Online data supplement
Animals – study design
Male Wistar rats were used in this study (n=63 Harlan, Horst, the Netherlands). Rats were
housed in pairs under controlled conditions (22C 12h/12h day/night cycle). Food and water
were available ad libitum. PAH was induced in 54 rats by a single injection of MCT dissolved
in sterile saline (8 mg/ml in 0.9% NaCl s.c.) at pH 7.4. Control rats (n=9) received saline s.c..
After 14 days, PAH rats were randomly assigned to daily oral treatment with Bosentan
(Actelion Pharmaceuticals, Allschwil, Switzerland) (300 mg/kg/day; n=9), Sildenafil (Pfizer
Inc. USA10 mg/kg/day; n=9), Fasudil (Mercachem 100 mg/kg/day; n=9), or the combinations
Fasudil+Bosentan (same dosages, n=9) or Fasudil+Sildenafil (same dosages, n=9). At day 28,
rats were euthanized by excision of the hearts under isoflurane anesthesia (>3.5 %). All
experiments were approved by the Institutional Animal Care and Use Committee conform the
Helsinki convention for the use and care of animals.
Echocardiography
Echocardiography was performed using an Aloka SSD4000 ultrasonographic system
(Biomedic, Almere, The Netherlands) equipped with a 13.5 MHz transducer. By means of
Doppler imaging of the aorta, heart rate (HR), and stroke volume (SV) were determined(1-3).
Subsequently cardiac output (CO) was calculated: CO= SV*HR/1000. In addition, in Mmodus RV end-systolic and end-diastolic diameters (RV EDD and ESD resp.), were measured
at mid papillary level as measures for RV dilatation. RV Fractional shortening (FS) was then
calculated: FS = ((EDD – ESD) / EDD) x 100%(4;5). In addition, Tricuspid Annular Plane
Systolic Excursion (TAPSE) was measured, by measuring the movement form the tricuspid
annulus to the apex, as previously described(1;2). Analyses were performed off-line (ImageArena 2.9.1, TomTec Imaging Systems, Unterschleissheim / Munich, Germany).
Hemodynamic measurements
RV systolic pressure (RVSP) was measured using a Millar pressure catheter (Millar, Texas,
USA) by direct insertion through the RV wall, after thoracotomy(4). Prior to the procedure
rats were intubated with a 16-gauge plastic tube and attached to a mechanical microventilator
(UNO, Zevenaar, the Netherlands), assuring a respiratory rate of 75/min with an IPPV/PEEP
of 15-5 mmHg. RVSP was measured for 20 seconds and averaged. During the procedure,
body temperature was monitored continuously and maintained at 37C with a heating pad.
RVSP measurements were discarded when systemic blood pressure decreased more than
15%.
PVR
was
estimated
by
Poiseuille’s
law:
[PVR]≈[mean
PAP]/[cardiac
output]≈(0.61×[systolic RV pressure]+2 mmHg)/[cardiac output] (2;6).
Quantitative histochemistry
To study the RV’s capacity to adapt to PAH in more detail, an extensive histochemical
analysis was performed on cardiac tissue. Animals were euthanized by excision of the hearts,
and hearts were perfused with Tyrode solution (120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,
2.0 mM Na2HPO4, 27 mM NaHCO3, 1 mM CaCl2, 10 mM glucose and 20 mM 2,3butanedione monoxime, equilibrated with 95% O2 and 5% CO2; pH 7.3–7.4 at 10°C) to
remove blood and prevent contraction(7). Hearts were then frozen in liquid nitrogen.
Cryosections (5µm) were air-dried for 10 minutes and stained. To determine the degree of
individual cardiomyocyte hypertrophy, sections were fixed for 10 min in 4% formaldehyde in
0.1 M sodium phosphate buffer, pH 7.4, and stained with Hematoxylin and Eosine. The cross-
sectional area of 40 randomly chosen cardiomyocytes in the RV was measured. In order to be
able to compare CSA from different hearts, we normalized CSA to a sarcomere length of 2
μm, by randomly measuring 5 stretches of sarcomeres in both ventricles. In addition, adjacent
sections were incubated for succinate dehydrogenase activity (SDHact) as described by des
Tombe et al(7) and measured in 40 cardiomyocytes of the RV at 660 nm, as a determinant for
the RV’s mitochondrial capacity. We also investigated the RV’s capillary density; sections
were stained for 60 min by using primary CD31- (1:35; sc-1506-R, Santa Cruz
Biotechnology, Santa Cruz CA) followed by appropriate secondary antibody staining as well
as WGA (glycocalyx) and DAPI (nuclei) counterstaining. Image acquisition was performed
on a Marianas digital imaging microscopy workstation (Intelligent Imaging Innovations (3i),
Denver CO). SlideBook imaging analysis software (SlideBook 4.2, 3i) was used to quantify
the number of capillaries per cardiomyocyte in at least three randomly chosen areas per
ventricle only selecting transversally sectioned cardiomyocytes(4;7). In addition, sections
were myoglobin concentration, which were quantified in 40 random RV cardiomyocytes(7;8).
The presence of Cytochrome c release was determined as an indicator of mitochondrial
integrity(9).
Results
For echocardiographic and haemodynamic results see main manuscript. Body mass at day 28
differed significantly between groups between groups (Fig S1; One way ANOVA P<0.0001).
We studied the effects of treatment on the adaptation of the RV to PAH in further detail by
measuring the number of capillaries per cardiomyocyte as a measure for oxygen supply, the
RV’s mitochondrial capacity by measuring SDH activity, and the concentration of myoglobin.
body mass day 28 (g)
400
300
200
100
Si
l
Fa
s
os
B
Fa
s
Fa
s
Si
l
os
B
C
T
M
C
on
tr
ol
0
Figure S1. Body mass at the day of sacrifice. Values are mean±SEM.
Compared to control MCT had no effect on the capillarization of the RV, the RV’s
mitochondrial capacity as reflected by SDH activity, or myoglobin concentration, nor did any
of the treatments affect any of these parameters (Fig S2 A,B,C).
B
Myoglobin (mM)
0.05
0.6
0.4
0.2
MCT
fa
s
si
l
bo
s
o
l
ac
eb
pl
nt
ro
Fa
s
l
os
Si
co
co
MCT
0.8
0.0
0.00
B
s
fa
si
l
bo
s
o
pl
ac
eb
co
nt
ro
l
0.0
0.10
o
0.5
0.15
l
1.0
0.20
ac
eb
1.5
1.0
0.25
pl
capillaries/cell
2.0
C
nt
ro
SDH activity (A660nm)
A
MCT
Figure S2
Additional information on RV adaptation was gathered by measuring the RV capillarization
(A), SDH activity (B), myoglobin concentration (C). In MCT rats, the number of capillaries
per cell, mitochondrial capacity and myoglobin concentration did not differ compared to
control. Fasudil, Bosentan or Sildenafil treatment had no effect on these parameters, as
compared to MCT, nor were they different compared to control. Bars represent meanSEM
(N=9), * p<0.05.
In addition, the effects of the combination of Fasudil with Bosentan or Sildenafil were similar
to Fasudil alone (table S1). Cytochrome C release was not found in any of the hearts studied.
Table S1
The effects of combination treatment on RV capillarization, mitochondrial capacity and O2
buffering
Fasudil
Fas+Bos
Fas+Sil
P value ANOVA
1.320.06
1.450.06
1.450.08
0.310
RV SDH activity(A660nm)
0.1870.017
0.2190.024
0.2070.012
0.461
RV myoglobin (mM)
0.7320.025
0.7920.036
0.7970.036
0.277
RV capillaries/myocyte
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