Download Materials and Methods

Supplementary Material
Inotropic effects of prostacyclins on the right ventricle are abolished
in isolated rat hearts with right ventricular hypertrophy and failure
Holmboe et al.
Supplementary Methods
Rat model of right heart hypertrophy and failure
Right ventricular (RV) hypertrophy and failure was induced by pulmonary trunk banding (PTB) as
previously described.1-3 Anesthesia was induced with 7 % sevofluran and maintained with 3.5%
sevoflurane (Abbot Scandinavia AB, Solona, Sweden) in a mixture of O2 1L/min and N2O 0.5
L/min. The rats were subjected to a thoracotomy revealing the heart. The pulmonary trunk was
banded with a titanium clip (Horizon Ligating Clips ref. 001200, Weck Closure Systems, USA)
using a horizon applier (Horizon Applier, small, cat. #HZ137081, Weck Closure Systems, USA).
The clip was compressed to an inner diameter of 1.0 mm or 0.5 mm generating either RV
hypertrophy (HYP) or manifest RV failure (FAIL). Rats without banding served as healthy controls.
To reduce post-operative pain, buprenorphine was injected subcutaneously (0.12 mg/kg) during the
procedure and applied to the drinking water (7.4 μg/mL) for three days postoperatively.
Echocardiography
Before isolating the hearts for the Langendorff, RV hypertrophy and function were evaluated using
a Vevo 2100 echocardiographic system (Visual Sonics, Toronto, Canada) with a 21 MHz linear
array transducer. M-mode was used to measure tricuspid annular plane systolic excursion (TAPSE),
a measure of RV systolic function correlating well to RV ejection fraction and survival in rats as
well as in humans.4,5 Cardiac output (CO) was calculated as CO = SVxHR =
(PAdiameter/2)×π×VTI×HR (PAdiameter: pulmonary artery diameter, VTI: velocity time integral, HR:
heart rate).6 The VTI was measured using pulsed wave doppler, parallel to the flow at the same
level as the measured diameter. Tricuspid regurgitation was evaluated using color Doppler. All
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analyses were made during expiration and are presented as a mean of three consecutive
measurements.
The Isolated perfused heart model – Langendorff
The rat hearts were isolated and connected to a pressure controlled Langendorff setup as previously
described.7 Anaesthesia was induced with a subcutaneous injection of a mix of midazolam (0.5
mg/kg), fentanyl citrate (0.158 mg/kg) and fluanisone (5 mg/kg). The aorta was cannulated and
retrograde perfusion was initiated before cutting out the heart and connecting it to the Langendorff
system (modified IH-SR type 844/1; Hugo Sachs Elektronik, Harvard Apparatus, MarchHugstetten, Germany). Krebs-Henseleit buffer ((mmol l−1) NaCl 118.5; KCl 4.7; NaHCO3 25.0,
glucosemonohydrate 11.0; MgSO4 1.2; CaCl2 2.4; KH2PO4, 1.2) was used as perfusion media,
gassed with 95 % O2 and 5 % CO2 to keep the pH at 7.4 and the heart oxygenized.
RV hemodynamic parameters were monitored using a fluid filled balloon introduced into the RV
and connected to a pressure transducer. The diastolic pressure was set to 5 mmHg and the hearts
were perfused with a constant pressure of 80 mmHg. Hemodynamic parameters were measured and
calculated with Notocord Hem Software (NOTOCORD Systems, Croissy, France). The rate
pressure product = HR×RV developed pressure, was used as a parameter indicative of cardiac work.
Expression of prostanoid receptors in the RV
RV tissue was excised from rats with normal hearts (n=8), hypertrophic RV (n=8) and RV failure
(n=8) and snapped frozen in liquid nitrogen. cDNA was synthesized from RNA isolated from the
RV tissue as described below.
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RNA isolation
RNA was isolated from the RV tissue using a commercial RNA purification kit according to the
manufactures instructions (NucleoSpin® RNA II, Macherey-Nagel, Düren, Germany). 30 mg of
tissue was homogenized (TissueLyser). 350 µL lysis buffer and 3.5 uL mercaptoethanol was added
and vortexed. The lysate was filtered using the NucleoSpin® Filter. 350 µL ethanol was added to
the lysate, loaded to the NucleoSpin® RNA II Column and centrifuged for 30 seconds at 11000 x g.
The column was transferred to a new tube and 350 µL desalting buffer was added and centrifuged
for 1 minute at 11000 x g. The column was incubated for 15 minutes with 95 µL DNase reaction
mixture to digest DNA. Finally the column was washed and centrifuged 3 times and RNA eluated
in 27 µL RNase-free H2O.
RNA concentration determination
The concentration of the RNA in the samples was determined using a spectrophotometer
(Eppendorf® BioPhotometer, Hamburg, Germany). The concentration was measured at wavelength
260 nm and the samples were diluted in RNase free H2O to a final concentration of 0.5 µg/mL.
Proper quality and integrity of a random sample of the RNA were confirmed using an Agilent 2100
Bioanalyzer (Agilent Technologies, Santa Clara, USA).
cDNA synthesis
cDNA was synthesized from the RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo
Fisher Scientific, Waltham, USA) according to manufacturers instructions. RNA, nuclease-free
water and random primer were mixed, centrifuged and incubated at 65 oC for 5 minutes. Reaction
buffer, RiboLock RNase inhibitor, dNTP mix and the reverse transcriptase were added and
incubated at 25 oC for 5 minutes followed by incubation at 42 oC for 60 minutes and finally at 70 oC
for 5 minutes to terminate the reaction.
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Quantitative Polymerase Chain Reaction (qPCR)
Primers (DNA Technology A/S, Risskov, Denmark) used in qPCR were designed for the β-myosin
heavy chain (β-MHC), sarco/endoplasmatic reticulum Ca2+-ATPase-2 (SERCA-2) and the rat
prostanoid receptor subtypes IP, EP1, EP2, EP3, EP4, DP1, TP and FP genes (Table s1). Before
samples were run, each primer was tested, and the PCR product sequenced to confirm the right
product was amplified. Sequencing was made on a 3130XL Genetic Analyzer, Applied
Biosystems® (Life Technologies, Waltham, USA). Amplification of the right products was
confirmed.
2 µL of each sample cDNA, water, SYBR Green Master Mix (Maxima SYBR Green qPCR Master
Mix (2X), Thermo Scientific, Waltham, USA), ROX solution and the primer specific for the gene
of interest were added to wells in duplicate. The samples were run in Mx3000p qPCR system
(Agilent Technologies, Santa Clara, USA) for 40 cycles of 95 oC for 30 seconds, 60 oC for 60
seconds and 72oC for 60 seconds.
Materials
Iloprost (Ilomedin) was purchased from Bayer Schering Pharma (Berlin-Wedding, Germany),
treprostinil (Remodulin) from NordicInfu Care AB (Nacka Strand, Sweden), epoprostenol
(Flolan) from GlaxoSmithKline (London, UK), and MRE-269 from Cayman Chemicals (Ann
Arbor, USA). Dobutamine (Dobutrex) was purchased from PharmaCoDane (Herlev, Denmark).
The DP1 receptor antagonist, BW A868C, was purchased from Sigma-Aldrich, USA, the EP2
receptor antagonist, PF04418948, from Pfizer Inc. (New York City, USA), and the IP receptor
antagonist, RO1138452, from Cayman Chemical (Ann Arbor, USA). The anaesthesia was made of
Dormicum®
(midazolam)
from
Matrix
Pharmaceuticals,
(Hellerup,
Denmark)
and
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Hypnorm® (fentanyl citrate and fluanisone) from VetaPharma Ltd, (Leeds, UK). Buprenorphine
(Termgesic®) was from RB Pharmaceuticals Limited (Slough, UK).
The concentrations of the prostacyclin mimetics used in this study was chosen to include a broad
range of concentrations starting from clinically relevant doses to supra-clinical doses. Thus are the
first 2 concentrations of iloprost and treprostinil, the first 3 concentrations of epoprostenol and the
first concentration of MRE-269 comparable to what is used in the clinic. This is based on previous
studies and calculated from information regarding plasma protein binding and pharmacokinetics of
each agent.8-13
The concentrations of the receptor antagonists were chosen based on previous studies. In vivo and
ex vivo studies have shown that 1 uM (and below) of BWA868C effectively inhibit DP1 receptors in
the rat.14,15 PF04418948 has also been demonstrated to effectively antagonise EP2 receptors at 1µM
(and below) in several previous in vitro studies.16-18
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Legends
Figure s1. Effect of dobutamine (0.1 μg/mL) on heart rate (A), dP/dtmax (B) and RV developed
pressure (C). Control n=30, Hypertrophic n=32, Failure n=32. Results are presented as mean±SD.
***p<0.001, ****p<0.001, Student’s t-test. RV: right ventricle.
Figure s2: Effects of increasing concentrations of treprostinil and dobutamine on RV function with
constant flow (13 mL/min), (n=7). Values are presented as mean +SD. RV: right ventricle. †p<0.05,
††p<0.01, compared to baseline. *p<0.05, **p<0.01, compared to treprostinil 150 ng/mL.
Figure s3: Effects of sodium nitroprusside on RV function. Control n=8, Hypertrophic n=8, Failure
n=8. Values are presented as mean +SD. †p<0.05, ††p<0.01, †††p<0.001, compared to baseline.
RV: right ventricle. Bpm: beats per minute.
Figure s4: Effects of increasing concentrations of treprostinil (n=8) and A-B: the DP1 receptor
antagonist, BW A868C (1 µM) (n=7) or C-D: the EP2 receptor antagonist, PF04418948 (n=8).
Values are presented as relative to mean of baseline normalized to 1, +SD. None of the two
antagonists altered the effect of treprostinil significantly, using 2-way ANOVA. †p<0.05, ††p<0.01,
†††p<0.001, compared to baseline. RV: right ventricle.
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