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Supplementary Methods
Echocardiography
After shaving the chest, transthoracic echocardiography was performed in the supine position
using echocardiography machine (Nemio, Toshiba Co., Tokyo, Japan) 1 week before and 3, 6, 9,
12, 15 and 20 weeks after the creation of the suprarenal aortic constriction (SAC) model. Rats
were lightly sedated with the lowest possible dose of isoflurane (initially 4%, then approximately
2-3%) mixed with oxygen. Images were acquired with a 9 MHz transducer connected to
echocardiography machine. Left ventricular (LV) septal wall thickness (LVSWT) and LV
posterior wall thickness (LVPWT) and the LV end diastolic (EDD) and systolic dimension (ESD)
were measured using M-mode echocardiography at the papillary muscle level. The LV ejection
fraction (EF) was calculated as (LVEDD2 - LVESD2) / LVEDD2. LV mass was estimated by a
formula which has been previously validated in small animals, and was adjusted based on body
weight: [LV mass = {(SWT + PWT + LVEDD)3 - (LVEDD)3} × 1.04]. Stroke volume (mL) was
calculated as: LV end diastolic volume - LV end systolic volume. LV fractional shortening (%)
was calculated as: 100 × {(LVEDD - LVESD) / LVEDD}. Cardiac output (mL/min) was
calculated as: stroke volume × heart rate.1
Exercise capacity
Maximal exercise capacity was evaluated using the Rota Rod Treadmill (Ugo Basile, Comerio,
Italy).2,3 Rats ran on a knurled drum to avoid falling off during its rotation. Rats were trained on
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2 occasions before the test to adjust the treadmill. Treadmill speed was gradually increased from
3 revolutions per minute (rpm) to 15 rpm every 1 minute, and maximum exercise time was
recorded. A trained observer recorded the immobility response due to exhaustion.
LV hemodynamic study
At the 3rd and 20th week after operation, rats were anesthetized with isoflurane and were placed
in the supine position. The right internal jugular vein was cannulated to administer fluids. Mean
arterial pressure was monitored and recorded via carotid artery cannulation during the
hemodynamic study. After an anterior thoracotomy, a microtip pressure-volume (PV) catheter
(SPR-838, Millar Instruments, Houston, TX, USA) was introduced into the LV cavity, via an
apical approach along the longitudinal axis of the heart until stable PV loops were obtained. PV
loops were acquired with the ventilator off for 5-10 seconds after 10 minutes of stabilization. The
sampling rate was 1,000/sec using the ARIA PV conductance system (Millar Instruments)
coupled to a PowerLab/4SP A/D converter (AD instruments; Mountain View, CA, USA) and
personal computer. Analyses of PV loops were performed using commercially available cardiac
PV analysis program, PVAN 3.5 (Millar Instruments). LV end-diastolic (LVEDP) and endsystolic pressure (LVESP), maximal slope of systolic pressure increment (+dP/dt) and the
decrease in diastolic pressure (-dP/dt) were calculated.2-4
Survival assessment
Survival analysis was performed for animals that survived the acute stage of SAC modeling
(within 3 weeks after surgical procedure) until 20 weeks after SAC modeling when a study was
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terminated. Numbers of rats in this analysis were 19, 18, and 14 for sham, SAC and SAC +
udenafil groups, respectively.
Histopathology and immunohistochemistry
After the hemodynamic measurements, heart, lung and liver were harvested. Heart was dissected
after it was flushed with isotonic saline and weighed. The LV was then fixed with 4%
paraformaldehyde and embedded in paraffin. The tissue was sectioned into 4 μm-thick sections,
and was stained with Masson’s trichrome to evaluate the degree of fibrosis. The expression of
phosphodiesterase
type
5
(PDE5)
in
the
myocardial
section
was
assessed
by
immunohistochemistry with PDE5 antibody (Abcam, ab14672). The expressions of matrix
metalloproteinase (MMP) -2 and -9 were assessed by immunohistochemistry with MMP
antibodies (MMP-2 with Santacruz 53630 and MMP-9 with Santacruz 6840). The degree of
apoptosis of myocardium was assessed by terminal deoxynuclotidyl transferase-mediated UTP
nick-end labeling (TUNEL) staining.
Serum cytokine measurement
An arterial blood sample was collected from the abdominal aorta immediately after the
hemodynamic measurements. After centrifugation (3,000 × g), the plasma was stored at -70 °C.
The serum inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10 and tumor necrosis
factor (TNF)-α were then measured using a bead-based multiplex assay.
Statistical analysis
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The results are expressed as mean ± standard deviation. The unpaired 2-sided Student’s t-test was
used to compare continuous variables between two groups. To compare the mean value of
continuous variables among three groups, analysis of variance (ANOVA) with Bonferroni’s posthoc analysis was used. The time course of parameter changes was compared between the groups
using a repeated measure ANOVA. Survival rate was evaluated by the Kaplan-Meier method. All
statistical analyses were performed using SPSS version 17.0 (IBM Co., Armonk, NY, USA), and
P-values < 0.05 were considered statistically significant.
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Supplementary Tables
Supplementary Table S1. Weights of heart, lung and liver of rats with 3 week-exposure to
sham and SAC operations (n = 4 in each group, 11 week old age)
Sham
SAC
P
Heart weight, g
1.28 ± 0.13
1.71 ± 0.34
0.011
Lung weight, g
1.88 ± 0.24
2.61 ± 0.64
0.016
Liver weight, g
14.26 ± 3.48
12.6 ± 3.15
0.368
Body weight, g
308 ± 28
303 ± 23
0.763
Heart/body weight, g/kg
4.20 ± 0.57
5.61 ± 0.89
0.004
Lung/body weight, g/kg
6.13 ± 0.70
8.60 ± 2.06
0.019
Liver/body weight, g/kg
41.33 ± 8.70
45.77 ± 7.27
0.321
SAC, suprarenal aortic constriction .
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Supplementary Table S2. Baseline echocardiographic parameters (n = 7 in each group, 8
week old age)
Parameter
Sham
SAC
SAC + udenafil
P
283 ± 19
267 ± 19
274 ± 23
0.373
LVSWT, mm
1.98 ± 0.14
1.97 ± 0.14
2.00 ± 0.18
0.946
LVEDD, mm
7.1 ± 0.4
7.0 ± 0.2
6.9 ± 0.1
0.702
2.88 ± 0.51
2.84 ± 0.42
2.75 ± 0.32
0.852
73 ± 4
75 ± 4
74 ± 4
0.703
Heart rate, /min
LVMI, g/kg
LVEF, %
SAC, suprarenal aortic constriction; LVSWT, left ventricular septal wall thickness; LVEDD, left
ventricular end-diastolic dimension; LVMI, left ventricular mass index; LVEF, left ventricular
ejection fraction.
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Supplementary Figure Legends
Supplementary Figure S1. Representative figures showing main processes of suprarenal
aortic constriction modeling
(A) Exposure of suprarenal abdominal aorta (white arrow), and (B) ligation of supra renal
abdominal aorta using 22-gauge needle.
Supplementary Figure S2. Representative figures showing luminal morphologies of control
and constricted abdominal aorta and quantification of luminal cross sectional areas
(A) Hematoxylin and eosin staining of rat abdominal aortas at the 3rd week after sham and SAC
operations, and (B) quantification of luminal cross sectional areas.
SAC, suprarenal aortic constriction. n = 4 in each group.
Supplementary Figure S3. Serial changes of body weights among 3 groups
SAC, suprarenal aortic constriction; UD, udenafil; NS, not significant.
Supplementary Figure S4. Serial changes of LV fractional shortening, stroke volume and
cardiac output among 3 groups
LV, left ventricle; SAC, suprarenal aortic constriction; UD, udenafil. *P < 0.05 for difference
from sham group; #P < 0.05 for difference from SAC group.
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Supplementary Figure S5. Gross examination and hematoxylin and eosin staining of left
ventricle at the 3rd week after operation
(A) Gross examination, and (B) hematoxylin and eosin staining of left ventricle at the 3rd week
after operation.
SAC, suprarenal aortic constriction; LV, left ventricle; RV, right ventricle. n = 4 in each group.
Supplementary Figure S6. Masson’s trichrome staining showing the extent of fibrosis of left
ventricle at the 3rd week after operation
Representative figures focusing interstitial fibrosis of left ventricle of sham (A) and SAC (B),
and representative figures focusing perivascular fibrosis of left ventricle of sham (C) and SAC
(D).
SAC, suprarenal aortic constriction. n = 4 in each group.
Supplementary Figure S7. Immunohistochemical staining showing the expression of
phosphodiesterase type 5 in myocardium at the 3rd week after operation
SAC, suprarenal aortic constriction. n = 4 in each group.
Supplementary Figure S8. Quantification of the result of TUNEL staining
TUNEL, terminal deoxynuclotidyl transferase-mediated UTP nick-end labeling; SAC, suprarenal
aortic constriction; UD, udenafil; HPF, high power field. n = 5 in each group.
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