SUPPLEMENT
METHODS
Electrophysiological study
Electrophysiological study was performed in the post-absorptive state under general
anesthesia. The venous access was obtained through the right femoral and left internal jugular
veins. A 10-pole catheter with 2-5-2 mm inter-electrode spacing (Daig Electrophysiology,
Minnetonka, MN) was advanced through the left internal jugular vein and positioned in the
coronary sinus (CS) with the proximal bipole positioned at the CS ostium in the left anterior
oblique view. A conventional transseptal puncture was performed using BRK1 needle and SL0
sheath to access the left atrium.
Surface electrocardiogram (ECG) and bipolar endocardial electrograms were
continuously monitored and stored on a computer based digital amplifier/recorder system for
off-line analysis (LabSystem Pro, Bard Electrophysiology, Lowell, MA, USA). Intracardiac
electrograms were filtered from 30 to 500 Hz, and measured with computer assisted calipers at
a sweep speed of 200 mm/s.
Electroanatomical mapping
Electroanatomic maps of right and left atrium were created in sinus rhythm. This was
performed using a 3.5-mm tip catheter (Navistar, Biosense Webster, Diamond Bar, CA, USA)
and CARTO XP mapping system (Biosense Webster, Diamond Bar, CA, USA). The
electroanatomic mapping system has been previously described in detail.(1) The accuracy of
the sensor position has been previously validated to 0.8 mm and 5°. In brief, the system records
the surface ECG and bipolar electrograms filtered at 30 to 400 Hz from the mapping and
reference catheters. Endocardial contact during point acquisition was facilitated by electrogram
stability, fluoroscopy, and the catheter icon on the CARTO system. Points were acquired in the
auto-freeze mode if the stability criteria in space (≤6mm) and local activation time (LAT; ≤5ms)
were met. Mapping was performed with an equal distribution of points using a fill-threshold of
15 mm. Editing of points was performed offline. Local activation time was manually annotated
to the peak of the largest amplitude deflection on bipolar electrograms. In the presence of
double potentials, the LAT was annotated at the largest potential. If the bipolar electrogram
displayed equivalent maximum positive and negative deflections, the maximum negative
deflection on the simultaneously acquired unipolar electrogram was used to annotate the LAT.
Points were excluded if they were not conforming to the surface ECG P-wave morphology or if
they were < 75% of the maximum voltage of the preceding electrogram.
Atrial conduction velocity
Isochronal activation maps (5-ms intervals) of the atria were created and regional conduction
velocity determined in the direction of the wave-front propagation (least isochronal crowding).
The CARTO system determines conduction velocity by expressing the distance between 2 points
as a function of the difference in local activation time. For analysis, each atrium was segmented
as previously described.(2) The mean conduction velocity for each segment was determined by
averaging the conduction velocity between 3-5 pairs of points, as previously described.(2)
Complex electrograms
The proportion of points demonstrating complex electrograms was determined using the
following definitions: 1) fractionated signals: complex activity of 50ms duration with 3 or more
deflections crossing baseline; and 2) double potentials: potentials separated by an isoelectric
interval and the total electrogram duration ≥50ms.
Atrial Voltage
For the purpose of evaluating regional bipolar voltage, the atria were segmented as above. Low
voltage areas were defined as three contiguous points with a bipolar voltage <0.5 mV.
Electrically silent areas (scar) was defined as three contiguous points with absence of
recordable activity or a bipolar voltage amplitude <0.05 mV (the noise level of the system). An
index of heterogeneity of the bipolar voltage amplitude was determined by calculating the
coefficient of variation of the different regions in each chamber.
Effective Refractory Period
Effective refractory period (ERP) testing was performed after the electroanatomic mapping.
Atrial ERPs were evaluated at twice the diastolic threshold at cycle lengths (CLs) of 450 and
300ms using an 8-beat drive train followed by an extra-stimuli (S2), starting with an S2 coupling
interval of 120ms increasing in 5ms increments. The ERP was defined as the longest coupling
interval failing to propagate to the atrium. ERP was measures from the following 7 sites: (1) RA
appendage, (2) RA lateral wall-upper, (3) RA free wall-lower, (4) proximal CS, (5) distal CS, (6)
LAA and (7) LA posterior wall. ERP was measured 3 times at each cycle length at each site and
averaged. If ERP varied by more than 10ms, an additional 2 measurements were made and the
total number averaged. ERP heterogeneity was determined by the coefficient of ERP variation
at each cycle length (CoV = SD/mean × 100%).
Fibrosis assessment
Morphometric analysis of Masson’s trichrome-stained sections was performed utilizing
‘analySIS LS Professional’ software (Olympus Soft Imaging Solutions, Münster, Germany) to
obtain a quantitative estimate of collagen within the tissue. Stained sections from LAPW and
LAA from of 6-8 animals per group were digitally captured (from 5 random fields per section
using NanoZoomer Digital Pathology System software (Hamamatsu Phonetics, Japan); at a
magnification of x5 with an area of Masson’s trichrome selected for its color range and the
proportional area of tissue with this range of color quantified.
Atrial TGFβ1
TGFβ1 concentration in atrial tissue was assessed with western blot. Tissue was sonicated in
homogenization buffer (20mM Tris-HCl, pH 7.4, 25oC; containing 2mM EDTA, 0.5mM EGTA,
1mM dithiothreitol, 50µg/ml leupeptin, 50µg/ml pepstatin A, 50µg/ml aprotinin and 0.1mM
phenylmethylsulphonyl fluoride). An equal volume of sample buffer (62.5mM Tris-HCl, pH 7.4,
containing 4% SDS, 10% glycerol, 10% β-mercaptoethanol and 0.002% bromophenol blue) was
then added and samples were heated to 80oC for 3 minutes; protein concentrations in each
sample were equalized according to the bicinchoninic acid assay.
Electrophoresis was performed using a Mini-PROTEAN® TGX™ precast gel system (Bio-Rad
Laboratories Pty Ltd, Gladesville, New South Wales, Australia). Denaturing polyacrylamide gels
(10% w/v) were employed for protein separation, which was undertaken as described
previously.(3) After separation, proteins were transferred to polyvinylidine fluoride (PVDF)
membranes for immunoprobing (Bio-Rad Laboratories Pty Ltd). Membranes were incubated
with antiserum for TGF-β1 (1:1000; Abcam; Cambridge UK) or actin (1:20000; Sigma Chemical
Company; Castle Hill, NSW, Australia) overnight, and labeling carried out using a two-step
detection procedure: first, biotinylated secondary antibody (Vector Laboratories; 1:1000; 30
minutes) was reacted with membranes and then streptavidin-peroxidase conjugate was applied
(1:1000; Pierce, Rockford, Il, USA). Positive antibody labeling was detected as described
previously (3) and quantification of detected proteins was achieved using the program, Adobe
PhotoShop CS2. Detection of β-actin was assessed in all samples as a positive gel-loading
control.
SUPPLEMENTARY REFERENCES
1.
Gepstein L, Hayam G, Ben-Haim SA. A novel method for nonfluoroscopic catheter-based
electroanatomical mapping of the heart. In vitro and in vivo accuracy results. Circulation
1997;95:1611-22.
2.
Sanders P, Morton JB, Davidson NC et al. Electrical remodeling of the atria in congestive
heart failure: electrophysiological and electroanatomic mapping in humans. Circulation
2003;108:1461-8.
3.
Wood JP, Chidlow G, Graham M, Osborne NN. Energy substrate requirements for
survival of rat retinal cells in culture: the importance of glucose and monocarboxylates. J
Neurochem 2005;93:686-97.