomotor responsiveness in aortae obtained from guinea
pigs with heart failure after chronic constriction of the
proximal aorta.
METHODS
Induction of aortic stenosis
Proximal aortic stenosis was induced in male Dunkin–
Hartley guinea pigs (600–800 g) by aortic banding using
a modification of the method described by Ling and
deBold [24]. In brief, animals were anaesthetized with a
bolus intraperitoneal dose (30 mg\kg) of methohexitone
sodium (Brietal4, Eli Lilly) and given subcutaneous
sulphadoxine and trimethoprim (Borgal4, Hoechst),
0.5 mg\kg, as an antibiotic. They were ventilated with 0.4
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litres\min O at a rate of 100 breaths\min using a
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Harvard ventilator throughout the procedure, and supplementary anaesthesia using 0.5 % halothane by inhalation was administered if required. Animals were
intubated with a curved 12-gauge intravenous cannula
(Sherwood Medical) using a purpose designed laryngoscope. A thoracotomy was performed in the third left
intercostal space, a section of the upper portion of the
ascending aorta was cleared of connective tissue and a
small high-density plastic clip with an internal diameter
of 1.99 mm placed on the vessel. The thoracotomy was
then closed. When animals showed signs of breathing
spontaneously they were extubated, given subcutaneous
injections of 0.006 mg\kg buprenorphine (Temgesic4,
Reckitt and Coleman) for analgesia, and allowed to
recover in a warm single cage. Further subcutaneous
doses of antibiotic were given as required. Sham-operated
animals underwent identical operative procedures but the
plastic clip was not placed on the aorta. Animals were
housed at 20p2 mC with a relative humidity of 50p5 %
and a 13-h light\11-h dark cycle. Animals received
Biosure RGP diet and fresh water ad libitum.
All animal work and surgery was performed in
accordance with the Home Office Guidance on the
Operation of Animals (Scientific Procedures) Act 1986,
HMSO, London.
Haemodynamic assessment
Animals were anaesthetized with a bolus intraperitoneal
dose (30 mg\kg) of pentobarbitone sodium (Sagatal4,
RMB Animal Health Ltd). Heart rate was calculated
from ECGs recorded using needle electrodes inserted
subcutaneously and connected to an ECG amplifier
(5340CA). ECGs were analysed after analogue to digital
conversion using Po-Ne-Mah Acquire Plus data acquisition and analysis software. Lead II, which shows
dominant R wave QRS complexes in this setting, was
used to measure R wave voltage and QRS and QTc
intervals. In each case, computer-generated validation
marks confirmed correct identification of interval
boundaries and wave peaks. QTc intervals were calculated as QT interval\N(R–R). Left ventricular pressure
was measured by direct puncture and the right carotid
artery was cannulated to measure systemic blood pressure using pressure transducers (SensoNor 840). Aortic
flow was measured using a flow probe placed on the
descending thoracic aorta connected to a Transonic T108
ultrasonic blood flow meter. All measurements were
displayed on a Lectromed recorder (frequency response
200 Hz to chart, 5 kHz to Po-Ne-Mah) and recorded and
analysed using a computer and Po-Ne-Mah Acquire Plus
data acquisition software (12-bit resolution ; sampling
rates were 250 Hz for arterial pressure and flow and 1
kHz for ECGs and left ventricular pressure). Peripheral
resistance was calculated as the mean carotid pressure\
aortic blood flow and expressed in dynes:s−&.
Increased distal aortic vasodilatation in proximal stenosis
Isolated aortic ring preparation
Animals were killed by cervical dislocation 149p6 days
after surgery and the thoracic aorta rapidly removed and
placed in a cold modified buffered Krebs–Henseleit
solution (pH l 7.4) containing 118 mM NaCl, 4.7 mM
KCl, 1.2 mM MgSO , 1.1 mM KH PO , 24 mM
%
# %
NaHCO , 2.5 mM CaCl , 9 mM glucose and 2 mM
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pyruvate, equilibrated with a 95 % O \5 % CO mixture.
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The aorta was carefully cleaned and cut into 2.5–3-mm
rings which were mounted in 10-ml organ baths and
maintained at 37 mC in Krebs–Henseleit solution gassed
with 95 % O \5 % CO . Preparations were allowed to
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equilibrate for 1 h during which the solution was periodically replaced and resting tension adjusted to 3 g.
Maximal constriction was assessed by contracting the
rings with a 60 mM K+ solution. The tissue was then
washed until resting tension was regained and constriction response curves for phenylephrine (10−(–
10−% M) and angiotensin II (10−(–3i10−' M) were constructed. An increase in tension was expressed as a
percentage of maximum constriction to a 60 mM K+
solution. Vasorelaxation was assessed in rings submaximally
preconstricted
with
phenylephrine
(3i10−& M) ; dose–response curves to cumulative doses
of acetylcholine (10−*–10−% M), isoprenaline (10−*–
10−% M), sodium nitroprusside (10−*–10−% M), forskolin
(10−*–10−% M) and cromakalim (10−*–10−% M) were
obtained. In all experiments, responses were allowed to
reach a steady state before continuing and tissue was
repeatedly washed until basal tension was achieved
between dose–response curves. Tension was measured
isometrically using tension transducers (UF1, Dynamometer) and recorded using a Lectromed (Multitrace 4)
chart recorder and amplifiers.
Compliance measurements
Animals were killed by cervical dislocation, a laparotomy
was performed and the abdominal aorta was located and
a section approximately 1.5 cm long isolated. This was
carefully cannulated using a Portex tube (0.5 mm internal
diameter and 1.0 mm external diameter), removed and
placed in warmed (38 mC) buffered Krebs–Henseleit
solution gassed with 95 % O \5 % CO (pH l 7.4).
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After flushing and filling with Krebs–Henseleit solution,
the other end of the section was tied with a 4\0 Mersilk
suture and the length of the unconstrained artery segment
was measured using a precalibrated eyepiece graticule in
a binocular microscope (Nikon, Telford, U.K.). The
cannulated, Krebs–Henseleit-filled section of aorta was
connected to the perfusion circuit as shown in Figure 1.
The compliance of the artery segment was determined
by measuring its response to a pulsatile pressure generated by an isolated feedback control pump specifically
designed for perfusion measurements. The mean pressure
was determined by the height of the reservoir and the
Figure 1 System used to measure dynamic compliance in
isolated aortae
A feedback-controlled stepping motor (6) generates pulsatile pressure by
compression and decompression of a silicon tube (2) in an enclosed chamber (1).
Pressure and flow changes induced were monitored for feedback control and for
measurement of systemic compliance.
desired pressure waveform was generated by altering the
pressure in the sealed chamber surrounding the flexible
tube (silastic tubing, 9 cm long, 4.2 mm internal diameter
and 6.0 mm external diameter ; Portex Ltd, Kent, U.K.)
connected with the arterial segment. This was done by
driving a 20 ml glass syringe with a stepper motor (RSstepping linear actuator-standard, RS Components Ltd,
Northants, U.K.) controlled by a feedback control
program running on a PC (Elonex PC486DX2, 50 MHz,
Elonex plc, London, U.K.). Pressure was measured using
a pressure transducer (Druck, PDCR75) connected to the
perfusion circuit and flow was measured using an in-line
ultrasonic flow probe and flowmeter (Transonic T101D).
Data were displayed using a Lectromed (Multitrace 4)
chart recorder and recorded and stored on the computer
via an analogue-to-digital converter with a sampling
frequency of 100 Hz. The difference between the measured pressure and the target pressure waveform was
calculated at each sampling time and used to determine
the direction and stepping rate of the motor necessary to
eliminate the difference. The pump was able to produce
the desired pressure waveforms up to a frequency of
about 2 Hz with suitable adjustment of the pump
conditions, particularly the volume of air introduced into
the sealed chamber which acted to smooth the higher
frequency perturbations introduced by the feedback
control system and stepper motor.
The pressure and flow data recorded as a result of the
pulsatile perfusion were used to calculate dynamic
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compliance. Several analogue models of the flow system
were considered and it was determined that the response
of the system could be modelled adequately by an RC
series circuit with C representing the compliance of the
artery and R the resistance of the tube used to cannulate
the artery. For this linear circuit :
Z(ω) oPq\oQq l Rj1\(iωC)
where oPq and oQq are the Fourier transforms of the
pressure and flow and ω is the frequency. R was
determined from steady-flow measurements to be
17.49p0.019 mmHg:min−":ml−" and was assumed to be
the same in all experiments. Z was determined for each
artery by calculating Z from the fast Fourier transforms
of P and Q. The dynamic compliance was then calculated
as
c (ω) l
1
ω(QzQ#kR#)"#
Note that the dynamic compliance at different frequencies can be obtained simultaneously by using a
pressure wave containing those frequency components.
In this study, triangular waves with a fundamental
frequency of 0.5 Hz were used because they contain
significant power at the even harmonics, i.e. 1.5 Hz,
2.5 Hz, etc. Measurements were also made over a range of
pulse pressures to test for pressure-dependent changes in
vessel compliance. In all cases the minimum pressure of
the triangular waveform was 30 mmHg and the peak
pressure was increased from 40 mmHg to 100 mmHg in
steps of 10 mmHg. These pressures embraced the normal
physiological pressures in the guinea pig [25].
Blood sampling and analysis
Aortic-constricted and sham-operated control animals
were anaesthetized and 10-ml blood samples were collected into cooled tubes by cardiac puncture and kept on
ice. Blood to be assayed for angiotensin II and atrial
natriuretic peptide (ANP) levels was collected with
EDTA anticoagulant and 100 units of the proteinase
inhibitor Trasylol4. Blood to be assayed for catecholamine levels was collected with EGTA and glutathione
anticoagulant. All blood samples were spun at 2500
rev.\min at 4 mC for 15 min and the plasma samples
obtained were stored at k70 mC until analysis. Plasma
ANP and angiotensin II levels were measured using
commercially available antibody assay kits (IDS Ltd,
Bolton, U.K.). ANP was extracted from plasma on a SepPak C-18 cartridge, eluted with acetic acid and ethanol,
evaporated under vacuum and reconstituted in assay
buffer. Samples were quantified by radioimmunoassay
using sheep anti-ANP antiserum and a "#&I-labelled
ANP tracer. Angiotensin II was extracted in ethanol,
# 1999 The Biochemical Society and the Medical Research Society
evaporated under vacuum, reconstituted in assay buffer
and assayed by a competitive radioimmunoassay using
rabbit anti-angiotensin II antiserum and a "#&I-labelled
angiotensin II tracer. The iodinated tracers were then
measured on a gamma counter (Cobra 50005, CanberraPackard, Berks, U.K.). Catecholamines were extracted
from plasma using a solvent extraction method [26].
Separation was achieved by HPLC on a 5 µmi
22 cmi0.46 cm ODS column (RP18, Brownlee Labs)
using a 15 % (v\v) acetonitrile in phosphate\acetate
buffer containing dodecyl sulphate as a mobile phase.
Catecholamines were detected by electrochemical detection (ESA Coulochem 5100 A) [27].
Morphometric studies
Light-microscopic morphometric methods were used to
estimate aortic wall thickness and to check for vascular
damage. Aortic rings were washed in Krebs–Henseleit
solution and immersion fixed in the relaxed state with a
buffered fixative containing 2 % formaldehyde and 2 %
glutaraldehyde for 24 h. Tissue was washed in sodium
cacodylate buffer and dehydrated with increasing concentrations of ethanol. Rings were embedded in paraffin
wax and transversely cut into 5-µm thick sections.
Sections were mounted, stained with Haematoxylin and
Eosin stain and analysis carried out using an image
analysis system (Seescan Solitaire Plus, Cambridge, U.K).
Statistical analysis
Values were expressed as meanspS.E.M. Data were
tested for deviations from Gaussian distribution using
the Kolmogorov–Smirnov test and were compared with
appropriate tests using analysis software (Prism v2.01,
GraphPad Software Inc., San Diego, CA, U.S.A.).
Dose–response curves were analysed by fitting sigmoidal
curves using non-linear regression analysis ; EC and
&!
maximum values were obtained for each experiment.
Statistical analysis of this EC and maximum data
&!
enabled us to compare dose–response curves. In all tests
values of P 0.05 were taken to indicate no significant
difference between the parameters under comparison.
RESULTS
Changes in organ weight
After chronic (149p6 days) aortic banding there was
evidence of marked changes in cardiovascular morphology (Table 1). Cardiac hypertrophy was indicated by a
72 % increase in heart weight to body weight ratio
compared with age- and weight-matched sham-operated
controls. This hypertrophy was observed in the left
ventricle (58 %), right ventricle (100 %) and atria (211 %).
In addition, lung weight to body weight ratio was
Increased distal aortic vasodilatation in proximal stenosis
Table 1
Organ weight data
Values are meanspS.E.M. Statistical significance : ***P
corresponding sham-operated control.
Body weight (g)
Heart/body weight (%)
Left ventricle/body weight (%)
Right ventricle/body weight (%)
Atria/body weight (%)
Lung/body weight (%)
Left kidney/body weight (%)
Right kidney/body weight (%)
0.001 compared with
Sham (n l 24)
Banded (n l 30)
1098p30
0.241p0.004
0.160p0.003
0.038p0.002
0.027p0.003
0.440p0.016
0.285p0.007
0.278p0.006
1093p19
0.414p0.018***
0.253p0.009***
0.076p0.007***
0.084p0.013***
0.710p0.047***
0.290p0.008
0.283p0.005
increased by 61 %, but kidney weights remained unchanged. These changes in organ weight to body weight
ratio were all significant (P 0.001) and represent an
actual increase in organ weight as the body weights of
banded and sham-operated control groups were not
significantly different (1098p30 g versus 1093p19 g).
Morphometric studies
Light-microscopic morphometric studies showed that
there were no statistically significant differences in
relaxed diameter (1081p21 versus 981p97 µm), wall
thickness (165p12 versus 137p16 µm) or wall thickness
to lumen ratio (0.23p0.02 versus 0.22p0.04) in aortae
from sham-operated control or banded animals. Examination of aortic sections taken after either compliance or
organ bath experiments showed that the endothelium
was still present.
Haemodynamic and ECG measurements
Haemodynamic results are shown in Table 2. Aortic
banding resulted in a left ventricular\carotid artery
Figure 2 Plasma levels of angiotensin II (Ang II), atrial
natriuretic peptide (ANP), noradrenaline (NA), adrenaline
(AD) and dopamine (DA) in sham-operated control and aorticbanded guinea pigs
*P 0.05 and **P 0.01 : significant difference between banded and
corresponding sham-operated control value.
systolic pressure gradient of 22.8p3.5 mmHg, increased
left ventricular systolic and end diastolic pressures,
reduced aortic flow and increased peripheral resistance
compared with control values, which were in the physiological range for guinea pigs [25]. The pressure gradient
across the banded aorta corresponds to a resistance of
67 800 dynes:s−":cm−& which represents approximately
67 % of the total peripheral resistance measured in the
banded group. In the sham-operated group aortic resistance was not significantly different from zero. Heart
rate derived from ECGs was unchanged. Analysis of
QRS configuration showed a significantly increased R
wave voltage (37 %) and QRS duration (19 %) compared
with controls. QTc intervals were also significantly
increased (11 %) in aortic-banded animals.
In vivo haemodynamic data
LV, left ventricular. Values are meanspS.E.M. Statistical significance : *P
with sham-operated control.
Table 2
Systolic carotid pressure (mmHg)
Diastolic carotid pressure (mmHg)
LV systolic pressure (mmHg)
LV end-diastolic pressure (mmHg)
Aortic flow (ml/min)
Peripheral vascular resistance (dynes:s−1:cm−5)
Aortic gradient (mmHg)
Heart rate (beats/min)
R-wave height (mv)
QRS interval (ms)
QTc interval (ms)
0.05, **P
0.01 and ***P
0.001 compared
Sham (n l 10)
Banded (n l 9)
53.9p4.9
31.6p4.3
54.9p5.1
3.4p0.8
52.5p4.7
63 651p6140
0.9p1.2
250p9
0.92p0.08
66.70p2.00
307.98p7.86
46.7p2.7
32.4p3.4
69.5p3.5*
7.5p1.1**
26.9p4.9**
102 046p12 240*
22.8p3.5***
256p13
1.26p0.09*
79.72p4.53*
341.80p11.91*
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M. P. Kingsbury and others
Figure 3 Dose–constriction responses to phenylephrine
(top) and angiotensin II (bottom) in isolated aortae obtained
from sham-operated control and aortic-banded guinea pigs
Table 3
Figure 4 Dose–relaxation responses to acetylcholine (top)
and isoprenaline (bottom) in preconstricted aortae obtained
from sham-operated control and aortic-banded guinea pigs
***P 0.001 : significant difference in maximum of fitted banded and
corresponding sham-operated control curves.
Dose–response curve data
Values are meanspS.E.M. Statistical significance : ***P
0.001 compared with corresponding sham-operated control.
EC50 (10−7 M)
Maximum (%)
Agent
Sham (n l 10)
Banded (n l 10)
Sham (n l 10)
Banded (n l 10)
Phenylephrine
Angiotensin II
Acetylcholine
Isoprenaline
Sodium nitroprusside
Forskolin
Cromakalim
100.2p4.9
24.2p1.2
34.1p0.6
21.0p0.4
106.6p0.9
122.6p2.5
57.0p1.5
109p4.9
23.4p0.3
83.2p0.9***
31.0p0.6***
110.9p0.9
121.3p1.8
59.8p1.4
0.8p0.2
3.8p1.4
3.5p0.3
9.2p1.0
1.4p0.1
9.8p1.1
11.4p1.6
0.6p0.1
3.5p0.4
2.7p0.2
7.4p0.8
0.9p0.1
12.9p1.0
10.0p1.3
# 1999 The Biochemical Society and the Medical Research Society
Increased distal aortic vasodilatation in proximal stenosis
Figure 6 Dynamic compliance measured using pulsatile
pressures at 0.5, 1.5 and 2.5 Hz in aortae obtained from
sham-operated control (top) and aortic-banded (bottom)
guinea pigs
plasma dopamine levels and an almost 20-fold increase in
plasma adrenaline levels in banded animals (Figure 2),
both these increases being statistically significant (P
0.01). Plasma noradrenaline levels were also increased
(106 %) in banded animals although this did not reach
statistical significance. Plasma ANP levels were increased
by 166 % (P 0.05) in banded animals while there was
no change in plasma angiotensin II levels (Figure 2).
Isolated aortic ring preparation
Figure 5 Dose–relaxation responses to sodium nitroprusside (top), forskolin (middle) and cromakalim (bottom)
in preconstricted aortae obtained from sham-operated control and aortic-banded guinea pigs
Blood sampling and analysis
Blood samples obtained from anaesthetized animals
showed large increases in circulating catecholamine levels
in banded animals. There was a three-fold increase in
In aortic rings taken from banded animals there was no
significant difference in the maximal constriction response to a 60 mM K+ solution (2.7p0.2 g ; n l 21)
compared with corresponding sham-operated controls
(3.5p0.3 g ; n l 13). The aortic rings contracted to
phenylephrine and angiotensin II in a dose-dependent
manner and the data were plotted as concentration–
response curves for both sham-operated control and
banded animals (Figure 3). The phenylephrine response
curve from banded animals was not significantly different
from corresponding controls, whereas the dose–response
curves for angiotensin II were almost superimposed.
There were no significant differences between rings from
control or banded animals in maximum response or EC
&!
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M. P. Kingsbury and others
values obtained from either phenylephrine or angiotensin
II dose–response curves (Table 3).
Aortic rings precontracted with 3i10−& M phenylephrine relaxed in response to isoprenaline and acetylcholine administration in a dose-dependent manner as
shown in Figure 4. Dose–response curves were
computer-fitted using non-linear regression. The maximum of the fitted curve for isoprenaline was increased by
48 % (P 0.001) in rings from banded animals compared
with controls and the curve maximum for acetylcholine
was increased by 144 % (P 0.001) compared with
controls. There were no significant differences in the
EC values for either isoprenaline or acetylcholine
&!
between banded and control groups (Table 3). As the
EC values are unchanged this describes a shift of the
&!
dose–response curves for banded animals up the y-axis
and thus an increase in vasodilator efficacy. Vasodilator
responses to sodium nitroprusside, forskolin and cromakalim were almost identical in aortae from sham-operated
control and aortic-constricted animals (Figure 5).
The compliance of vessels from both banded and
sham-operated control animals decreased with increasing
frequency (Figure 6). The compliance also decreased
slightly with increases in the range of the pressure
waveform. There were, however, no significant differences in the dynamic compliance between vessels from
banded and sham-operated control animals. There was a
trend towards a decrease in compliance in aortic segments
from banded animals over all pressure ranges at a
frequency of 0.5 Hz although this did not reach statistical
significance.
DISCUSSION
The main findings of this study are that chronic proximal
aortic constriction resulted in severe cardiac hypertrophy
and cardiac failure. These changes were accompanied by
increased aortic vasodilator responsiveness to acetylcholine and isoprenaline, while constrictor responses,
dynamic compliance and gross morphology were unchanged. To investigate possible mechanisms for the
altered vasodilatation, dose responses to forskolin, sodium nitroprusside and cromakalim were constructed.
The similar responses to these agents suggest that the
mechanism is unlikely to involve altered responsiveness
to cyclic AMP or nitric oxide or to be a general alteration
in vasodilator responsiveness. The markedly different
response to acetylcholine suggests that the effect is likely
to be mediated by endothelium-mediated vasodilatation.
Given the large increase in plasma adrenaline concentration down-regulation of β -adrenoceptors might be
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expected in aortic-constricted animals and therefore a
diminished β -mediated vasodilator response. The
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marked increase in the vasodilator response to iso# 1999 The Biochemical Society and the Medical Research Society
Figure 7 Dose–relaxation responses to acetylcholine (top),
isoprenaline (middle) and sodium nitroprusside (bottom) in
the presence of L-NAME (3i10−5 M) in preconstricted aortae
obtained from sham-operated control and aortic-banded
guinea pigs
***P 0.001 : significant difference in maximum of fitted banded curves in the
presence and absence of L-NAME. There was no significant difference between the
maximum of sham-operated control curves and banded curves in the presence of
L-NAME.
Increased distal aortic vasodilatation in proximal stenosis
prenaline is consistent with increased endotheliummediated vasodilatation [28,29]. This is further supported
by attenuation of this response in rings in the presence of
NG-nitro-L-arginine methyl ester (L-NAME), 3i10−& M
(Figure 7).
Aortic constriction is widely used as a method of
inducing left ventricular hypertrophy. In the present
studies chronic banding of the proximal aorta resulted in
substantial enlargement of all the cardiac chambers and of
the lungs. The increases observed in R wave voltage, and
QRS and QTc duration, are consistent with left ventricular hypertrophy [30]. The presence of cardiac failure
is indicated by the marked increase in lung weights,
reduced aortic flow, increased peripheral resistance and
elevated left ventricular end-diastolic pressure. In these
experiments haemodynamic measurements and plasma
samples were taken during general anaesthesia and
therefore may not reflect levels in conscious animals. The
striking differences in plasma ANP, noradrenaline,
adrenaline and dopamine are in keeping with the presence
of heart failure [30–33], although angiotensin II levels
were unchanged, unlike other studies. To avoid interference from endogenous humoral and autonomic
stimulation in these experiments, constrictor and vasodilator responses were studied in vitro.
Vasoconstrictor responses to 60 mM K+, phenylephrine and angiotensin II were similar in aortae obtained
from control and banded animals. In contrast, vasodilator
responses to acetylcholine and isoprenaline were increased in aortae from banded animals. These findings
differ from a previous study [34], which explored
constrictor and vasodilator responses in isolated aortae
taken proximal and distal to an abdominal constriction
in rats. That study found increased constrictor responses
and reduced vasodilator responses proximal to aortic
constriction, but there was no change in vasodilator
responsiveness in segments distal to the constriction.
Several factors may account for these differences ; in the
present study the constriction was to the ascending aorta
and it was associated with features of both cardiac
hypertrophy and failure. It is possible, therefore, that
aortic constriction of the abdominal aorta is closer to the
human syndrome of coarctation and its complication of
systemic hypertension, whereas ascending aortic constriction mimics the hypotensive and vasodilator complications of aortic valve stenosis. The position of the
aortic band did not allow sufficient proximal aortic tissue
for study in these experiments.
Abnormal vasoconstriction at rest and during exercise
are recognized features of heart failure [11,14,15] and the
present findings of increased aortic vasodilator responses
appear paradoxical. These results cannot be applied
generally to the arterial circulation and it will be
important to clarify whether left ventricular outflow
obstruction alters the effects of heart failure on other
vessels, particularly resistance arterioles, as this may have
an important bearing on responses to treatment. Syncope
and hypotension are recognized complications of left
ventricular outflow obstruction. Suggested mechanisms
for this include carotid sinus hyper-reactivity [35],
arrhythmias [36,37], acute left ventricular failure [38] and
vasodilatation mediated by left ventricular baroreceptor
activation. The present findings raise the possibility that
abnormal locally mediated vasodilatation may be a
contributory factor.
Arterial compliance is an important element in left
ventricular hydraulic load and we were interested to
explore whether an increase in aortic compliance might
occur distal to the site of constriction. We reasoned that
proximal constriction would blunt the impact of ventricular ejection of the descending aorta resulting in some
degree of atrophy and possibly increased compliance.
Our results show that the morphology and compliance of
the distal aorta were unchanged. Several methods have
been used to measure arterial compliance [39,40]. The
method used here was based on a feedback control
system and measurements of instantaneous pressure and
flow in the isolated aortae. Compliance declined with
increasing perfusion pressure and with increasing pulsation frequency, changes which have been observed
previously in small porcine coronary arteries [41] and the
resistance vessels of spontaneously hypertensive rats [42].
A tendency for compliance at 0.5 Hz to be lower in the
aortic-constricted group was not significant. These
findings therefore suggest that alterations in aortic
compliance do not contribute to the haemodynamic
consequences of aortic stenosis.
Conclusions
Chronic constriction of the ascending aorta in guinea
pigs resulted in left ventricular hypertrophy and heart
failure. Aortic rings distal to the aortic constriction
showed similar constrictor responses to angiotensin II
and phenylephrine. In contrast, vasodilator responses to
acetylcholine and isoprenaline were increased while those
to forskolin, sodium nitroprusside and cromokalim were
unchanged. These findings indicate increased endothelium-mediated vasodilatation in aortic tissue distal to
a proximal constriction. We hypothesize that this may
contribute to hypotension and syncope in patients with
left ventricular outflow obstruction.
Limitations of the present study
To the best of our knowledge this is the first study to
examine vasodilator and compliance properties of the
distal aorta after chronic constriction of the ascending
aorta. The present model differs from aortic valve stenosis
in that the constriction is above the origin of the coronary
arteries. Although studying isolated arteries avoids interference from endogenous constrictor and dilator
# 1999 The Biochemical Society and the Medical Research Society
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M. P. Kingsbury and others
effects, it only allows responses to pharmacological
stimuli and neuronal stimulation cannot be explored.
Further work is needed to determine whether the changes
observed here are a general feature of the arterial
circulation and the mediators involved.
ACKNOWLEDGMENTS
We would like to thank Ms Lorraine Lawrence for her
assistance with the histology and Ms Laura Watson for
her work with the blood sample analysis.
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Received 9 June 1998/23 September 1998; accepted 1 October 1998
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