Assessment of myocardial fibrosis by endoventricular electromechanical mapping in
experimental nonischemic cardiomyopathy.
The International Journal of Cardiovascular Imaging
Short title Electromechanical mapping in NICM
Peter J Psaltis, MBBS, PhD;1,2 Angelo Carbone, BSc;1 Darryl P Leong, MBBS;1 Dennis H
Lau, MBBS;1 Adam J Nelson, BMSc;1 Tim Kuchel, BVSc, MVS;3 Troy Jantzen, BSc,
PhD;4 Jim Manavis, BSc;5 Kerry Williams;1 Prashanthan Sanders, MBBS, PhD;1 Stan
Gronthos, BSc, MSc, PhD;2 Andrew CW Zannettino, BSc, PhD;2 Stephen G Worthley,
MBBS, PhD.1
1
Cardiovascular Research Centre, Royal Adelaide Hospital and Departments of Medicine
and Physiology, University of Adelaide, Australia
2
Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and
Veterinary Science & Centre for Stem Cell Research, University of Adelaide, Australia
3
Veterinary Services Division, Institute of Medical and Veterinary Science, Adelaide,
Australia
4
Biosense-Webster, Johnson & Johnson Medical Pty Ltd, Australia
5
Hanson Institute Centre for Neurological Diseases, Institute of Medical and Veterinary
Science, Adelaide, Australia
Address for Correspondence
1
Stephen G Worthley
Cardiovascular Investigation Unit, Royal Adelaide Hospital
North Terrace, Adelaide, South Australia, Australia 5000
Telephone: +61-8-82225608
Fax: +61-8-82222454
E-mail: stephen.worthley@adelaide.edu.au
2
Supplementary Methods
General anesthesia and post-procedural care
Anesthetic induction was achieved by mask inhalation of isoflurane (4% in 100%
oxygen), followed by endotracheal intubation and maintenance of anesthesia by
inhalation of a mixture of isoflurane (2-3%) in 100% oxygen. Animals were
mechanically ventilated with tidal volume of 10 ml/kg, to maintain end tidal CO2 at
approximately 40 mmHg. Additional monitoring under anesthesia included pulse
oximetry, limb lead electrocardiography and intra-arterial assessment of blood pressure.
Post-operative care after invasive surgeries included subcutaneous administration of 1-2
mg/kg Ketoprofen for analgesia and 0.1 mL/kg Terramycin (oxytetracycline 200 mg/mL)
for antibiotic prophylaxis.
Cardiac magnetic resonance
Prior to CMR, anesthetized sheep had the wool over their left parasternal region
shaved and the skin thoroughly cleansed with warm water, detergent and ethanol, to
improve electrocardiogram lead adherence. They were then positioned in dorsal
recumbence inside the CMR scanner and mechanical ventilation was commenced to ensure
adequate breath-holds during image acquisition.
The following parameters were used for the cine sequences: repetition time
(TR)/echo time (TE) 52.05 ms/1.74 ms; flip angle 70°; matrix 256x150; 25 phases per
cardiac cycle; FOV 380 mm with slice thickness 6 mm and inter-slice gap 4 mm through
the ventricles. Heart rate was typically 90-100 bpm (R-R’ interval 667-600 msec) during
imaging and therefore the breath-hold times were between 8 and 12 sec for all animals.
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For delayed contrast-enhanced imaging, a segmented T1-weighted inversionrecovery (turboFLASH) sequence was used with the following parameters: TR 2 cardiac
cycles; TE 3.4 ms; α 25°; FOV 380 mm; acquisition matrix 256x150; typical voxel size
1.9x1.4x6 mm. The inversion delay (TI) was determined using an inversion recovery scout
sequence, and was lengthened during scanning in order to maintain optimal nulling of
normal myocardium. Typically, the TI was between 260 and 320 msec. Standard and phase
sensitive imaging were performed, although the standard, delayed contrast-enhanced,
inversion recovery gradient echo-based (FLASH) sequences were used for analyses.
Ventricular ejection fractions and chamber volumes were determined in blinded
fashion, with Argus software. End-diastolic and end-systolic images were chosen as the
maximal and minimal, mid-ventricular, cross-sectional areas in a cinematic display. Short
axis endocardial and epicardial borders were traced manually for each slice in enddiastole and end-systole. These areas were multiplied by the slice thickness (10 mm) and
added together to obtain the EDV and ESV, respectively. Papillary muscles were
included for the volume measurements. Care was taken not to include atrial slices at endsystole secondary to apical movement of the base of the heart during LV contraction.
Ejection fraction was calculated by the formula: EF (%) = (EDV-ESV)/EDV x100.
Transthoracic echocardiography
Transthoracic echocardiography (Acuson XP-128, 4 MHz probe, Siemens
Medical Systems, PA, USA) was performed serially during animal studies to monitor
changes in LV dimensions and contractile function, especially during and after
doxorubicin dosing. Images were obtained once animals had been fully anesthetized and
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mechanically ventilated for 30 min and placed in the right lateral decubitus position. Two
dimensional-guided M-mode measurements of LV end-diastolic dimension (EDD) and
end-systolic dimension (ESD) were taken from right parasternal short axis views, just
basal to the insertion of the papillary muscles. For each parameter, measurements from
three separate consecutive cardiac cycles were averaged. Left ventricular fractional
shortening (FS) was derived from the equation: FS (%) = (EDD-ESD)/EDD x100.
Cardiac catheterization and hemodynamic measurements
An 8 Fr sheath was used to access the left external jugular vein and a 7.5 Fr
thermodilution catheter was passed for obtaining right atrial, right ventricular, pulmonary
arterial and pulmonary capillary wedge pressures which were recorded in end-expiration.
Cardiac outputs were obtained at end-tidal CO2 40 mmHg, from triplicate thermodilution
measurements, using iced 5% dextrose boluses (10 mL).
Left ventricular systolic and end-diastolic pressure measurements were obtained
using a 5 Fr pigtail catheter passed retrogradely into the LV from the femoral arterial
access site.
Masson’s trichrome staining
Sections were initially dewaxed, taken to distilled water and then stained with
Celestin Blue for 5 min. After rinsing with distilled water, they were stained with
haematoxylin for 5 min and then washed with running tap water for 10 min. They were
then placed in Biebrich scarlet-acid fuchsin for 8 min and again rinsed in distilled water.
Following this, the sections were treated with Phosphomolybdic-phosphotungstic acid
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solution for 15 min, placed into Aniline blue solution for a further 5 min and then rinsed
in distilled water. Finally, they were differentiated in 1% acetic acid for 3-5 min, then
dehydrated, cleared and mounted.
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Supplementary Figure Legends
Supplementary Figure 1. Left ventricular segmentation for DE-CMR
For the purpose of DE-CMR analysis, horizontal slices were used from the basal (a), mid
(b) and distal LV levels. As was the case for NOGA® XP and histology, segmentation
was performed by dividing the basal and mid-ventricular levels into four segments each
(septum (S) 120°, anterior (A) 80°, lateral (L) 80° and posterior (P) 80°), while the distal
level was analyzed in its entirety. The margins of the interventricular septum were used
to guide segmental orientation.
Supplementary Figure 2. Left ventricular segmentation for histology
Formalin-fixed sheep hearts were sliced transversely through the left ventricle, at 1 cm
intervals commencing at the apex. Bold line markings show the surfaces used for
processing basal, mid and distal ventricular levels (a). Further segmentation of the
myocardium was performed as per the nine-segment strategy used by NOGA® XP. The
basal (b) and mid (c) ventricular levels were divided into four regions each,
corresponding to the septum (S: 120°), anterior (A: 80°), lateral (L: 80°) and posterior (P:
80°) segments. The distal level was analyzed in its entirety. As for DE-CMR, the margins
of the interventricular septum were used to assist with segmental orientation.
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