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J. Exp. Med.
The Rockefeller University Press • XXXXXXXXXX/2002/02/375/07 $5.00
Volume 195, Number 3, Fe
uary 4, XXXXXXXXXX–381
http:
www.jem.org/cgi/content/full/195/3/375
Brief Definitive Report
375
Evidence for a Role of Mast Cells in the Evolution to
Congestive Heart Failure
Masatake Hara,
1
Koh Ono,
1
Myung-Woo Hwang,
1
Atsushi Iwasaki,
1
Masaharu Okada,
1
Kazuki Nakatani,
2
Shigetake Sasayama,
1
and Akira Matsumori
1
1
Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto
XXXXXXXXXX, Japan
2
Second Department of Anatomy, Osaka City University Medical School, Osaka XXXXXXXXXX, Japan
Abstract
Mast cells are believed to be involved in the pathophysiology of heart failure, but their precise
ole in the process is unknown. This study examined the role of mast cells in the progression of
heart failure, using mast cell-deficient (WBB6F1-W/W
v
) mice and their congenic controls
(wild-type [WT] mice). Systolic pressure overload was produced by banding of the abdominal
aorta, and cardiac function was monitored over 15 wk. At 4 wk after aortic constriction, car-
diac hypertrophy with preserved left ventricular performance (compensated hypertrophy) was
observed in both W/W
v
and WT mice. Thereafter, left ventricular performance gradually de-
creased in WT mice, and pulmonary congestion became apparent at 15 wk (decompensated
hypertrophy). In contrast, decompensation of cardiac function did not occur in W/W
v
mice;
left ventricular performance was preserved throughout, and pulmonary congestion was not
observed. Perivascular fi
osis and upregulation of mast cell chymase were all less apparent in
W/W
v
mice. Treatment with tranilast, a mast cell–stabilizing agent, also prevented the evolu-
tion from compensated hypertrophy to heart failure. These observations suggest that mast cells
play a critical role in the progression of heart failure. Stabilization of mast cells may represent a
new approach in the management of heart failure.
Key words: heart failure • mast cells • left ventricular hypertrophy • pressure overload • chymase
Introduction
When the heart is exposed to pressure overload, cardiac hy-
pertrophy develops to preserve its function by normalizing
chamber wall stress (1). If mechanical overload persists, the
hypertrophied heart dilates and its contractile function de-
creases, resulting in congestive heart failure (1). The mech-
anism of transition from compensated hypertrophy to heart
failure has not been clarified (2).
Mast cells are found in the human heart (3), and have
een implicated in cardiovascular diseases (4,
5). They
were increased in both hypertrophied (5) and failing hearts
(6). However, their role in the pathophysiology of cardiac
hypertrophy and failure is unclear. We have recently ob-
served that mast cells cause apoptosis of cardiac myocytes
and proliferation of nonmyocytes in vitro (7). As loss of
cardiac myocytes and proliferation of nonmyocytes both
esult in cardiac dysfunction (1), we hypothesized that
myocardial mast cells may be implicated in the progression
of heart failure.
This study was performed to examine whether mast cells
play a role in the evolution from compensated hypertrophy
to heart failure in a murine model of systolic pressure over-
load, using W/c-kit mutant WBB6F1-W/W
v
mice, in
which mast cells are nearly absent, and tranilast, a mast cell–
stabilizing agent.
Materials and Methods
All experiments were performed in 9-wk-old male mice, ob-
tained from Shizuoka Agricultural Cooperation Association, and
treated in accordance with local institutional guidelines at all
stages of the experiments.
Experiment 1.
Male W/W
v
mice (
n
�
25), or their normal
male littermates, WBB6F1-
�
�
(wild-type [WT]) mice (
n
�
24), were exposed to 15 wk of pressure overload produced by
anding of the abdominal aorta with minor modifications of a
Address co
espondence to Akira Matsumori, Department of Cardiovas-
cular Medicine, Kyoto University Graduate School of Medicine, 54
Kawaracho Shogoin, Sakyo-ku, Kyoto XXXXXXXXXX, Japan. Phone: 81-75-
XXXXXXXXXX; Fax: XXXXXXXXXX; E-mail: XXXXXXXXXX
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ow
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ctober 2021
376
Mast Cells in Heart Failure
method described previously (8). The mice were anesthetized by
intraperitoneal injection of a mixture of ketamine, 100 mg/kg,
and xylazine, 5 mg/kg. The abdominal aorta was banded at the
suprarenal level with 5–0 silk suture material ligated around the
vessel and a 26-gauge needle, following which the needle was
withdrawn. In addition, 14 W/W
v
mice and 11 WT mice under-
went identical surgical procedures, except for banding of the ab-
dominal aorta (sham-operated controls). At 4 wk after operation,
10 W/W
v
mice and 10 WT mice were killed to examine the role
of mast cells in compensated hypertrophy. Thereafter, the re-
mainder of the mice were followed with serial echocardiography,
and killed at 15 wk to examine the role of mast cells in congestive
heart failure analyses.
Mast Cell Reconstitution of W/W
v
Mice.
Mast cell reconstitution
of W/W
v
mice was performed as described previously (9). Bone
ma
ow cells from femurs of male WT mice were cultured for 3
wk in WEHI-3 conditioned medium. To generate W/W
v
�
MC
mice, adoptive transfer of mast cells (
�
98% purity) into the hearts
of W/W
v
mice was achieved by intravenous injection of 5
�
10
6
mast cells, 2 d before aortic banding. The mice were killed 15 wk
after mast cell reconstitution and aortic banding. The density of
cardiac mast cells was confirmed by staining with toluidine
lue.
Bone Ma
ow Reconstitution of W/W
v
Mice.
The bone ma
ow
econstitution method has been described previously (10).
Briefly, WT mice were killed by cervical dislocation, and the
one ma
ow was flushed with RPMI 1640 culture medium.
Bone ma
ow cells (3
�
10
7
) were injected intravenously into
W/W
v
mice 2 d before aortic banding. W/W
v
mice with recon-
stituted bone ma
ow were killed 15 wk later. Hematocrit was
measured to confirm successful reconstitution.
Morphologic and Echocardiographic Studies.
After measurement
of their body mass, the animals were anesthetized with ketamine
(50 mg/kg) and xylazine (2.5 mg/kg). Transthoracic echocar-
diography was performed with a cardiac ultrasound recorde
(Toshiba Power Vision), using a 7.5-MHz transducer. After the
acquisition of high quality two-dimensional images, M-mode im-
ages of the left ventricle were recorded. Measurements of left
ventricular enddiastolic (LVDd) and endsystolic (LVDs) internal
dimensions were performed by the leading edge-to-leading edge
convention adopted by the American Society of Echocar-
diography. Percent fractional shortening (%FS) was calculated as
%FS
�
([LVDd
�
LVDs]/LVDd)
�
100.
Blood Pressure and Heart Rate Monitoring.
The hemodynamic
effects of aortic banding were monitored via the right carotid ar-
tery exposed through a cervical incision and isolated by blunt dis-
section as described by Rockman et al XXXXXXXXXXThe lungs were
dried for 120 min at 60
�
C and weighed again. The lung wate
content (LW) was calculated as LW
�
lung weight (wet)
�
lung
weight (dry).
The pressure gradient across the aortic constriction was mea-
sured directly at 4 wk after operation with a 24 gauge polyethyl-
ene tube (TERUMO), inserted into the infrarenal abdominal
aorta, then advanced through the stenosis to measure blood pres-
sure at the suprarenal level. Pressure gradient was calculated as
(systolic blood pressure at the suprarenal level)
�
(systolic blood
pressure at the infrarenal level).
Histological Analysis.
We examined 15 banded W/W
v
mice,
14 banded WT mice, 10 sham-operated W/W
v
mice, and 9
sham-operated WT mice. The hearts were fixed with 10% for-
malin for histological examinations. The fixed hearts were im-
edded in paraffin, sectioned in 2-
�
m thick slices, and stained
with hematoxylin-eosin for overall morphology, or with Sirius
ed F3BA (0.1% solution in saturated aqueous picric acid) to al-
low a clear discrimination between cardiac myocytes and collagen
matrix (12). Changes in perivascular fi
osis were ascertained by
elating the area of perivascular fi
osis to the total vessel area as
described previously (13).
For transmission electron microscopy, heart specimens were
fixed with Karnovsky solution (3% glutaraldehyde and 1.6%
paraformaldehyde in 0.1 mol/liter phosphate buffer [PB], pH 7.4)
overnight at 4
�
C and then were cut into 1-mm thick sections.
They were postfixed in 1% osmium tetroxide in PB overnight at
4
�
C, dehydrated in ethanol series, and embedded in Polybed
(Polysciences Inc.). 70-nm thick ultrathin sections were stained
with saturated uranyl acetate and lead citrate, and observed unde
a JEM-1200EX electron microscope (JEOL) at 100 kV.
Measurement of Plasma Angiotensin II Level and Renin Activity.
The abdomen of eight WBB6F1-W/W
v
mice and seven WT
mice was opened under anesthesia with ketamine and xylazine
when killed at 15 wk after aortic banding. Blood was rapidly ob-
tained by puncture of the inferior vena cava, transfe
ed to chilled
tubes containing aprotinin (1,000 kallidinogenase inactivato
units per milliliter) and Na
2
EDTA (1 mg/ml), and immediately
centrifuged at 4
�
C. Plasma samples were stored at
�
80
�
C until
angiotensin II measurement by ELISA. Plasma renin activity
(PRA) was determined using RENIN-RIABEAD (Dainabot).
Quantitative Reverse Transcription PCR Analysis.
We examined
five mice of each groups. Total RNA was isolated from the left
ventricle by the acid guanidinium thiocyanate-phenol-chloro-
form method. Real-time quantitative PCR (TaqMan PCR) us-
ing an ABI PRISM 7700 Sequence Detection System and Taq-
Man PCR Core Reagent Kit (PerkinElmer) was performed ac-
cording to the manufacturer’s protocol. 1
�
l of the first strand
cDNA was used in the following assay. The following forward
(F) and reverse (R) oligonucleotides, and probes (P) were used
for the quantification of mouse mast cell protease