Download Left Ventricular Eccentric Remodeling and Matrix Loss Are Mediated

Journal of the American College of Cardiology
© 2007 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 49, No. 7, 2007
ISSN 0735-1097/07/$32.00
doi:10.1016/j.jacc.2006.06.083
PRECLINICAL STUDIES
Left Ventricular Eccentric Remodeling and
Matrix Loss Are Mediated by Bradykinin and Precede
Cardiomyocyte Elongation in Rats With Volume Overload
Thomas D. Ryan, MD, PHD,* Emily C. Rothstein, PHD,* Inmaculada Aban, PHD,†
Jose A. Tallaj, MD,‡§ Ahsan Husain, PHD,*‡ Pamela A. Lucchesi, PHD,* Louis J. Dell’Italia, MD*‡§
Birmingham, Alabama
Objectives
We hypothesized that left ventricular (LV) remodeling and matrix loss in volume overload (VO) are mediated by
bradykinin (BK) and exacerbated by chronic angiotensin-converting enzyme (ACE) inhibition.
Background
Chronic ACE inhibition increases anti-fibrotic BK and does not attenuate LV remodeling in pure VO. The relative
contribution of changes in extracellular matrix versus cardiomyocyte elongation in acute and chronic LV chamber
remodeling during VO is unknown.
Methods
Echocardiography, LV collagen content, and isolated cardiomyocytes were studied in rats after aortocaval fistula
(ACF) of 12 h, 2 and 5 days, and 4, 8, and 15 weeks. We also studied ACF rats after BK2 receptor (BK2R) blockade (2 days) or ACE inhibition (4 weeks).
Results
At 2 days after ACF, LV end-diastolic dimension (LVEDD)/wall thickness was increased, and LV interstitial collagen was decreased by 50% without cardiomyocyte elongation. The BK2R blockade prevented collagen loss and
normalized LVEDD/wall thickness. From 4 to 15 weeks after ACF, interstitial collagen decreased by 30% and left
ventricular end-systolic (LVES) dimension increased despite normal LVES pressure and isolated cardiomyocyte
function. The ACE inhibition did not decrease LVEDD/wall thickness, further decreased LV interstitial collagen,
and did not improve LV fractional shortening despite decreased LVES pressure.
Conclusions
Immediately after ACF induction, eccentric LV remodeling is mediated by interstitial collagen loss without cardiomyocyte elongation. Acute BK2R blockade prevents eccentric LV remodeling and improves function. Chronic ACE
inhibition does not prevent eccentric LV remodeling or improve function. These findings suggest that ACE inhibitormediated increase in LV BK exacerbates matrix loss and explains why ACE inhibition is ineffective in VO. (J Am Coll
Cardiol 2007;49:811–21) © 2007 by the American College of Cardiology Foundation
When subjected to a volume overload, the mammalian left
ventricular (LV) chamber dilates and remodels in an eccentric manner. It is generally believed that eccentric remodeling occurs by cardiomyocyte elongation and hypertrophy
that serves to normalize left ventricular end-diastolic dimension (LVEDD) to wall thickness ratio (1). Eccentric
hypertrophy therefore accommodates the increased preload
From the *Departments of Physiology and Biophysics, †Department of Biostatistics,
and ‡Department of Medicine, Division of Cardiovascular Disease, University of
Alabama at Birmingham, Birmingham, Alabama; and the §Birmingham Veteran
Affairs Medical Center, Birmingham, Alabama. Dr. Lucchesi is currently at the
Department of Pharmacology, Louisiana State University, New Orleans, Louisiana.
Dr. Rothstein is currently a Senior Imaging Scientist, Integrative Biology, Eli Lilly
and Company, Indianapolis, Indiana. Supported by National Institutes of Health
grants R01HL60707 and R01HL54816 to Dr. Dell’Italia, R01HL063318 to Dr.
Lucchesi, and 5T32HL07918-05 to Dr. Ryan, and Specialized Center for Clinically
Oriented Research in Cardiac Dysfunction grant P50HL077100. Drs. Ryan and
Rothstein contributed equally to this work.
Manuscript received March 28, 2006; revised manuscript received May 25, 2006,
accepted June 19, 2006.
because end-systolic stress is normal in a compensated
volume overload (1,2). During the compensated phase of
volume overload, forward cardiac output is maintained by a
greater-than-normal fractional shortening and stroke volume (2). In fact, normal or slightly reduced fractional shortSee page 822
ening and/or an increase in the LVEDD/wall thickness is
associated with a poor outcome in patients with volume
overload caused by mitral or aortic valvular regurgitation (2).
Cardiomyocyte elongation and dysfunction are accepted as
crucial events during the LV remodeling and decompensation
response to volume overload (1).
Angiotensin-converting enzyme (ACE) inhibitors are
widely used to treat heart failure; however, there is emerging
evidence showing that ACE inhibitors do not effectively
attenuate eccentric remodeling during pure volume overload.
Ryan et al.
Left Ventricular Remodeling in Volume Overload
11
14
8
7
8
6
8
7
8
7
8
Values are mean ⫾ SEM. *Based on ranks. †Based on variance component analysis. ‡p ⬍ 0.008 (⫽ 0.05/6) versus age-matched sham (SHM).
ACF ⫽ aortocaval fistula; dP/dtmax ⫽ rate of left ventricular pressure decline; LV ⫽ left ventricular; MAP ⫽ mean arterial pressure.
LV ⫺ dP/dtmax*
(mm Hg/s)
8
5,560 ⫾ 379
LV ⫹ dP/dtmax†
(mm Hg/s)
n
5,251 ⫾ 372
6,325 ⫾ 247
5,505 ⫾ 193
5,718 ⫾ 250
5,491 ⫾ 503
(n ⫽ 7)
6,393 ⫾ 417
(n ⫽ 5)
4,622 ⫾ 132
5,122 ⫾ 264
4,932 ⫾ 117
4,697 ⫾ 152
90 ⫾ 5
7,176 ⫾ 582
MAP* (mm Hg)
4,995 ⫾ 185
7,559 ⫾ 219
112 ⫾ 10
121 ⫾ 6
8,225 ⫾ 202
7,989 ⫾ 262
99 ⫾ 2
101 ⫾ 3
7,741 ⫾ 510
7,956 ⫾ 332
(n ⫽ 7)
94 ⫾ 6
114 ⫾ 10
7,989 ⫾ 342
(n ⫽ 5)
7,547 ⫾ 172
74 ⫾ 2‡
85 ⫾ 3
7,170 ⫾ 282
7,463 ⫾ 199
71 ⫾ 2
76 ⫾ 2
70 ⫾ 2‡
6,767 ⫾ 391
SHM
260 ⫾ 18
289 ⫾ 5‡
ACF
SHM
240 ⫾ 10
296 ⫾ 7
ACF
SHM
259 ⫾ 6
ACF
348 ⫾ 11‡
SHM
285 ⫾ 15
7,202 ⫾ 349
ACF
SHM
243 ⫾ 9
247 ⫾ 3
ACF
SHM
ACF
251 ⫾ 14
15-week
8-week
4-week
5-day
2-day
12-h
Hemodynamic Measurements in Rats With ACF and Age-Matched Sham Rats
Table 1
Animal preparation. Abdominal ACF was performed in
male Sprague-Dawley rats (200 to 250 g) as previously
described (4). Age-matched sham- and ACF-operated rats
were generated for echocardiographic and hemodynamic
study at 12 h; 2 and 5 days; and 4, 8, and 15 weeks, after
which time they were killed and tissues were collected for
morphometry, immunohistochemistry, and protein analysis
(n values given in Table 1). A separate group of rats was
killed at similar time points for isolated myocyte studies
(n values given in Table 2). In a third group of animals, the
BK receptor type 2 (BK2R) antagonist Hoe 140 (0.5
mg/kg/day, subcutaneous, Sigma/RBI, Natick, Massachusetts) was started after ACF or sham surgery, and animals
were killed at 2 days (n values given in Table 3). A subset of
rats (n ⫽ 8) was treated with angiotensin II (1 ␮g/kg/min,
osmotic mini-pump, Bachem Bioscience Inc., King of
Prussia, Pennsylvania) for 2 days after ACF to generate a
blood pressure increase similar to that with ACF ⫹ Hoe
140. In a final group, the ACE inhibitor was started in
the drinking water (ramipril at 10 mg/kg/day, Hoechst,
Frankfurt, Germany) after induction of ACF or sham,
Hemodynamic Measurements in Rats With ACF and Age-Matched Sham Rats
Methods
291 ⫾ 7
In addition to converting angiotensin I to angiotensin II, ACE
binds and cleaves bradykinin
ACE ⴝ angiotensin(BK) to inactive fragments with
converting enzyme
an affinity several-fold greater
ACF ⴝ aortocaval fistula
than that for angiotensin I (3).
BK ⴝ bradykinin
We previously showed an inBK2R ⴝ BK receptor type 2
crease in LV interstitial fluid BK
LV ⴝ left ventricle/
during the volume overload of
ventricular
aortocaval fistula (ACF) that is
LVED ⴝ left ventricular
further increased by ACE inhiend-diastolic
bition (4). Bradykinin has been
LVEDD ⴝ left ventricular
shown to decrease collagen proend-diastolic dimension
duction and increase matrix
LVES ⴝ left ventricular
metalloproteinase (MMP) expresend-systolic
sion in cultured fibroblasts (5–7).
LVESD ⴝ left ventricular
We hypothesized that the antifiend-systolic dimension
brotic response in acute volume
MAP ⴝ mean arterial
overload, leading to adverse LV
pressure
remodeling and dysfunction, inMMP ⴝ matrix
dependent of cardiomyocyte remetalloproteinase
modeling, is directly mediated by
TIMP ⴝ tissue inhibitor of
matrix metalloproteinase
BK. Here we show that the critical early event in LV remodeling
VCFr ⴝ velocity of
circumferential shortening
and dysfunction is BK-mediated
dissolution of the collagen matrix, and not cardiomyocyte elongation. Additionally, chronic ACE inhibition does not
attenuate LV remodeling or improve systolic function during volume overload, explaining why ACE inhibitors, which
increase antifibrotic BK, are ineffective in the treatment of
volume overload.
Abbreviations
and Acronyms
251 ⫾ 6
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February 20, 2007:811–21
Heart rate* (beats/min)
812
Ryan et al.
Left Ventricular Remodeling in Volume Overload
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813
Isolated Cardiomyocyte Function in Age-Matched Sham and ACF Rats
Table 2
Isolated Cardiomyocyte Function in Age-Matched Sham and ACF Rats
2-day
5-day
4-week
8-week
15-week
SHM
ACF
SHM
ACF
SHM
ACF
SHM
ACF
SHM
ACF
Cell length* (␮m)
131 ⫾ 4
132 ⫾ 8
120 ⫾ 6
123 ⫾ 6
134 ⫾ 2
144 ⫾ 4
146 ⫾ 4
162 ⫾ 4†
143 ⫾ 4
176 ⫾ 5†
Shortening* (%)
11.5 ⫾ 0.6
13.4 ⫾ 0.8
10.5 ⫾ 0.9
11.4 ⫾ 0.8
11.1 ⫾ 0.7
13.5 ⫾ 0.9
11.8 ⫾ 0.5
11.6 ⫾ 0.7
12.6 ⫾ 0.9
9.8 ⫾ 0.7
⫹dL/dt (␮m/s)
218 ⫾ 23
284 ⫾ 24
204 ⫾ 20
227 ⫾ 30
195 ⫾ 11
263 ⫾ 24
241 ⫾ 17
279 ⫾ 20
231 ⫾ 19
239 ⫾ 19
⫺dL/dt (␮m/s)
295 ⫾ 25
348 ⫾ 44
244 ⫾ 24
282 ⫾ 35
297 ⫾ 35
381 ⫾ 37
301 ⫾ 20
358 ⫾ 28
313 ⫾ 31
292 ⫾ 25
96 ⫾ 10
87 ⫾ 8
81 ⫾ 4
110 ⫾ 4†
114 ⫾ 9
100 ⫾ 7
103 ⫾ 7
94 ⫾ 4
130 ⫾ 8
119 ⫾ 7
7
6
7
9
9
13
15
15
16
Time to 50%
relaxation† (ms)
n
18
Values are mean ⫾ SEM. *Based on rank. †p ⬍ 0.01 versus age-matched SHM.
dL/dt ⫽ change in length with time; other abbreviations as in Table 1.
and animals were killed at 4 weeks. Hemodynamic and
echocardiographic measurements were made on all drugtreated animals. This protocol was approved by the
Animal Resource Program at the University of Alabama
at Birmingham.
Hemodynamic and echocardiographic measurements.
Rats were anesthetized with ketamine (80 mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitoneally). High-fidelity
LV pressures (SPR-249A Millar Mikro-Tip catheter transducer, Millar Instruments, Houston, Texas) were recorded
concurrently with echocardiography (Agilent Sonos 5500,
Philips, Bothell, Washington). The LV wall stress and function were calculated as described previously (8).
Collagen analysis. Hearts were immersion fixed in 10%
buffered formalin, embedded in paraffin, sectioned at 3-␮m
thickness, and stained with picric acid Sirius red F3BA.
Interstitial collagen volume percent was evaluated with light
microscopy at high power (40⫻ objective, 1,220⫻ video
screen magnification) in 30 to 40 randomly selected fields.
Collagen fiber thickness was measured using laser-confocal
images (40⫻ objective) of 15-␮m-thick sections and fluorescein isothiocyanate-labeled phalloidin. Image analysis
was performed using a line orthogonal grid overlay at 250 ⫻
250 ␮m for each field (approximately 5 fields per animal).
Average width was calculated at each grid intersection
point.
Adult rat LV myocyte isolation and measurement of
single-cell contractility. Left ventricular myocytes were
isolated by cardiac retrograde aortic perfusion as previously
described (9), with minor modifications. Briefly, hearts from
anesthetized rats (phenobarbital 50 mg/kg intraperitoneally)
were rapidly dissected and perfused using a Langendorf
apparatus with Kreb’s buffer (NaCl 118 mM, KCl 4.7 mM,
NaHCO3 25 mM, Ca Cl2 1.8 mM, Mg2SO4 1.2 mM,
KH2PO4 1.2 mM, glucose 11), followed by Ca2⫹-free
Kreb’s buffer, and Ca2⫹ free-Kreb’s buffer containing 100
U/ml type II collagenase (Worthington Biochemical, Lakewood, New Jersey). Left ventricular tissue was isolated and
further digested in collagenase/body surface area (BSA)
Kreb’s buffer containing 25 ␮M CaCl2. The resulting cells
were resuspended in perfusion buffer with 3% BSA, in
which the concentration of CaCl2 was stepwise increased to
1 mM. Rod-shaped cells were resuspended in Medium199
(Sigma, St. Louis, Missouri) supplemented with 5 mM
creatine, 2 mM L-carnitine, and 5 mM taurine and plated
on laminin-coated glass coverslips.
Following two 1-h incubation periods, with medium
changed between hours, coverslips were placed in a perfu-
Measurements
Effect
of Hoe 140
in Control
on Hemodynamic
Rats (2-Day
and
SHM)
Echocardiographic
and Rats 2 Days After ACF
Effect of Hoe 140 on Hemodynamic and Echocardiographic
Table 3
Measurements in Control Rats (2-Day SHM) and Rats 2 Days After ACF
SHM ⴙ Hoe 140
ACF ⴙ Hoe 140
SHM Untreated
ACF Untreated
259 ⫾ 6
296 ⫾ 7†
75 ⫾ 2
71 ⫾ 2
98 ⫾ 4†‡
6⫾1
7 ⫾ 2†
7⫾1
55 ⫾ 3
55 ⫾ 2
76 ⫾ 3†‡
76 ⫾ 7†‡
LV ⫹ dP/dtmax* (mm Hg/s)
6,767 ⫾ 391
7,463 ⫾ 199
9,902 ⫾ 1,006†
9,814 ⫾ 1,353
LV ⫺ dP/dtmax* (mm Hg/s)
4,698 ⫾ 152
4,932 ⫾ 117
7,160 ⫾ 826†
6,087 ⫾ 1,118
Heart rate* (beats/min)
MAP* (mm Hg)
LVEDP* (mm Hg)
LVESP (mm Hg)
1 ⫾ 0.1
311 ⫾ 19
280 ⫾ 25
90 ⫾ 10
LVEDD (mm)
6.9 ⫾ 0.2
8.0 ⫾ 0.2†
6.8 ⫾ 0.3‡
7.3 ⫾ 0.3
LVESD* (mm)
4.0 ⫾ 0.2
4.1 ⫾ 0.2
3.1 ⫾ 0.4
3.2 ⫾ 0.4
LVEDD/wall thickness (mm/mm)
4.6 ⫾ 0.2
6.0 ⫾ 0.3†
4.1 ⫾ 0.4‡
4.8 ⫾ 0.4
LV fractional shortening* (%)
41 ⫾ 2
49 ⫾ 1†
55 ⫾ 4†
57 ⫾ 4
Heart weight/body weight* (g/g ⫻ 103)
3.3 ⫾ 0.0
3.7 ⫾ 0.1†
3.4 ⫾ 0.1‡
3.4 ⫾ 0.1‡
7
6
n
7
8
Values are mean ⫾ SEM. *Based on variance component analysis. †p ⬍ 0.008 (⫽ 0.05/6) versus SHM. ‡p ⬍ 0.008 versus ACF.
LVED ⫽ left ventricular end-diastolic; LVEDD ⫽ left ventricular end-diastolic dimension; LVEDP ⫽ left ventricular end-diastolic pressure; LVES ⫽
left ventricular end-systolic; LVESD ⫽ left ventricular end-systolic dimension; LVESP ⫽ left ventricular end-systolic pressure; other abbreviations as
in Table 1.
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Ryan et al.
Left Ventricular Remodeling in Volume Overload
sion chamber with internal stimulating platinum electrodes
(Warner Instruments, Hamden, Connecticut) attached to a
Grass stimulator, and imaged using an inverted microscope
(Olympus America Inc, Melville, New York) and CCD
camera (Philips, Andover, Massachusetts). A rod-shaped
cell without blebbing or spontaneous contractions was
chosen for imaging. The chamber was perfused with Tyrode
basic salt solution and paced (3-ms duration) for 2 min at a
starting frequency of 0.5 Hz. The stimulating frequency was
stepwise increased from 0.5 to 3 Hz. The contractility of
each cell was recorded with a video edge detection system
(Crescent Electronics, Sandy, Utah) and analyzed using
Digi-Med System Integrator Model 200/1 and Cell Data
Analysis software program (MicroMed Technology, Inc.,
Houston, Texas). At each frequency, data were analyzed
after 1 min of pacing.
MMP analysis. Left ventricular tissue was homogenized in
lysis buffer (0.2% Triton X-100, 10 ␮g/ml aprotinin, 10
␮g/ml leupeptin, 0.5 mM PMSF, 500 ␮M Na3VO4) and
centrifuged at 14,000 rpm for 10 min at 4°C. Protein
concentrations were assessed with a bicinchonic acid assay.
Western blot analysis was performed with monoclonal anti-rat
MMP-13, monoclonal anti-rat tissue inhibitor of MMP-1
(TIMP-1), and rabbit polyclonal anti-rat TIMP-4 (all antibodies Chemicon, Temecula, California). Membranes were
washed and treated with horseradish peroxidase-conjugated
secondary antibodies (goat anti-mouse or anti-rabbit immunoglobulin G). Immunoreactive bands were visualized using
enhanced chemiluminescence reagents exposed to Hyperfilm at the linear range of film density. Films were scanned,
and densitometric analysis was performed with National
Institutes of Health image software.
Statistical analyses. All statistical analyses were done using
SAS software (SAS Institute Inc., Cary, North Carolina).
Generalized linear models (two-factor with interaction and
one-factor) were used to analyze all variables. We used the
Kolmogorov-Smirnov test and the Levene test on the residuals
to check normality and constancy of variance, respectively. If all
p values for testing these assumptions were ⬎0.05, we considered the model a good fit. Otherwise, we did a transformation
(natural logarithm or ranks) and then chose the appropriate
transformation that would resolve the assumption(s) violated.
Problems of non-normality and/or nonconstant variance of
the error terms were observed in a majority of the variables.
If transformations worked, p values for these goodnessof-fit tests were all ⬎0.10. Transformations applied to the
variables are indicated in the tables and figures.
In a few variables, neither log nor rank transformation
remedied the goodness-of-fit problem observed. In these cases,
we used the Kruskal-Wallis test for 1-factor designs and
variance component analyses (via SAS PROC MIXED) to
accommodate the unequal variances across the factor combinations for 2-factor designs. All tests were performed using a
5% significance level. The Bonferroni method was used in the
pair-wise comparisons for each variable to ensure an overall 5%
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
significance level. Appropriate decision rules based on the
Bonferroni adjustment are indicated in the tables and figures.
Results
LV remodeling in ACF. Twelve hours after the induction of volume overload, LVEDD had increased (p ⬍
0.05) by 20%, whereas LVEDD/wall thickness was
increased by 30% at 2 days after ACF (Figs. 1A and 1B).
There was no increase in cardiomyocyte length at either
time (Fig. 1C). Both LVEDD and LVEDD/wall thickness were increased by 40% at 4 to 15 weeks after ACF
(Figs. 1A and 1B). Mean LV wall thickness decreased (p
⬍ 0.05) at 15 weeks after ACF (Fig. 1D). Cardiomyocyte
length increased by 9% and 18% above shams only at the
8- and 15-week post-ACF time points, respectively (Fig.
1C), whereas LV weight, which had increased by 40% at
4 weeks after ACF, did not increase further after this
point (Fig. 1E).
Acutely, there was a 75% decrease in collagen volume
percent 12 h after ACF that persisted at day 2 (Figs. 2A to
2E). Collagen volume percent returned to sham values by 5
days after ACF (Fig. 2D); however, the average diameter of
collagen fibers at 5 days was increased compared with sham
(Fig. 2F). Chronically, collagen volume percent was decreased 30% in ACF rats at 4, 8, and 15 weeks (data not
shown).
Indexes of systolic function in ACF. Mean arterial pressure was decreased and heart rate increased both 12 h and 5
days after induction of ACF, but did not differ from shams
at 4, 8, and 15 weeks after ACF (Table 1). Left ventricular
end-systolic dimension (LVESD) did not differ from shams
at 12 h and 2 and 5 days after ACF. However, at 4 weeks
after ACF, LVESD increased by 40% and remained elevated in 8- and 15-week post-ACF rats despite the fact that
left ventricular end-systolic (LVES) pressure did not differ
from sham (Figs. 3A and 3B). Left ventricular fractional
shortening and velocity of circumferential shortening
(VCFr) were not different from sham values until 15 weeks
after ACF, when there was a decrease in VCFr concomitant
with an increase in LVES stress (Figs. 3C to 3E). This
decrease in VCFr occurred in the face of normal cardiomyocyte
fractional shortening and velocity of shortening (Table 2).
Indexes of diastolic function in ACF. Left ventricular
end-diastolic (LVED) pressure and LVED wall stress (Figs.
4A and 4B) were increased at 2 days and remained elevated
throughout the time course, whereas lung wet weight (Fig.
4C) was increased at 4 to 15 weeks and right ventricular
weight (Fig. 4E) was increased from 5 days after ACF
onward. Isolated cardiomyocyte relaxation velocity and time
to 50% relaxation did not differ in ACF and age-matched
shams across all time points, except at 5 days after ACF
(Table 2), whereas LV ⫺ rate of left ventricular pressure
decline (dP/dtmax) in ACF did not differ from shams at all
time points (Table 1).
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
Figure 1
Ryan et al.
Left Ventricular Remodeling in Volume Overload
815
LV Remodeling in Age-Matched Sham and ACF Rats
Left ventricular (LV) remodeling in aortocaval fistula (ACF) (open circles) at 12 h; 2 and 5 days; and 4, 8, and 15 weeks compared with age-matched shams (closed
circles). Left ventricular end-diastolic dimension (LVEDD) (A), LVEDD/wall thickness (B), isolated cardiomyocyte length (C), left ventricular end-diastolic (LVED) wall
thickness (D), and LV wet weight (E). Alternating black and white boxes each represent one 24-h period. Data are presented as mean ⫾ SEM, n values given in Tables
1 and 2 except: 4-week sham LVEDD, LVEDD/wall thickness, LVED wall thickness (n ⫽ 9); 4-week ACF echocardiographic parameters and LV wet weight (n ⫽ 9); 8-week
sham echocardiographic parameters and LV wet weight (n ⫽ 8); 15-week sham LV wet weight (n ⫽ 12); and 15-week ACF LV wet weight (n ⫽ 9). *p ⬍ 0.008
(⫽ 0.05/6) for sham versus ACF at each time of the 6 time points except for isolated cardiomyocyte length, where p ⬍ 0.01 (⫽ 0.05/5) for each of the 5 time points.
Analyses were based on ranks of all the variables in this figure.
Hoe 140 and LV remodeling and function in 2-day ACF
rats. Hoe 140 prevented the decrease in LV interstitial
collagen (Fig. 5A) and the increase in LVEDD and
LVEDD/wall thickness at 2 days after ACF (Table 3).
There was an increase in LVES pressure in both sham ⫹
Hoe 140 and ACF ⫹ Hoe 140 rats (Table 3); however,
LVES wall stress did not differ among all groups (data
not shown). Left ventricular fractional shortening was
increased in sham ⫹ Hoe 140 and ACF ⫹ Hoe 140
despite an increased left ventricular end-systolic pressure
compared with untreated sham and ACF cases (Table 3).
Left ventricular weight/body weight did not increase in
either ACF group; total heart weight/body weight increased in ACF versus age-matched shams. In contrast,
total heart weight/body weight did not increase in ACF ⫹
Hoe 140 versus sham ⫹ Hoe 140 and sham rats (Table 3). At
816
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
hypertrophy in ACF ⫹ angiotensin II when compared
with the ACF ⫹ Hoe 140 (Table 4).
Effect of ramipril on LV remodeling and function in
4-week ACF rats. Ramipril decreased LVES pressure and
heart weight/body weight in 4-week post-ACF rats. However, the decrease in LVES pressure was not accompanied
by an improvement in fractional shortening, LV dilation,
and LVEDD/wall thickness (Table 5). Ramipril was associated with a further decrease in LV interstitial collagen
compared with untreated ACF (Fig. 6).
Discussion
Figure 2
Representative Examples of LV Interstitial
Collagen in Control and ACF Rats
Representative examples of LV interstitial collagen in control (CTL) (n ⫽ 8) (A)
and ACF at 12 h (n ⫽ 5) (B), 2 days (n ⫽ 8) (C), and 5 days (n ⫽ 4) (D).
Mean collagen volume percent in 2-day CTL vs. ACF at 12 h, 2 days, and 5
days, where *p ⬍ 0.0167 (⫽ 0.05/3) using variance component analysis (E).
Mean collagen fiber width at 5 days of ACF versus CTL (F) where *p ⬍ 0.05
using t test. Data are presented as mean ⫾ SEM. Abbreviations as in Figure 1.
2 days after ACF, MMP-13 (rodent collagenase) was
increased, whereas TIMP-1 and -4 protein levels were not
altered. Hoe 140 prevented the increase in MMP-13 but
had no effect on TIMP-1 and -4 levels (Figs. 5B to 5D).
To assess whether the effects of Hoe 140 were through
a direct mechanism on the heart or an indirect mechanism through increased blood pressure, we infused angiotensin II and achieved a similar increase in MAP as
seen with Hoe 140 infusion (Table 4). In comparison
with ACF alone, angiotensin II partially prevented the
decrease in collagen and the increase in LVEDD/wall
thickness. However, there was an increase in cardiac
The current study provides new insights into the mechanism of eccentric remodeling in response to the volume
overload of ACF by combining LV chamber, cardiomyocyte, and interstitial collagen analyses. First, the critical,
very early event (12 h to 2 days) in LV remodeling is
BK-mediated dissolution of the collagen matrix, and not
elongation of the cardiomyocyte. Second, further adverse
LV remodeling and LV dysfunction are seen at the
4-week stage of volume overload in the presence of
normal cardiomyocyte shape and function. Third, treatment with ACE inhibitor in the first 4 weeks after ACF
does not attenuate LV remodeling or improve function
and further decreases collagen relative to ACF alone.
These findings explain why ACE inhibitors, which increase antifibrotic BK, are ineffective in attenuating LV
remodeling in a pure volume overload.
BK-mediated collagen dissolution exacerbates acute LV
remodeling. Important sequential changes in LV chamber and cardiomyocyte remodeling with ACF are shown
in Figure 1. Within 2 days, there is an increase in
LVEDD and LVEDD/wall thickness with no change in
cardiomyocyte length. At the same time, there is a
marked decrease in interstitial collagen, showing that
cardiomyocyte elongation is not the earliest step in LV
eccentric remodeling in response to volume overload.
This rapid decrease in interstitial collagen is likely to be
multifactorial. Simple stretch of cardiac fibroblasts in
vitro increases collagenase activity (10) and membrane
type 1-MMP expression (11). In addition, we and others
have found an increase in mast cells and BK in this early
phase of ACF (4,12). Mast cell secretory products such as
trypsin, chymase, stromelysin (MMP-3), and cytokines,
such as tumor necrosis factor-␣, are potent in vitro
activators of MMPs (13–16). Inhibition of ACE in ACF
rats significantly increased interstitial fluid BK but did
not attenuate increases in mast cell density, which was
blocked by the addition of BK2R blockade, supporting
the role of BK as an inflammatory mediator (4).
In addition to its effects on inflammatory cell recruitment, BK decreases collagen I and III mRNA production
in cardiac fibroblasts (5), modulates MMP activation
through the plasminogen activator system (17), and
increases MMP-2 (6) and MMP-9 (7) expression. Two
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
Figure 3
817
Indexes of LV Systolic Function in Age-Matched Sham and ACF Rats
Left ventricular (LV) function in ACF (open circles) at 12 h; 2 and 5 days; and 4, 8, and 15 weeks compared with age-matched shams (closed circles). Left ventricular
end-systolic (LVES) pressure (A), left ventricular end-systolic dimension (LVESD) (B), LV fractional shortening (FS) (C), LVES wall stress (␴) (D), and LV velocity of circumferential shortening (VCFr) (E). Alternating black and white boxes each represent one 24-h period. Data are presented as mean ⫾ SEM, n values given in Table 1
except: 4-week sham LVESD, LV fractional shortening, and LV VCFr (n ⫽ 9); 8-week sham LVESD, LV fractional shortening, and LV VCFr (n ⫽ 8). *p ⬍ 0.008 (⫽ 0.05/6)
sham versus ACF at each of the 6 time points. Analyses for left ventricular end-systolic pressure (LVESP), LVESD, and LVVCFr were based on their ranks, whereas analyses for LV fractional shortening and LVES ␴ were based on their natural logarithms.
days after the induction of ACF, MMP-13 levels are
increased in the absence of changes in TIMP-1 and -4
levels (Fig. 5) and collagen I and III expression (data not
shown), suggesting that the decrease in interstitial collagen is caused by increased degradation, but not decreased
synthesis. This acute loss of collagen in ACF stands in
stark contrast to a marked increase in collagen synthesis
that has been reported after 2 days of acute pressure
overload in the rat (18). Thus, an acute inflammatory
response [increased BK and mast cell density (4)] coupled
with a stretch stimulus, which occurs in the absence of
pressure overload, may induce a rapid loss of the collagen
matrix.
Blockade of BK2R prevents an increase in MMP-13 and
a decrease of interstitial collagen, and normalizes LVEDD/
wall thickness at 2 days of ACF stress. Further, LVES
pressure increases as LVESD trends down 25% vs. untreated ACF rats (Table 3), suggesting that BK2R blockade
improves LV function. This functional improvement could
be a consequence of more efficient transmission of forces
818
Figure 4
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
Indexes of Diastolic LV Remodeling in Age-Matched Sham and ACF Rats
Indexes of diastolic LV remodeling in ACF (open circles) at 12 h; 2 and 5 days; and 4, 8, and 15 weeks compared with age-matched shams (closed circles). LVED pressure (A), LVED wall stress (␴) (B), lung wet weight (C), and right ventricular (RV) wet weight (D). Alternating black and white boxes each represent one 24-h period.
Data are presented as mean ⫾ SEM, n values given in Table 1 except: 4-week sham LVED pressure and LVED ␴ (n ⫽ 5); 4-week sham lung wet weight and RV wet
weight (n ⫽ 8); 4-week ACF lung wet weight and RV wet weight (n ⫽ 9); 8-week sham lung wet weight and RV wet weight (n ⫽ 8); 15-week sham lung wet weight and RV
wet weight (n ⫽ 12); and 15-week ACF lung wet weight and RV wet weight (n ⫽ 9). *p ⬍ 0.008 (⫽ 0.05/6) sham versus ACF at each of the 6 time points. Analyses
were based on ranks of all the variables in this figure. Abbreviations as in Figure 1.
between adjacent myocytes and myofibers because of a
preservation of the matrix. Interestingly, antagonism of
BK2R in ACF animals is associated with a decrease in
cardiac hypertrophy despite the increase in MAP. To test
whether the local cardiac effects of BK2R blockade are
important, or whether functional and remodeling improvements during ACF are attributable to the increase in blood
pressure caused by BK2R blockade, we examined the effect
of a hypertensive challenge that was similar in magnitude to
that seen with Hoe 140 treatment. Using angiotensin II
infusion, we achieved an increase in blood pressure at 2 days
after ACF equivalent to that seen with Hoe 140 treatment.
Similar to Hoe 140, angiotensin II increased collagen matrix
and normalized LVEDD/wall thickness compared with
ACF alone; unlike Hoe 140, it also increased cardiac
hypertrophy. Because BK is upregulated acutely in the LV
during ACF, it is tempting to speculate that its blockade in
the heart prevents the loss of collagen through a direct
mechanism rather than through an indirect effect on blood
pressure.
At 5 days after ACF, the collagen volume percent returns
to normal; however, this rebound is abnormal because there
are fewer collagen fibers and these fibers are thicker than
normal (Fig. 2). This rebound in collagen occurs in the
absence of an increase in MAP, and may also be a response
to a local increase in cardiac interstitial angiotensin II (4).
The change in collagen structure could be a temporary
adaptive response because it is associated with a cessation of
further LV dilation at 5 days. However, at 4 weeks after
ACF there is a subsequent decrease in extracellular matrix
and a marked increase in LVEDD/wall thickness.
Adverse LV remodeling and dysfunction in chronic
volume overload. Another important finding of this study
is that LV systolic and diastolic dysfunction (4 to 15 weeks)
is not related to cardiomyocyte dysfunction. There is evidence of LV systolic dysfunction as early as the 4-week stage
of ACF (Fig. 3), manifested by an increase in LVESD,
whereas LV fractional shortening does not increase despite
an increase in preload and normal LVES wall stress. At 4
weeks after ACF, LVESD is increased in the presence of
normal LVES pressure, resulting in a significant increase in
LVES wall stress at 15-week ACF. It is also important to
note that there is no incremental increase in LV hypertrophy 4 weeks after induction of ACF, despite an increase in
LVES wall stress at 15 weeks. In addition, ACF LV wall
thickness is decreased and cardiomyocytes are elongated at
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
Figure 5
819
Indexes of Collagen Homeostasis in Hoe 140-Treated Sham and ACF Rats
Collagen volume percent (A), matrix metalloproteinase (MMP)-13 (B), tissue inhibitor of matrix metalloproteinase (TIMP)-1 (C), and TIMP-4 (D) levels at 2 days in
sham (SHM) and ACF rats with and without Hoe 140 treatment. Data are presented as mean ⫾ SEM, n values given in Table 3. *p ⬍ 0.008 (⫽ 0.05/6) versus SHM;
†p ⬍ 0.008 versus ACF. Collagen volume and MMP-13 were analyzed via variance component, whereas TIMP-1 and -4 were analyzed using a Kruskal-Wallis test. Abbreviations as in Figure 1.
15 weeks, accentuating adverse eccentric LV remodeling
and contributing to a decrease in LV VCFr, all in the face
of normal cardiomyocyte function. At a similar ACF time
point, Wang et al. (19) found enhanced LV ␤1-receptor
density and increased basal activity. Thus, the disparity
between isolated myocyte function in vitro and LV chamber
function in vivo could be caused by loss of collagen
structural support.
Hemodynamic
Untreated
ACF,and
2-Day
Morphometric
ACF ⴙ HoeParameters
140, and 2-Day
in 2-Day
ACF ⴙ Ang II
Hemodynamic and Morphometric Parameters in 2-Day
Table 4
Untreated ACF, 2-Day ACF ⴙ Hoe 140, and 2-Day ACF ⴙ Ang II
ACF Untreated
ACF ⴙ Hoe 140
ACF ⴙ Ang II
296 ⫾ 19
280 ⫾ 62
308 ⫾ 10
MAP* (mm Hg)
71 ⫾ 2
90 ⫾ 10
91 ⫾ 5†
LVEDP (mm Hg)
5⫾1
7⫾1
9⫾2
LVEDD (mm)
8.0 ⫾ 0.2
7.3 ⫾ 0.3
7.8 ⫾ 0.4
LV posterior wall thickness (mm)
1.3 ⫾ 0.1
1.6 ⫾ 0.1
1.8 ⫾ 0.1†
LVESD* (mm)
4.1 ⫾ 0.2
3.2 ⫾ 0.4
3.7 ⫾ 0.2
LVEDD/wall thickness (mm/mm)
6.0 ⫾ 0.3
4.8 ⫾ 0.4
4.4 ⫾ 0.4†
LV fractional shortening (%)
49 ⫾ 1
57 ⫾ 4
51 ⫾ 3
Heart weight/body weight (g/g ⫻ 103)
3.7 ⫾ 0.1
3.4 ⫾ 0.1†
4.0 ⫾ 0.1‡
Collagen volume %
2.1 ⫾ 0.1
4.1 ⫾ 0.3† (n ⫽ 8)
3.2 ⫾ 0.3†‡
8
6
8
Heart rate* (beats/min)
n
Values are mean ⫾ SEM. *Based on ranks. †p ⬍ 0.0167 (⫽ 0.05/3) versus ACF untreated. ‡p ⬍ 0.0167 versus ACF ⫹ Hoe 140.
Ang II ⫽ angiotensin II; other abbreviations as in Tables 1 and 3.
820
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
Measurements
Effect
of RAM on
in Hemodynamic
Control Rats (4-Week
and Echocardiographic
SHM) and Rats After 4 Weeks of ACF
Effect of RAM on Hemodynamic and Echocardiographic
Table 5
Measurements in Control Rats (4-Week SHM) and Rats After 4 Weeks of ACF
ACF Untreated
SHM ⴙ RAM
Heart rate* (beats/min)
260 ⫾ 18
291 ⫾ 7 (n ⫽ 8)
282 ⫾ 12
MAP (mm Hg)
114 ⫾ 10
94 ⫾ 6 (n ⫽ 8)
108 ⫾ 7
83 ⫾ 4†
13 ⫾ 1 (n ⫽ 7)
12 ⫾ 2
13 ⫾ 2
SHM Untreated
LVEDP (mm Hg)
6 ⫾ 1 (n ⫽ 5)
ACF ⴙ RAM
299 ⫾ 5
100 ⫾ 10
82 ⫾ 5 (n ⫽ 8)
91 ⫾ 8
LV ⫹ dP/dtmax (mm Hg/s)
7,989 ⫾ 342
7,956 ⫾ 332 (n ⫽ 7)
7,607 ⫾ 278
7,464 ⫾ 166
LV ⫺ dP/dtmax (mm Hg/s)
6,393 ⫾ 417
5,491 ⫾ 503 (n ⫽ 7)
6,067 ⫾ 258
5,452 ⫾ 254
LVESP (mm Hg)
64 ⫾ 5†
LVEDD (mm)
7.5 ⫾ 0.1 (n ⫽ 9)
9.9 ⫾ 0.2†
7.7 ⫾ 0.2
9.4 ⫾ 0.5
LVESD‡ (mm)
4.2 ⫾ 0.3 (n ⫽ 9)
5.5 ⫾ 0.3†
5.1 ⫾ 0.3
5.5 ⫾ 0.5†
LVEDD/wall thickness‡ (mm/mm)
3.9 ⫾ 0.3 (n ⫽ 9)
5.6 ⫾ 0.3†
4.7 ⫾ 0.2
6.1 ⫾ 0.5†
LV fractional shortening‡ (%)
44 ⫾ 4 (n ⫽ 9)
44 ⫾ 2
34 ⫾ 3
43 ⫾ 3
Heart weight/body weight (g/g ⫻ 103)
3.1 ⫾ 0.1 (n ⫽ 8)
4.9 ⫾ 0.2†
2.5 ⫾ 0.1§
4.0 ⫾ 0.2†§储
n
6
9
6
7
Values are mean ⫾ SEM. *Based on Kruskal-Wallis test. †p ⬍ 0.008 (⫽ 0.05/6) versus SHM. ‡Based on ranks. §p ⬍ 0.008 versus ACF. 储p ⬍ 0.008 versus SHM ⫹ RAM.
RAM ⫽ ramipril; other abbreviations as in Tables 1 and 3.
Left ventricular end-diastolic pressure and wall stress
are elevated as early as 2 days after ACF and throughout
the entire time course (Fig. 4). Lung wet-weight is also
increased, indicative of decreased LV compliance despite
the decrease in interstitial collagen and normal indexes of
cardiomyocyte relaxation throughout 15 weeks after ACF
(Table 2). Previous studies in ACF rats at 6 weeks
showed an increase in collagen cross-linkage and no
change in collagen content, resulting in a stiffer LV
chamber (20). Thus, a simple assessment of collagen
content without regard to collagen cross-linking or subtype distribution, especially in volume overload, could
obscure changes in chamber compliance. Our results
suggest that the progressive LV remodeling and systolic
Figure 6
Volume Percent of Collagen in
Ramipril-Treated Sham and ACF Rats
Collagen volume percent in 4-week sham (SHM) and aortocaval fistula (ACF),
4-week sham ⫹ ramipril, and 4-week ACF ⫹ ramipril. Data are presented as
mean ⫾ SEM, n values given in Table 5. *p ⬍ 0.008 (⫽ 0.05/6) versus
sham, †p ⬍ 0.008 versus ACF, ‡p ⬍ 0.008 versus sham ⫹ ramipril. Variance
components analysis was used.
dysfunction, which is disproportionate to cardiomyocyte
remodeling, results from dissolution of the collagen
matrix.
Chronic ACE inhibition does not attenuate LV remodeling in volume overload. We found that ACE inhibition increased collagen loss at 4 weeks after ACF (Fig. 6)
and did not attenuate adverse LV chamber remodeling as
summarized in Table 5. With ACF ⫹ ACE inhibitor
treatment, LVEDD/wall thickness and LVED wall stress
remained elevated and LVESD increased relative to
ACF-untreated rats, even though there was a decrease in
LVES pressure and cardiac hypertrophy. The failure to
improve LV remodeling and function may be caused by
an ACE inhibitor-induced loss of supporting collagen
structure that results in further cardiomyocyte elongation.
Our BK2R blockade studies suggest that ACE inhibitorinduced increase in BK contributes to the acute adverse
remodeling seen in volume overload after ACF. However, we do not show the role of long-term BK2R
activation in the heart on the volume overloaded LV, and
therefore, the mechanism of the long-term deleterious
effects of chronic ACE inhibition remains uncertain. We
have shown a similar loss of collagen and MMP activity
in early and late phases of mitral regurgitation in the dog
(21). Indeed, ACE inhibitor or angiotensin II receptor
blockade failed to attenuate LV remodeling in dogs with
mitral regurgitation (8,22), and we have shown that such
therapy results in further cardiomyocyte elongation (8).
Finally, ACE inhibitor or angiotensin II receptor blockade have not been shown to be beneficial in patients with
pure volume overload due to valvular regurgitation (2).
Summary. Here we show that an increase in LVEDD/
wall thickness, previously thought to be caused by myocyte elongation, may have less to do with myocyte length
and more to do with matrix changes that affect the
collagen scaffolding connecting individual myocytes and
collections of myocytes that comprise the laminar structure of the heart (23). We also show that the BK-
Ryan et al.
Left Ventricular Remodeling in Volume Overload
JACC Vol. 49, No. 7, 2007
February 20, 2007:811–21
mediated change in the extracellular matrix is an early
event that sets in motion adverse LV remodeling and
function. This study has important implications for the
BK-enhancing effects of ACE inhibitor therapy in pure
volume overload states. The role of matrix degradation in
volume overload challenges current concepts regarding
the pathophysiology of eccentric LV remodeling and
explains why ACE inhibitors are effective at reversing LV
remodeling during pressure overload but are ineffective in
treating volume overload.
Acknowledgments
The authors thank Dr. Joanna Morrison for editorial
assistance and Wayne E. Bradley and Pamela C. Powell for
technical expertise.
Reprint requests and correspondence: Dr. Louis J. Dell’Italia,
Center for Heart Failure Research, University of Alabama at
Birmingham, 432 BMR2, 901 19th Street South, Birmingham,
Alabama 35294-2180. E-mail: [email protected].
9.
10.
11.
12.
13.
14.
15.
16.
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