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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:1082–1094, 1997
Vol. 283, No. 3
Printed in U.S.A.
Angiotensin-Converting Enzyme Inhibition and Angiotensin II
Subtype-1 Receptor Blockade during the Progression of Left
Ventricular Dysfunction: Differential Effects on Myocyte
Contractile Processes1
FRANCIS G. SPINALE, HENRY H. HOLZGREFE, RUPAK MUKHERJEE, MARIA L. WEBB, R. BARRY HIRD,
MARTYN J. CAVALLO, JAMES R. POWELL and WILLIAM H. KOSTER
Accepted for publication August 21, 1997
ABSTRACT
Inhibition of the angiotensin-converting enzyme (ACE) in the
setting of chronic left ventricular (LV) dysfunction has been
demonstrated to have beneficial effects on survival and symptoms. However, whether ACE inhibition has direct effects on
myocyte contractile processes and if these effects are mediated primarily through the AT1 angiotensin-II receptor subtype
remains unclear. The present project examined the relationship
between changes in LV and myocyte function and beta adrenergic receptor transduction in four groups of six dogs each: (1)
Rapid Pace: LV failure induced by chronic rapid pacing (4
weeks; 216 6 2 bpm); (2) Rapid Pace/ACEI: concomitant ACE
inhibition (ACEI: fosinopril 30 mg/kg b.i.d.) with chronic pacing;
(3) Rapid Pace/AT1 Block: concomitant AT1 Ang-II receptor
blockade [Irbesartan: SR 47436(BMS-186295) 30 mg/kg b.i.d.]
with chronic pacing; and (4) Control: sham controls. With Rapid
Pace, the LV end-diastolic volume increased by 62% and the
ejection fraction decreased by 53% from control. With Rapid
Pace/ACEI, the LV end-diastolic volume was reduced by 24%
and the ejection fraction increased by 26% from Rapid Pace
only values. Rapid Pace/AT1 Block did not improve LV geom-
Angiotensin-converting enzyme is a membrane-bound metalloexopeptidase which cleaves angiotensin I to angiotensin
II (Antonaccio and Wright, 1990). The major actions of angiotensin II include increased vascular tone, enhanced sympathetic nerve activation and modulation in the activity of
other neurohormonal systems (Antonaccio and Wright,
1990). In addition to systemic production of Ang-II, studies
Received for publication April 1, 1997.
1
Supported by National Institutes of Health grant HL-45024 (F.G.S.), a
Basic Research Grant from Bristol Myers Research Institute (F.G.S.), American Heart Association Grant-in-Aid (F.G.S.) and an MUSC Post-Doctoral Research Award (R.B.H.). F.G.S. is an Established Investigator of the American
Heart Association.
etry or function from Rapid Pace values. Myocyte contractile
function decreased by 40% with Rapid Pace and increased
from this value by 32% with Rapid Pace/ACEI. Rapid Pace/AT1
Block had no effect on myocyte function when compared with
Rapid Pace values. With Rapid Pace/ACEI, beta receptor density and cyclic AMP production were normalized and associated with an improvement in myocyte beta adrenergic response
compared with Rapid Pace only. Although Rapid Pace/AT1 also
normalized beta receptor density, cyclic AMP production was
unchanged and myocyte beta adrenergic response was reduced by 15% compared with Rapid Pace only. ACE inhibition
with chronic rapid pacing improved LV and myocyte geometry
and function, and normalized beta receptor density and cyclic
AMP production. However, AT1 Ang-II receptor blockade with
chronic rapid pacing failed to provide similar protective effects
on LV and myocyte geometry and function. These unique findings suggest that the effects of ACE inhibition on LV geometry
and myocyte contractile processes in the setting of developing
LV failure are not primarily caused by modulation of AT1 Ang-II
receptor activation.
have demonstrated that Ang-II can be produced within the
myocardium through a local ACE system as well as by serine
proteases (Baker et al., 1992; Dzau, 1988; Ehring et al., 1994;
Gavras, 1994; Gohlke et al. 1994; Hirsch et al., 1991; Lindpaintner and Ganten, 1991; Maisel et al., 1989; Nolly et al.,
1994; Schunkert et al., 1993; Urata et al., 1990, 1993; Weber
et al., 1994). Although an area of investigation, it appears
that a major mode of action of angiotensin II is through a
specific receptor subtype, the AT1 Ang-II receptor (Dudley et
al., 1990; Lopez et al., 1994; Sechi et al., 1992; Urata et al.,
1989). Clinical trials have demonstrated that chronic ACE
inhibition improved symptoms and survival in patients with
LV dysfunction because of a wide range of etiologies (The
ABBREVIATIONS: ACE, angiotensin-converting enzyme; Ang-II, angiotensin II; AT1 Ang-II, angiotensin-II subtype-1 receptor; LV, left ventricle;
AMP, adenosine monophosphate; ANF, atrial natriuretic factor; EGTA, ethylene glycol bis(b-aminoethyl ether)N,N9-tetraacetic acid.
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Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina (F.G.S., R.M., R.B.H., M.J.C.) and Bristol
Myers Squibb Research Institute, Princeton, New Jersey (H.H.H., M.L.W., J.R.P., W.H.K.)
1997
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ACE inhibition or AT1 Ang-II receptor blockade in the setting
of progressive LV dysfunction will have significant and differential effects on myocyte contractile processes and the
beta receptor transduction system.
Methods
This study directly examined the effects of chronic ACE inhibition
and AT1 Ang-II receptor blockade on myocyte contractile processes
with the development of LV dysfunction caused by chronic rapid
pacing. Accordingly, ACE inhibition or AT1 Ang-II receptor blockade
was begun at the initiation of chronic rapid pacing and continued
throughout the pacing period. LV function, geometry and neurohormonal profiles were serially monitored during the entire pacing
protocol. At the termination of the study, isolated myocyte contractile function, beta adrenergic receptor transduction and function
were examined.
Model of pacing-induced LV dysfunction. Twenty-four adult
mongrel dogs of either sex (9–16 months of age, 15–25 kg, Hazelton,
Kalamazoo, MI) were used in this study. The animals were instrumented chronically to serially measure LV and arterial pressures as
well as obtain plasma samples. In addition, a pacemaker and stimulating electrode were implanted to produce rapid right ventricular
pacing. The animals were induced with thiopental (2 mg/kg, Pentothal, Abbot Labs, Chicago, IL), intubated and ventilated with 100%
oxygen. Maintaining a surgical plane of anesthesia with 1% to 3%
isoflurane (Aurthan, Anaquest, Madison, WI), a left thoracotomy
was performed and a shielded stimulating electrode was sutured
onto the right ventricular outflow tract, connected to a programmable pacemaker modified for programming heart rates up to 300
beats/min (Spectrax 5985, Medtronic, Inc., Minneapolis, MN) and
buried in a subcutaneous pocket. A previously calibrated microtipped transducer (model p5-X4, Konigsberg Instruments, Pasadena, CA) was placed into the LV chamber through a small incision
at the apex. The connection of the LV transducer was tunneled and
externalized in the suprascapular region of each animal. The pericardium was left open, the incision closed and the pleural space
evacuated of air. Next, the right carotid artery was exposed and a
vascular access port (model GPV, 9F, Access Technologies, Skokie,
IL) was placed in the artery, advanced to the aortic arch and sutured
in place for subsequent arterial blood pressure measurements and
blood sampling. The animals were allowed a 14-day recovery period
at which time proper operation of all implanted instrumentation was
confirmed. All animals used in this study were treated and cared for
in accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (National Research Council.
1985: NIH publication no. 86–23).
Experimental design. After recovery from the surgical procedure, baseline LV pressure and dimensions and arterial pressure
were measured, and plasma samples were obtained for each dog as
described in the following sections. The pacemakers were activated
for rapid ventricular pacing (216 6 2 bpm), and 1:1 capture confirmed by electrocardiography. The dogs were then randomly assigned to one of four treatment protocols: (1) ACE Inhibition: Dogs
were administered the ACE inhibitor, fosinopril (30 mg/kg p.o. b.i.d.),
during the pacing period (n 5 6). (2) AT1 Ang-II Receptor Blockade:
Dogs were administered the AT1 Ang-II receptor antagonist, Irbesartan [SR 47436(BMS-186295) Sanofi-Recherche, France/Bristol
Myers Squibb, NJ] at a dose of 30 mg/kg p.o. b.i.d. during the pacing
period (n 5 6). (3) Rapid Pacing Only: Dogs were given gelatin
capsules during the pacing period (n 5 6). (4) Sham Control: These
dogs were instrumented and cared for in a fashion identical with the
groups described with the exception of activation of the pacemaker
and drug treatment (n 5 6). Simultaneous electrocardiograms and
ventricular pressure recordings were performed frequently during
the 28-day pacing protocol to ensure proper operation of the pacemaker and the presence of 1:1 conduction. At weekly intervals, the
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CONCENSUS Trial Study Group, 1987; The SOLVD Investigators, 1991). However, despite these past clinical reports it
is still unclear whether the mechanisms of action of ACE
inhibition in the setting of LV dysfunction are caused by
global hemodynamic effects (changes in loading conditions),
reduced production of angiotensin II with subsequent diminished AT1 Ang-II receptor activation or modulation in the
activity of alternative neurohormonal systems and enzymatic
pathways. To begin addressing this issue, several studies
have been performed in which ACE inhibition was instituted
in experimental models of chronic LV dysfunction (McDonald
et al., 1994; Sabbah et al., 1994; Spinale et al., 1995). These
studies clearly demonstrated that ACE inhibition had direct
and beneficial effects on LV geometry and function. In addition, McDonald et al. (1994) demonstrated that, in a model of
LV dysfunction caused by myocardial injury, chronic therapy
with an alpha-1 receptor antagonist did not provide similar
beneficial effects on LV geometry and function when compared with ACE inhibition. Thus, the findings of these past
reports suggest that the mechanisms for the beneficial effects
of ACE inhibition in the setting of LV dysfunction are the
results of direct myocardial effects, rather than changes in
systemic loading conditions. However, whether chronic ACE
inhibition in the setting of developing LV dysfunction has
direct effects on myocyte contractile processes, and whether
these effects are mediated specifically through the AT1
Ang-II receptor remains unknown. Accordingly, the overall
goal of the present study was to determine the direct and
potentially differential effects of chronic ACE inhibition or
specific AT1 Ang-II receptor blockade on LV function and
geometry, and LV myocyte contractile processes in a model of
progressive LV dysfunction.
Past reports from this laboratory and others have demonstrated that chronic pacing-induced tachycardia in animals
caused LV dilation and dysfunction and activation of several
neurohormonal and sarcolemmal systems (Armstrong et al.,
1986; Cory et al., 1993; Eble and Spinale, 1995; Finckh et al.,
1991; Kim et al., 1994; Margulies et al., 1991; Perreault et al.,
1992; Ping and Hammond, 1994; Roth et al., 1993; Spinale et
al., 1990, 1992a,b, 1994, 1995; Travill et al., 1992; Williams et
al., 1994). Specifically, this laboratory has previously demonstrated that chronic pacing-induced tachycardia resulted
in decreased isolated myocyte contractile function (Spinale et
al., 1992a, b, 1994). In addition, the development of tachycardia-induced LV dysfunction is associated with down-regulation of beta adrenergic receptors, blunted beta adrenergic
responsiveness and alterations in the content and mRNA
expression of components of the beta adrenergic receptor
system (Ping and Hammond, 1994; Roth et al., 1993; Spinale
et al., 1994). This animal model of chronic rapid pacing produces similar functional and neurohormonal alterations
which have been observed previously in patients with severe
LV dysfunction (Benedict et al., 1993; Bristow et al., 1986;
Eschenhagen et al., 1992). In a recent report from this laboratory, concomitant ACE inhibition with chronic rapid pacing
improved LV function and geometry (Spinale et al., 1995).
Thus, chronic ACE inhibition in this model of pacing-induced
LV dysfunction appears to result in effects similar to those
observed in clinical studies (Antonaccio and Wright, 1990;
Lindpaintner and Ganten, 1991; The SOLVD Investigators,
1991). Accordingly, this model of pacing-induced LV dysfunction was used to test the central hypothesis that concomitant
ACE Inhibition or Ang-II Blockade
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Spinale et al.
tubes, frozen in a dry ice/methanol bath and stored at 280°C until
the time of assay. Norepinephrine concentration, atrial natriuretic
peptide levels, cyclic GMP content and plasma renin activity were
determined from these plasma samples. Plasma norepinephrine was
measured by high-performance liquid chromatography and normalized to picograms per milliliter of plasma (Goldstein et al., 1986). For
the atrial natriuretic peptide and cyclic GMP assays, the plasma was
first eluted over a cation-exchange column (C-18 Sep-Pak, Waters
Associates, Milford, MA). Standardized radioimmunoassay procedures were performed to determine atrial natriuretic peptide concentrations, cyclic GMP levels and plasma renin activity (Peninsula
Laboratories, Belmont, CA). All plasma assays were performed in
duplicate.
Myocyte isolation and myocardial sample preparation.
Four weeks after the institution of the protocols described above, the
dogs were brought to the laboratory, and a final series of LV function
measurements and plasma samples were obtained. The animals
were then anesthetized as described under “Neurohormonal Measurements,” a sternotomy performed and the heart quickly extirpated and placed in a phosphate-buffered ice slush. The great vessels, atria and right ventricle were carefully trimmed away, and the
LV weighed. The region of the LV free wall incorporating the circumflex artery (5 3 5 cm) was excised and prepared for myocyte
isolation. The posterior region of the LV free wall (4 3 4 cm) was
snap frozen in liquid nitrogen for subsequent sarcolemmal preparation. The region of the left ventricular free wall comprising the left
anterior descending artery (3 3 5 cm) was cannulated and prepared
for perfusion fixation.
Myocytes were isolated from the LV free wall with methods described by this laboratory previously (Mukherjee et al., 1993; Spinale
et al. 1992a,b, 1994).The left circumflex coronary artery was perfused with a collagenase solution (0.5 mg/ml, Worthington, type II;
146 U/mg) for 35 min. The tissue was then minced into 2-mm sections and gently agitated. After 15 min, the supernatant was removed, filtered and the cells allowed to settle. The myocyte pellet
was then resuspended in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (Gibco Laboratories, Grand Island, NY). With
use of this myocyte isolation method, a high yield (75 6 4%) of viable
myocytes was routinely obtained for the myocyte contractile function
measurements as described under “Myocyte Contractile Function
Measurements.”
The LV section for microscopic analysis was perfused with a buffered sodium cacodylate solution containing 2% paraformaldehyde,
2% glutaraldehyde solution (pH 7.4, 325 mOsM) for 20 min with a
perfusion pressure of 100 mm Hg. Full-thickness LV samples (1 cm
in thickness) were embedded in paraffin, sectioned at 4 mm in thickness and stained with hematoxylin and eosin. These sections were
imaged using an epi-florescence illuminator with a rhodamine filter
at a magnification of 10003. Myocytes in a cross-sectional orientation were digitized and analyzed with an image analysis system
(Zeiss/Kontron, IBAS, Germany). Only those myocytes in which the
nucleus was centrally located within the cell were digitized and
analyzed to ensure uniformity in cardiocyte profile measurements.
By use of this approach, the myocyte cross-sectional area could be
determined in situ.
Myocyte contractile function measurements. Isolated myocytes were placed in a thermostatically controlled chamber (37°C)
fitted with a coverslip on the bottom for imaging on an inverted
microscope (Sedival, Jena, Germany). The volume of the chamber
was 2.5 ml and contained two stimulating platinum electrodes. The
myocytes were imaged using a 203 long working distance objective.
Myocyte contractions were elicited by field stimulating the tissue
chamber at 1 Hz (S11, Grass Instruments, Quincy, MA) by current
pulses of 5-ms duration and voltages 10% above the contraction
threshold. The polarity of the stimulating electrodes was alternated
at every pulse to prevent the build-up of electrochemical byproducts.
Myocyte contractions were imaged by use of a charge-coupled device
with noninterlaced scan rate of 240 Hz (GPCD60, Panasonic, Secau-
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dogs were brought to the laboratory and the pacemaker was deactivated. After a 30-min stabilization period, LV pressures were recorded and echocardiographic measurements were obtained as described in the next section. To determine changes in neurohormonal
status with the progression of pacing-induced LV dysfunction,
plasma samples were obtained immediately after the LV function
measurements. After the LV function studies and plasma collection,
the pacemaker was reactivated (with the exception of the sham
controls). At the conclusion of the 28-day pacing protocol, the dogs
were returned to the laboratory for terminal study as described in
the next section.
Dosage rationale. The AT1 Ang-II receptor antagonist chosen for
the present study is a selective nonpeptide AT1 Ang-II receptor
antagonist and has been characterized previously (Cazaubon et al.,
1993; van den Meiracker et al., 1995). The pharmacological activity
of the specific dosage schedule used in the present study was fully
characterized in preliminary Ang-I and Ang-II dose-response studies. Dogs (n 5 3) were administered the AT1 Ang-II receptor antagonist (30 mg/kg b.i.d.) for 72 h to achieve steady-state plasma levels,
and Ang-II pressor studies were then performed. The pressor response to intravenous infusion of Ang-II (100 ng/kg) was reduced by
90% at 2 h after the morning dose on the fourth day compared with
untreated baseline values. At 12 h after the morning dose, Ang-II
pressor response was reduced by 33% from untreated base-line values. In four dogs, oral administration of the ACE inhibitor fosinopril
at 30 mg/kg reduced the Ang-I pressor response by 80%. Thus, the
dosage regimen used in the present study (30 mg/kg b.i.d.) provided
a pharmacological profile consistent with specific effects of AT1
Ang-II receptor blockade (McDonald et al., 1994; van den Meiracker
et al., 1995) and ACE inhibition. More importantly, this dosing
regimen had no effects on resting mean arterial blood pressure.
Thus, the confounding influences of differences in systemic hemodynamics could be minimized and provide for more meaningful comparisons of the direct effects of the different treatments on LV and
myocyte function in the setting of developing LV failure.
LV function measurements. Indices of LV systolic and diastolic
function were obtained at base line and at weekly intervals during
the 28-day pacing period using simultaneously recorded pressure
and echocardiographic measurements described previously (Laurenceau and Malergue, 1981; Tomita et al., 1991; Zile et al., 1992). All
measurements were performed in a darkened room with the animal
resting quietly in a sling. The arterial access port was punctured
with a 22-gauge Huber point needle (Access Technologies, Skokie,
IL) connected to a fluid-filled catheter. Pressures from the fluid-filled
aortic catheter were obtained with use of an externally calibrated
transducer (Statham P23ID, Gould, Oxnard, CA). The electrocardiogram and pressure waveforms were recorded by use of a multichannel recorder (Gould, TA4000, Irvine, CA) as well as digitized on
computer for subsequent analysis at a sampling frequency of 250 Hz
(PO-NE-MAH, Storrs, CT). Two-dimensional and M-mode echocardiographic studies (ATL Ultramark 7, 3.5 MHz transducer, Bothell,
WA) were used to image the LV from a right parasternal approach.
LV volumes and ejection fractions were computed from the twodimensional and M-mode echocardiographic recordings (Tomita et
al., 1991; Zile et al., 1992). Peak positive and negative (dP/dt) and
peak systolic wall stress were computed using methods described
previously (Tomita et al., 1991). Finally, LV mass was computed
from the two-dimensional targeted echocardiographic recordings using previously validated methods (Zile et al., 1992).
Neurohormonal measurements. To examine the relationship
between changes in neurohormonal status which accompany
changes in LV function with chronic rapid pacing, blood samples
were drawn at the conclusion of each LV function study. With the
animal resting quietly, 35 cc of blood was drawn from the arterial
access port into tubes containing ethylenediaminetetraacetic acid
(1.5 mg/ml), sodium azide (0.2 mg/ml) and aprotinin (1.15 trypsininhibiting units/ml). The blood samples were immediately centrifuged (2000 3 g, 10 min, 4°C), the plasma decanted into separate
Vol. 283
1997
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Results
In the present study, six dogs were successfully studied in
each of the following treatment groups: (1) 28 days of rapid
ventricular pacing and concomitant ACE inhibition, (2) 28
days of rapid pacing with concomitant AT1 Ang-II receptor
blockade, (3) 28 days of rapid pacing with no drug (gelatin
capsule only) and (4) sham-operated controls (no pacing or
drug administration). Myocytes were successfully harvested
from all animals at terminal study with no differences in the
yield of viable myocytes among groups (P . .75).
LV function with chronic rapid pacing; effects of
ACE inhibition or AT1 Ang-II blockade. The weekly
changes in LV end-diastolic volume, ejection fraction and
peak wall stress with chronic rapid pacing are shown in
figure 1. LV end-diastolic volume significantly increased in a
time-dependent fashion with each week of rapid pacing when
compared with sham controls or base-line values (P , .05). In
the rapid pacing only group, LV ejection fraction significantly
decreased from baseline values after 1 week of pacing (P ,
.05) and continued to decline with each week of pacing. After
2 weeks of rapid pacing, LV end-diastolic volume had increased from baseline values in the ACE inhibition and AT1
Ang-II receptor blockade group. However, with concomitant
ACE inhibition and rapid pacing, LV end-diastolic volume
was significantly lower than with rapid pacing alone values
for the entire pacing protocol (P , .05). After 1 week of
pacing, LV ejection fraction was significantly lower in the
ACE inhibition and AT1 Ang-II receptor blockade groups
than in baseline value or sham control groups (P , .05) and
declined with each week of pacing. After 4 weeks of rapid
pacing, the LV ejection fraction was higher in the ACE inhibition group than in the rapid pacing alone group (P 5 .038).
LV peak wall stress significantly increased with each week in
the rapid pacing only group when compared with the sham
control group or base-line values (P , .05). With concomitant
ACE inhibition and rapid pacing, LV wall stress was not
significantly different from base-line or sham control values
after 1 week of pacing (P 5 .417). In the ACE inhibitor and
rapid pacing group, LV peak wall stress remained significantly lower than in the rapid pacing only group for the
entire pacing protocol (P , .05). A summary of LV function
and hemodynamics obtained in sham controls, after 28 days
of rapid pacing and 28 days of rapid pacing with concomitant
ACE inhibition or AT1 Ang-II receptor blockade is presented
in table 1. Resting heart rate was increased and mean arterial pressure was reduced in the rapid pacing only group
when compared with sham controls. Concomitant ACE inhibition or AT1 Ang-II receptor blockade and rapid pacing
resulted in a significant reduction in mean arterial pressure
when compared with sham controls, but was not significantly
different from the rapid pacing only group (P 5 .261). After 4
weeks of rapid pacing, LV peak systolic pressure and peak
1dP/dt were significantly lower with rapid pacing than with
the control group, irrespective of drug treatment.
Neurohormonal changes with rapid pacing and ACE
inhibition or AT1 Ang-II blockade. A summary of weekly
changes in plasma norepinephrine, ANF and cyclic GMP are
presented in figure 2. Plasma norepinephrine significantly
increased from baseline values after 1 week in all of the dogs
undergoing chronic rapid pacing when compared with sham
controls or baseline values (P , .05). However, these 1-week
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cus, NJ). Myocyte motion signals were captured with the cell parallel
to the video raster lines, and this video signal was input through an
edge detector system (Crescent Electronics, Sandy, UT). The changes
in light intensity at the myocyte edges were used to track myocyte
motion (Mukherjee et al., 1993). The distance between the left and
right myocyte edges was converted into a voltage signal, digitized
and entered into a computer (80386, Zenith Data Systems, St. Joseph, MI) for subsequent analysis. Stimulated myocytes were allowed a 5-min stabilization period after which contraction data for
each myocyte were recorded from a minimum of 20 consecutive
contractions. Parameters computed from the digitized contraction
profiles included: percentage shortening (%), peak velocity of shortening (mm/s), peak velocity of relengthening (mm/s), total contraction
duration (ms) and time to peak contraction (ms). After collection of
baseline indices of myocyte function, measurements were then performed in the presence of 25 nM (2)-isoproterenol (Spinale et al.,
1994).
Beta adrenergic receptor system. To determine whether
changes occurred in the beta adrenergic receptor system or the
Na1,K1-ATPase system with concomitant ACE inhibition or AT1
Ang-II receptor blockade with chronic rapid pacing, membrane preparations were made by ultracentrifugation methods described previously (Bristow et al., 1986; Ping and Hammond, 1994; Roth et al.,
1993; Spinale et al., 1994). After sarcolemmal membrane preparation, protein content was determined by use of a standardized colorimetric assay (Bio-Rad Protein Assay, Bio-Rad, Richmond, CA). The
samples were then quick frozen and stored at 280°C until the time
of biochemical assay. Beta adrenergic receptor antagonist binding
studies were performed in the presence of six concentrations of
[125I]cyanopindolol (ICYP;74 Bq/mmol, Amersham Corp., Arlington
Heights, IL) from 0.015 to 0.75 nM (Bristow et al., 1986; Spinale et
al., 1994). A standard Scatchard linear regression analysis was performed on the amount of bound/free ligand with an r2 . 0.90 as the
criterion for acceptability of the data. With this analysis, the maximal number of binding sites Bmax expressed as femtomoles per
milligram of protein, and the equilibrium dissociation constant KD
(pM) were computed (Bristow et al., 1986; Roth et al., 1993; Spinale
et al., 1994). Adenylate cyclase activity was determined by timed
cyclic AMP production in aliquots of 30 to 50 mg/100 ml of membrane
preparation by previously described methods (Spinale et al., 1994).
Reactions were terminated by placing the tubes in an ice-cold bath
followed by centrifugation at 6,500 3 g for 5 min. Pellets were
resuspended in 0.5 ml buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM
EGTA, 10 mM phenylmethylsulfonyl fluoride, 2.8 mM EGTA), boiled
for 5 min and then centrifuged at 6,500 3 g for 10 min. The supernatant was assayed for cyclic AMP content by a competitive radiolabeled assay (RIA Kit, Advanced Magnetics Inc., Cambridge, MA).
Adenylate cyclase activity was determined at baseline as well as in
the presence of either 1023 M (2) isoproterenol or 100 mM forskolin.
Results were expressed as picomoles of cyclic AMP produced per
milligram of sarcolemmal protein per minute. All measurements
were performed in duplicate.
Data analysis. Indices of LV and myocyte function were compared among the treatment groups by analysis of variance. Analysis
of the morphological data was performed with the average measurements obtained for each animal, and the groups were compared by
analysis of variance. If the analysis of variance revealed significant
differences, pairwise tests of individual group means were compared
with Bonferroni probabilities (Steel and Torrie, 1980). The critical
values obtained from the Bonferonni probabilities were adjusted for
the multiple comparisons performed with respect to the LV and
myocyte function data. For comparisons in neurohormonal values
between groups, the Mann-Whitney rank-sum test was used (Steel
and Torrie, 1980). All statistical procedures were performed with the
BMDP statistical software package (BMDP Statistical Software Inc.,
Los Angeles, CA). Results are presented as mean 6 S.E.M. Values of
P , .05 were considered to be statistically significant.
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Spinale et al.
Vol. 283
plasma norepinephrine values were lower in both the ACE
inhibition and AT1 Ang-II receptor blockade groups than in
the rapid pacing only group (P , .05). With longer durations
of pacing, plasma norepinephrine values appeared to plateau, but remained significantly elevated from baseline values. In the ACE inhibition and AT1 Ang-II receptor blockade
groups, plasma norepinephrine remained higher than baseline values throughout the pacing protocol, but were consistently lower than rapid pacing only values (P , .05). After 1
week of rapid pacing, plasma ANF and cyclic GMP concentrations significantly increased from baseline values and remained elevated throughout the pacing protocol. With rapid
pacing and concomitant ACE inhibition or AT1 Ang-II receptor blockade, plasma ANF values were variable during the
pacing protocol. Plasma ANF increased from baseline values
with both ACE inhibition or AT1 Ang-II receptor blockade
after 1 and 2 weeks of pacing, but remained significantly
lower than with rapid pacing only values (P , .05). With
either concomitant ACE inhibition or AT1 Ang-II receptor
blockade and chronic rapid pacing, cyclic GMP was not significantly increased from baseline values. In the rapid pacing
only group, plasma renin activity remained unchanged from
sham control values after 28 days of rapid pacing (3.2 6 0.9
vs. 3.1 6 0.9 pmol/ml/h, respectively). With rapid pacing and
concomitant ACE inhibition or AT1 Ang-II receptor blockade,
plasma renin activity was higher than with the sham control
and rapid pacing only groups (6.9 6 1.6 and 7.27 6 1.9
pmol/ml/h, respectively, P , .05).
LV myocardial structure with rapid pacing and ACE
inhibition or AT1 Ang-II receptor blockade. LV mass
obtained at autopsy for the four groups of dogs is summarized
in table 1. There was no significant change in LV mass in the
chronic rapid pacing group when compared with the sham
control group. The LV mass/body weight ratios obtained in
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Fig. 1. Changes in LV end-diastolic volume (EDV), ejection fraction (EF) and
peak wall stress plotted with respect to
week 0 (baseline) values in controls, with
chronic rapid pacing, with chronic pacing and concomitant ACE inhibition and
with chronic rapid pacing and concomitant AT1 Ang-II receptor blockade (AII
blockade). (top panel) The LV end-diastolic volume significantly increased with
each week of rapid pacing and appeared
to plateau by week 4. LV end-diastolic
volume was significantly lower in the
concomitant ACE inhibition group at
each week of pacing when compared
with the rapid pacing only group (P ,
.05). LV end-diastolic volume was lower
with AT1 Ang-II receptor blockade at
weeks 1 and 2 when compared with
rapid pacing only values (P 5 .05). However by weeks 3 and 4, LV end-diastolic
volumes were similar in the concomitant
AT1 Ang-II receptor blockade group and
the rapid pacing only group (P . .30).
(middle panel) The LV ejection fraction
decreased in a time-dependent manner
with each week of pacing irrespective of
drug treatment (P , .05). However, at
weeks 3 and 4, a higher LV ejection fraction was observed in the rapid pacing
and ACE inhibition group when compared with rapid pacing alone values
(P , .05). There was no significant difference in the LV ejection fraction with
concomitant AT1 Ang-II receptor blockade when compared with the rapid pacing only values at any time point (P .
.50). (bottom panel) In untreated dogs,
LV wall stress increased significantly
with each week of pacing (P , .05). In
contrast, a significant reduction in LV
wall stress was observed with either
concomitant ACE inhibition or AT1 Ang-II
receptor blockade at weeks 2 to 4 of
chronic rapid pacing (P , .05). See table
1 for week 4 summary results.
1997
ACE Inhibition or Ang-II Blockade
1087
TABLE 1
LV function and mass with chronic rapid pacing: effects of ACE inhibition and AT1 Ang-II receptor blockadea
Rapid Pacingb
Rapid Pacing
and ACE
Inhibitionc
Rapid Pacing
and Ang-II
Blockaded
85 6 5
117.5 6 6.3
139.6 6 8.6
9.1 6 0.4
3142 6 292
52.4 6 3.4
1.0 6 0.1
122 6 10
71.8 6 1.9
124 6 12*
99.3 6 4.8*
113.8 6 4.4*
17.1 6 2.9*
1857 6 150*
83.8 6 8.4*
0.82 6 0.03*
189 6 13*
34.3 6 3.0*
108 6 7
92.9 6 3.4*
109.9 6 3.8*
15.2 6 1.3*
1694 6 91*
65.2 6 3.4*†
0.83 6 0.03*
166 6 8*†
42.7 6 2.8*†
116 6 5
96.7 6 2.1*
111.9 6 2.1*
12.9 6 1.9*
1835 6 137*
76.8 6 6.9*
0.83 6 0.03*
179 6 12*
36.4 6 3.2*
111 6 6
19.3 6 0.8
5.6 6 0.2
19.4 6 0.4
5.9 6 0.2
6
114 6 7
19.6 6 1.2
5.8 6 0.3
19.6 6 0.5
5.8 6 0.4
6
95 6 4*
18.1 6 0.5
5.2 6 0.3
18.3 6 0.4
5.1 6 0.2*
6
106 6 4
17.2 6 0.4
6.5 6 0.2
18.4 6 0.3
6.0 6 0.2
6
All values are presented as mean 6 S.E.M. * P , .05 vs. sham control; † P , .05 vs. rapid pacing only.
Rapid pacing: 28 days of right ventricular pacing, 220 bpm.
Rapid pacing and ACE inhibition.
d
Rapid pacing and AT1 Ang-II receptor blockade.
a
b
c
the present study for the control and rapid pacing groups
were all within normal limits for dogs of this size and were
not significantly different between groups (Bienvenu and
Drolet, 1991). Absolute LV mass was lower in the group with
rapid pacing and concomitant ACE inhibition than in the
sham control group (P 5 .025). When LV mass was normalized to tibial length, LV mass/tibial length was lower in the
rapid pacing and ACE inhibitor group than in the sham
control group (P 5 .041). In the rapid pacing and concomitant
AT1 Ang-II receptor blockade group, LV mass did not change
from control or rapid pacing only values (P . .50). LV myocardial structure and composition was examined by morphometric analysis of perfusion fixed myocardial sections. Myocyte cross-sectional area was computed from a minimum of
300 myocyte profiles from each group. The frequency distribution for this parameter is shown in figure 3. Myocyte
cross-sectional area was 297 6 6 mm2 in the sham control
group and decreased to 249 6 5 mm2 with chronic rapid
pacing (P , .05). With concomitant ACE inhibition and rapid
pacing, myocyte cross-sectional area was decreased from
both sham control and rapid pacing only values (207 6 4
mm2, P , .05). Concomitant AT1 Ang-II receptor blockade
caused changes in myocyte cross-sectional area similar to
rapid pacing only values (256 6 5 mm2, P 5 .37).
Beta adrenergic receptor system: effects of ACE inhibition or AT1 Ang-II receptor blockade. Beta receptor
density decreased significantly with chronic rapid pacing
with no change in affinity (table 2). In contrast, beta adrenergic receptor density remained unchanged with chronic
rapid pacing and concomitant ACE inhibition or AT1 Ang-II
receptor blockade. Although beta receptor affinity remained
unchanged in the ACE inhibition group, beta receptor affinity
was significantly increased in the AT1 Ang-II receptor blockade group. In the control group, cyclic AMP production significantly increased from basal levels after beta receptor
stimulation and with direct adenylate cyclase activation with
forskolin. Basal cyclic AMP production was reduced in the
chronic rapid pacing only group when compared with controls. In addition, cyclic AMP production was reduced by
approximately 50% in the rapid pacing group either after
beta receptor stimulation or by adenylate cyclase activation.
Concomitant ACE inhibition during rapid pacing resulted in
a normalization of cyclic AMP production both after beta
receptor stimulation and by direct activation of adenylate
cyclase. In contrast, cyclic AMP production remained significantly reduced in the AT1 Ang-II receptor blockade group.
Myocyte contractile function and chronic pacing:
ACE inhibition or AT1 Ang-II receptor blockade. A
summary of isolated myocyte resting length and baseline
contractile function is presented in table 3. Representative
contraction profiles of isolated myocytes taken from sham
controls, with chronic rapid pacing, and chronic rapid pacing
with concomitant ACE inhibition or AT1 Ang-II receptor
blockade are shown in figure 4. Isolated myocyte resting
length significantly increased from control values in all three
groups of dogs with rapid pacing. Isolated myocyte length
was lower in the groups with concomitant ACE inhibition or
AT1 Ang-II receptor blockade than in rapid pacing alone
values. Myocyte percent and velocity of shortening significantly decreased from control values in all of the rapid pacing
groups. However, in the rapid pacing and concomitant ACE
inhibition group, myocyte percent and velocity of shortening
were higher than in the rapid pacing only group or the group
with rapid pacing and AT1 Ang-II receptor blockade. The
velocity of myocyte lengthening was lower in all three groups
of dogs with chronic rapid pacing. In the rapid pacing and
concomitant ACE inhibition group, the velocity of myocyte
lengthening was significantly higher than in the rapid pacing
group or the rapid pacing group with concomitant AT1 Ang-II
receptor blockade. The time to peak myocyte contraction and
total duration of contraction were prolonged in the rapid
pacing group without drug treatment. The time to peak myocyte contraction was more prolonged in all rapid pacing
groups, irrespective of drug treatment. In the rapid pacing
and AT1 Ang-II blockade group, the total duration of contraction was similar to control values. Myocyte contractile function was examined in the presence of the beta adrenergic
agonist, isoproterenol (table 3). Isolated myocyte contractile
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Left ventricular function
Resting heart rate (bpm)
Mean arterial pressure (mm Hg)
LV systolic pressure (mm Hg)
LV end-diastolic pressure (mm Hg)
LV peak 1 dP/dt (mm Hg/s)
LV end-diastolic volume (cc)
LV posterior wall thickness (cm)
LV peak wall stress (g/cm2)
LV ejection fraction (%)
Left ventricular mass
LV mass (g)
Body weight (kg)
LV mass/body weight (g/kg)
Tibial length (cm)
LV mass/tibial length (g/cm)
Sample size (n)
Control
1088
Spinale et al.
Vol. 283
function in the presence of isoproterenol was significantly
lower in all three rapid pacing groups. However, beta adrenergic responsiveness was significantly greater in the group
with rapid pacing and ACE inhibition than in the group with
rapid pacing alone or with concomitant AT1 Ang-II receptor
blockade. In fact, myocyte function after beta adrenergic
stimulation in the AT1 Ang-II receptor blockade and rapid
pacing group was lower than the rapid pacing alone values.
Discussion
ACE inhibition has provided beneficial effects on symptoms and survival in patients with LV dysfunction (Spinale et
al., 1995; The CONCENSUS Study Group, 1987; The SOLVD
Investigators, 1991). However, the cellular and molecular
events that occur within the LV myocardium with chronic
ACE inhibition and LV dysfunction are not fully understood.
ACE is an important determinant of Ang-II production both
systemically and at the myocardial level (Antonaccio and
Wright, 1990; Baker et al., 1992; Dzau, 1988; Gavras, 1994;
Lindpaintner and Ganten, 1991). Although the distribution,
types and function of Ang-II receptors is an area of active
research, AT1 Ang-II has been studied the most intensively
and appears to mediate numerous physiological responses
(Dudley et al., 1990; Lopez et al., 1994; Sechi et al., 1992;
Urata et al., 1989). In addition, the AT1 Ang-II receptor has
been prevalent within the myocardium (Dudley et al., 1990;
Lopez et al., 1994; Sechi et al., 1992; Urata et al., 1989).
However, it remains unclear whether the effects of ACE
inhibition in the setting of LV dysfunction are caused primarily by diminished activation of the AT1 Ang-II receptor or
by alternative mechanisms. To address these issues, the
present study quantified changes in myocyte function and
the beta adrenergic system after either ACE inhibition or
AT1 Ang-II receptor blockade during the development of LV
dysfunction caused by chronic rapid pacing. The important
findings from the present study were 2-fold. First, ACE in-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Fig. 2. Serial changes in plasma norepinephrine, ANF and cyclic GMP in controls, with chronic rapid pacing, with
chronic pacing and concomitant ACE inhibition and chronic rapid pacing with
concomitant AT1 Ang-II receptor blockade. (top panel) Plasma norepinephrine
significantly increased from baseline values in the rapid pacing only group (P ,
.05) and appeared to plateau with longer
durations of pacing. Plasma norepinephrine concentrations were significantly
lower with ACE inhibition or with AT1
Ang-II receptor blockade when compared with rapid pacing only values (P ,
.05). (middle panel) Plasma levels of ANF
were significantly increased after 1 week
of rapid pacing (P , .05) and remained
elevated for the entire 4-week pacing
protocol. With concomitant ACE inhibition or AT1 Ang-II receptor blockade,
plasma ANF values were somewhat variable during the pacing protocol. ANF
was increased from baseline values after
2 and 4 weeks of pacing in both the ACE
inhibition and AT1 Ang-II receptor blockade groups (P , .05). After 4 weeks of
pacing, plasma ANF were lower in both
drug treatment groups than with pacing
alone values (P , .05) (bottom panel)
Plasma cyclic GMP levels increased after 1 week in the rapid pacing only group
(P , .05) and remained elevated for the
remainder of the rapid pacing protocol.
In contrast, there was no significant increase in plasma cyclic GMP levels with
either concomitant ACE inhibition or AT1
Ang-II receptor blockade during the pacing period (P . .50).
1997
ACE Inhibition or Ang-II Blockade
1089
hibition reduced the degree of LV dilation associated with
chronic rapid pacing, improved myocyte function and normalized beta receptor density and cyclic AMP production.
Second, AT1 Ang-II receptor blockade did not prevent the
development of LV dilation and dysfunction which invariably
occurs with chronic rapid pacing. Moreover, concomitant AT1
Ang-II receptor blockade did not result in significant improvement in myocyte contractile function or beta adrenergic
responsiveness. Therefore, the results from this study demonstrated that contributory mechanisms for the beneficial
effects of ACE inhibition in a model of LV dysfunction with
respect to myocyte contractile processes are not mediated
solely through the AT1 Ang-II receptor subtype.
ANF is a peptide hormone of cardiac origin, and ANF
receptor activation results in the generation of cyclic GMP
(Margulies et al., 1991). Consistent with past reports (Mar-
gulies et al., 1991; Travill et al., 1992), chronic rapid pacing
caused an early and persistent elevation in plasma levels of
ANF and cyclic GMP. Concomitant ACE inhibition or AT1
Ang-II receptor blockade caused a significant reduction in
plasma ANF or cyclic GMP when compared with untreated
dogs by chronic rapid pacing. Although beyond the scope of
the present study, potential mechanisms for this reduction in
ANF and cyclic GMP levels with chronic ACE inhibition or
AT1 Ang-II receptor blockade include diminished local ANF
production caused by modulation of local neuroendocrine
function, and enhanced ANF degradation. The development
and progression of LV dysfunction is associated with increased plasma catecholamine levels (Armstrong et al., 1986;
Benedict et al., 1993; Bristow et al., 1986; Eble and Spinale,
1994; Eschenhagen et al., 1992; Margulies et al., 1991; McDonald et al., 1994; Roth et al., 1993; Sabbah et al., 1994;
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Fig. 3. Frequency distribution of the
myocyte cross-sectional area from perfusion-fixed LV myocardial sections
taken from sham controls after 28 days
of rapid ventricular pacing, rapid pacing
and concomitant ACE inhibition and
rapid pacing with concomitant AT1
Ang-II receptor blockade. Myocyte
cross-sectional area values were fitted
to a Gaussian distribution (solid lines).
Chronic rapid pacing resulted in a significant decline in the myocyte cross-sectional area compared with controls (P ,
.05). A further decline from rapid pacing
alone values was observed with concomitant ACE inhibition and rapid pacing
(P , .05). There was no significant difference in the myocyte cross-sectional
area between the rapid pacing only
group and the concomitant AT1 Ang-II
receptor blockade group (P . .65). Summary statistics for this index of myocyte
geometry are presented under “Results.”
1090
Spinale et al.
Vol. 283
TABLE 2
Changes in the beta adrenergic and Na1,K1-ATPase systems with pacing-induced LV dysfunction: effects of ACE inhibition and AT1
Ang-II receptor blockadea
Beta adrenergic system
Beta receptor density (fmol/mg)
Beta receptor dissociation constant (KD, pM)
Adenylate cyclase activity Basal (pmol/cAMP/min)
Isoproterenol (pmol/cAMP/min)e
Forskolin (pmol/cAMP/min)f
Sample size (n)
Control
Rapid Pacingb
Rapid Pacing and
ACE Inhibitionc
Rapid Pacing and
Ang-II Blockaded
257 6 22
84 6 2
210 6 12
390 6 14#
657 6 74#
6
153 6 11*
88 6 4
154 6 17*
182 6 20*
380 6 47*#
6
244 6 13†
91 6 5
141 6 15*
331 6 29†#
595 6 681#
6
231 6 15†
106 6 10*
137 6 19*
211 6 28*§#
425 6 55*‡#
6
a
All values presented as mean 6 S.E.M. * P , .05 vs. sham control; † P , .05 vs. rapid pacing only; § P , .05 vs. rapid pacing and ACE inhibition; # P , .05 vs.
basal values.
b
Rapid pacing: 28 days of right ventricular pacing, 220 bpm.
c
Rapid pacing and ACE inhibition.
d
Rapid pacing and AT1 Ang-II receptor blockade.
e
Measured in the presence of 1023 M (2)-isoproterenol.
f
Measured in the presence of 100 mM forskolin.
adrenergic responsiveness was significantly reduced with the
development of pacing-induced LV dysfunction. Contributory
mechanisms for the blunted myocyte beta adrenergic response included a reduction in beta adrenergic receptor density and diminished cyclic AMP production. Concomitant
ACE inhibition with chronic pacing improved myocyte beta
adrenergic responsiveness. Results from the present study
suggest that contributory mechanisms for the improved myocyte beta adrenergic response with ACE inhibition included a
normalization of beta adrenergic receptor density and cyclic
AMP production. Maisel et al. (1989) demonstrated that the
ACE inhibitor captopril normalized beta receptor density and
transduction in the setting of cardiac hypertrophy induced by
chronic isoproterenol administration. Taken together, the
results from these past reports and the present study suggest
that a contributory mechanism for the beneficial effects of
ACE inhibition in the setting of progressive LV dysfunction is
the modulation of beta adrenergic receptor density and transduction. Although concomitant ACE inhibition with chronic
rapid pacing prevented the reduction in beta receptor density
and cyclic AMP production, myocyte response to beta adrenergic stimulation remained lower than normal myocytes. The
persistent defect in myocyte beta adrenergic response with
ACE inhibition is probably caused by several factors. First,
alterations in the content and activity of the guanine nucleotide-binding regulatory protein complex (G-protein complex) associated with the beta adrenergic receptor transduction system have occurred with the development of LV
dysfunction caused by chronic rapid pacing (Ping and Hammond, 1994; Roth et al., 1993; Spinale et al., 1994). Second,
findings from the present study as well as past reports have
demonstrated that pacing-induced LV dysfunction is associated with abnormalities in Na1,K1-ATPase density and
function (Kim et al., 1994; Spinale et al., 1992a). The present
study demonstrated that concomitant ACE inhibition with
chronic rapid pacing did not completely prevent these abnormalities in the Na1,K1-ATPase system. Finally, down-regulation of Ca11 transport systems within the sarcoplasmic
reticulum and alterations in Ca11 homeostasis have occurred with the development of LV dysfunction caused by
chronic rapid pacing (Cory et al., 1993; Perreault et al., 1992).
Thus, potential mechanisms for the failure of ACE inhibition
and chronic rapid pacing to normalize myocyte function and
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Spinale et al., 1994; Travill et al., 1992). In the present study,
both concomitant ACE inhibition or AT1 Ang-II receptor
blockade caused an equivalent and significant reduction in
circulating plasma norepinephrine when compared with
chronic rapid pacing only values. Consistent with this observation, Sabbah and colleagues (1994) demonstrated that
chronic ACE inhibition attenuated the increase in plasma
norepinephrine which occurred in the setting of progressive
LV dysfunction caused by coronary embolization. It has been
demonstrated previously that a chronic elevation in circulating catecholamines and persistent activation of the beta receptor system causes a reduction in beta receptor density
(Bristow et al., 1986). Thus in the present study, the normalization of beta receptor density which was observed with
either concomitant ACE inhibition or AT1 Ang-II receptor
blockade and rapid pacing was probably caused, at least in
part, by the reduction in plasma norepinephrine levels.
In the present study, chronic rapid pacing in dogs increased myocyte resting length and reduced myocyte crosssectional area. Concomitant ACE inhibition with chronic
rapid pacing was associated with a reduction in myocyte
length from rapid pacing only values and a further reduction
in cross-sectional area. Although concomitant AT1 Ang-II
receptor blockade reduced myocyte length from rapid pacing
only values, there was no significant change in myocyte
cross-sectional area. Thus, a contributory mechanism for the
reduction in LV end-diastolic volume with ACE inhibition
and chronic rapid pacing was the direct and selective effects
on myocyte geometry. This laboratory has demonstrated previously that the development of LV dysfunction caused by
chronic rapid pacing is associated with a significant reduction in the contractile performance of isolated myocytes (Spinale et al., 1992b). Consistent with a recent report (Spinale et
al., 1995), chronic ACE inhibition increased indices of myocyte contractile performance by more than 30% from rapid
pacing only values. Thus, in addition to the changes in myocyte geometry, concomitant ACE inhibition with chronic
rapid pacing caused a significant improvement in contractile
function. To quantitate more carefully the ability of the isolated myocyte to respond to an inotropic stimulus, the
present study examined myocyte function in the presence of
the beta adrenergic receptor agonist isoproterenol. Consistent with past reports (Spinale et al., 1994), myocyte beta
1997
ACE Inhibition or Ang-II Blockade
TABLE 3
Isolated myoCyte contractile performance with pacing-induced
LV dysfunction: Effects of ACE Inhibition and AT1 Ang-II receptor
blockadea
Base Line
150.7 6 1.0
179.6 6 1.4*
167.6 6 1.2*†
170.1 6 1.3*†
25 nM Isoproterenol
148.6 6 1.4
174.6 6 1.8*
164.5 6 1.6*†
168.8 6 2.4*†
4.28 6 0.09
2.43 6 0.07*
3.33 6 0.08*†
2.61 6 0.07*
10.38 6 0.23#
7.49 6 0.22*#
8.39 6 0.21*†#
6.19 6 0.35*†#
63.3 6 1.5
38.1 6 1.2*
49.5 6 1.3*†
41.1 6 1.1*
192.6 6 6.2#
145.2 6 5.2*#
156.1 6 4.8*†#
120.3 6 8.1*†#
67.4 6 1.9
35.0 6 1.3*
48.3 6 1.6*†
37.2 6 1.2*
208.6 6 6.7#
145.4 6 5.7*#
161.8 6 5.4*†#
114.4 6 7.9*†#
187.8 6 1.8
214.6 6 3.5*
208.9 6 2.0*
198.7 6 2.5*
165.0 6 2.0#
181.2 6 2.3*#
176.3 6 1.9*#
165.6 6 2.8†#
360.4 6 4.0
423.1 6 5.6*
411.5 6 3.8*
390.8 6 5.3*†
316.4 6 4.9#
370.8 6 5.4*#
361.7 6 4.9*#
334.9 6 6.9†#
414
469
455
338
220
227
231
120
a
All values presented as mean 6 S.E.M. * P , .05 vs. control; † P , .05 vs.
rapid pacing only; # P , .05 vs. base line.
b
Rapid pacing: 28 days of right ventricular pacing, 220 bpm.
c
Rapid pacing and ACE inhibition.
d
Rapid pacing and AT1 Ang-II receptor blockade.
beta adrenergic responsiveness include persistent defects in
sarcolemmal function and alterations in Ca11 homeostasis.
Based on the findings of the present study, future studies
which more carefully examine myocyte Ca11 homeostasis
after chronic ACE inhibition and chronic rapid pacing would
help clarify this issue. In the present study, concomitant AT1
Ang-II receptor blockade prevented the reduction in beta
adrenergic receptor density but failed to normalize cyclic
AMP production. With concomitant AT1 Ang-II receptor
blockade and chronic rapid pacing, cyclic AMP production
could not be returned to normal levels either by beta adrenergic receptor stimulation or by direct activation of adenylate
cyclase. These persistent defects in cyclic AMP production
with AT1 Ang-II receptor blockade were associated with a
reduction in myocyte beta adrenergic responsiveness from
both normal and chronic rapid pacing values. Activation of
the AT1 Ang-II receptor caused a reduction in cyclic AMP
production and activation of G-proteins independent of the
beta adrenergic receptor system (Allen et al., 1988; Antonaccio and Wright, 1990; Baker and Singer, 1988; Baker et al.,
1992). Characterization of AT1 Ang-II receptor activity and
G-protein structure and function with chronic rapid pacing
and either ACE inhibition or AT1 Ang-II receptor blockade
were beyond the scope of the present study. However, the
important and unique findings from this portion of the study
are 2-fold. First, these results suggest that the protective
effects of chronic ACE inhibition in this model of LV dysfunction with respect to beta adrenergic contractile responsiveness and transduction are not solely caused by modulation of
AT1 Ang-II receptor activation. Second, as opposed to ACE
inhibition, chronic AT1 Ang-II receptor blockade with pacinginduced LV dysfunction appeared to have differential effects
on beta adrenergic receptor transduction.
A unique finding of the present study was that chronic
ACE inhibition or specific AT1 Ang-II receptor blockade administered at equivalent and subhypotensive doses did not
provide equivalent effects on LV and myocyte geometry and
function in a model of developing LV dysfunction. Thus, the
beneficial effects of concomitant ACE inhibition with chronic
rapid pacing were probably at least partly the result of alternative receptor pathways and enzymatic processes other
than that of preventing myocardial Ang-II formation. It has
been well established that ACE inhibitors have inhibitory
effects on other enzyme systems such as bradykinin production, neurotensin, and substance P (Antonaccio and Wright,
1990; Gavras, 1994; Levens et al., 1992). Thus, the beneficial
effects of ACE inhibition on LV and myocyte function observed in the present study may be caused by the modulation
of these active peptide systems. There is significant evidence
to suggest that kallikrein-kinin proteolytic cascade systems
exist within the myocardium (Ehring et al., 1994; Gavras,
1994; Gohlke et al., 1994; Nolly et al., 1994; Weber et al.,
1994). Bradykinin, a nonapeptide which is produced by the
kallikrein cascade, has been implicated to play a direct role
in myocardial remodeling and functional recovery from myocardial ischemia (Ehring et al., 1994; Weber et al., 1994).
Moreover, ACE inhibition seems to prevent the rapid degradation of bradykinin and thereby potentiates the beneficial
effects of this peptide in the environment of myocardial ischemia (Ehring et al., 1994). McDonald and colleagues (1995)
demonstrated that in a canine model of myocardial injury the
beneficial effects of ACE inhibition could be attenuated by
the administration of a bradykinin antagonist. Thus, a contributory mechanism for the beneficial effects of concomitant
ACE inhibition which were observed in the present study
may be caused by enhanced bradykinin levels within the
myocardium. Future studies which use specific bradykinin
and AT1 Ang-II receptor antagonists in this model of chronic
LV dysfunction will be necessary to elucidate the interdependence and functional significance of the myocardial Ang-II
and bradykinin forming pathways with the progression of LV
failure.
A limitation of the present study is that in this model of
chronic rapid pacing, a significant increase in plasma renin
activity did not occur in the untreated dogs. This is in contrast to previous reports (Eble and Spinale, 1995; Margulies
et al., 1991; Travill et al., 1992), and suggests that significant
activation of systemic neurohormonal systems such as the
renin-angiotensin system had not occurred. Thus, the poten-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Resting length (mm)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Percent shortening (%)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Shortening velocity (mm/s)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Relengthening velocity (mm/s)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Time to peak contraction (ms)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Total contraction duration (ms)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
Number of cells (n)
Control
Rapid pacingb
Rapid pacing and ACEc
Rapid pacing and Ang-II
blockaded
1091
1092
Spinale et al.
Vol. 283
tial differential effects between ACE inhibition and AT1
Ang-II receptor blockade in which the progression of LV
dysfunction was accompanied by activation of the endocrinehumoral renin angiotensin system could not be determined.
Future studies would be appropriate in which the direct
effects of ACE inhibition or AT1 Ang-II receptor blockade are
instituted in this model of LV dysfunction and in which more
severe hemodynamic compromise with subsequent activation
of the systemic renin-angiotensin system occurs. The present
study used an identical dosage protocol for ACE inhibition
and AT1 Ang-II receptor blockade. This dose of AT1 Ang-II
receptor antagonist was chosen because inhibition of the
Ang-II pressor response was achieved without secondary systemic hemodynamic effects. This dosage strategy was chosen
to directly compare potential direct and differential effects of
chronic ACE inhibition and AT1 Ang-II receptor blockade on
myocyte contractile processes. It has been recently reported
that approximately a 10- fold higher dose of AT1 Ang-II
receptor antagonist is required to achieve an equivalent
blood pressure reduction when compared with ACE inhibition (van den Meiracker et al., 1995). Thus, it must be recognized that a much higher dose of AT1 Ang-II receptor
blockade may be necessary to potentially provide additional
beneficial effects in the setting of LV dysfunction. Mean
arterial pressure in dogs with either ACE inhibition or AT1
Ang-II receptor blockade was similar to untreated dogs undergoing chronic rapid pacing. Thus, the direct and differential effects of either concomitant ACE inhibition or AT1
Ang-II receptor blockade with chronic rapid pacing on LV
geometry and myocyte contractile processes were probably
not caused by differences in systemic loading conditions.
However, although not statistically significant, mean arterial
pressure was lower in the ACE inhibition group than in the
AT1 Ang-II receptor blockade or rapid pacing group. Thus,
the possibility remains that more favorable LV loading conditions were achieved in the ACE inhibition group than in
the AT1 Ang-II receptor antagonist group, which would in
turn influence overall LV pump function. In the present
study, plasma renin activity was not significantly increased
in the dogs undergoing chronic rapid pacing. This was probably because of the duration of rapid pacing and the degree of
LV dysfunction which had been induced in this model (Travill et al., 1992). These observations suggest that the differences in LV and myocyte geometry and function and the beta
adrenergic receptor system, which were observed with ACE
inhibition or AT1 Ang-II receptor blockade with pacing-in-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
Fig. 4. Representative isolated myocyte contraction profiles taken from sham controls after 28 days of rapid ventricular pacing, rapid pacing with
concomitant ACE inhibition and rapid pacing with AT1 Ang-II receptor blockade. Isolated myocyte length significantly increased with chronic rapid
pacing. With concomitant ACE inhibition or AT1 Ang-II receptor blockade, myocyte length was reduced from rapid pacing alone values. Statistics
for indices of myocyte contractile performance obtained at baseline and after beta adrenergic receptor stimulation are summarized in table 3.
1997
Acknowledgments
The authors express their appreciation to Dr. Michael Antonaccio
for the valuable advice and discussion provided during the execution
of this project.
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point in time. Thus, serial changes in LV myocardial and
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Ang-II receptor blockade in this model of LV dysfunction
were not addressed. Nevertheless, the present study demonstrated that concomitant ACE inhibition in a model of
chronic rapid pacing-induced LV dysfunction improved LV
and myocyte geometry and function, and normalized beta
receptor density and cyclic AMP production. Concomitant
AT1 Ang-II receptor blockade with chronic rapid pacing did
not provide similar effects on LV and myocyte geometry and
function. Therefore, the findings from the present study suggest that the beneficial effects of ACE inhibition on LV geometry and myocyte contractile function are not primarily
caused by modulation of AT1 Ang-II receptor activation but
rather by alternative mechanisms.
ACE Inhibition or Ang-II Blockade
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Send reprint requests to: Francis G. Spinale, MD, PhD, Division of Cardiothoracic Surgery, RM 418 CSB, 171 Ashley Avenue, Medical University of
South Carolina, Charleston, SC 29425.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 11, 2017
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