Online supplemental material
TITLE: Atrial fibrillation is associated with the fibrotic remodeling of adipose tissue in the
subepicardium of human and sheep atria
AUTHORS: Peter Haemers, Hadhami Hamdi, Kevin Guedj, Nadine Suffee, Patrick
Farahmand, Natasa Popovic, Piet Claus, Pascal LePrince, Antonino Nicoletti, Jose Jalife,
Carmen Wolke, Uwe Lendeckel, Pierre Jaïs, Rik Willems, Stéphane N. Hatem
1
Methods
Sheep model of atrial fibrillation
Atrial fibrillation was induced in sheep by long-term rapid atrial pacing (RAP), as previously
described.1-6 In brief, a neurostimulator (Itrel, Medtronic, Minneapolis, USA) was implanted and used
for continuous pacing of the atria at 900 bpm, resulting in atrial capture at approximately 300 bpm, for
at least 15 weeks to induce AF. All sheep received the same standard diet. This investigation
conformed to the Guide for the Care and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the local Ethics
Committee (Ethische Commissie Dierproeven, K.U. Leuven, Leuven, Belgium).
Cardiac magnetic resonance imaging in sheep
The AF (n=11) and sham (n=5) sheep used to assess cardiac adipose tissue volume were
sacrificed at a fixed end point of 16 weeks. One week before sacrifice the neurostimulator was
surgically replaced by a regular pacemaker in the AF group. At this moment, RAP was stopped,
allowing the assessment of AF burden by AF detection software of a dual chamber pacemaker
(Adapta, Medtronic, Minneapolis, USA). Before cardiac MRI at 16weeks, electrical reconversion was
performed if no spontaneous reconversion occurred during this last monitoring week.
Adipose tissue was assessed by cardiac magnetic resonance imaging (MRI) on a 3T scanner
(TRIO-TIM, Siemens, Erlangen, Germany) with a 8-channel phased-array coil. Adipose tissue volume
was assessed before starting RAP (0 week) and at 16 weeks. All scans were performed under general
anesthesia with isoflurane (1%), after pre-medication with ketamine (10 mg/kg IM) and xylazine (0.2
mg/kg IM). Mechanical ventilation allowed breath holding during image acquisition. Sequential short
axis images were acquired with a 4 mm slice thickness and no interslice gap. Slices were aligned at 90
degrees to the long axis of the left ventricle. Image acquisition started at the lowest part of adipose
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tissue associated with the ventricular apex, up to the most proximal atrial fat deposition (defined as the
mid part of the pulmonary artery on the long axis view). An ECG-gated inversion recovery sequence
with a long inversion time (800ms) and gradient echo readout resulted in a high contrast between
adipose and other tissues. Heart rate was kept constant in all animals by atrial pacing at 90 beats per
minute, ensuring constant image quality between the different time points and animals.
Pericardial adipose tissue was assessed at ventricular, atrial and left atrial levels. Adipose
tissue was measured semi-automatically in a blinded fashion using custom made software in Matlab
(Matworks, Natick, MA). First, a region of interest including all cardiac adipose tissue was delineated
in all short axis images. Secondly, within this region of interest adipose tissue was segmented by
adjusting the gray scale threshold (Supplemental figure 2A). Adipose volume was calculated by the
summation of disk method. Ventricular adipose tissue was measured from the apex up to the mitral
valve plane and atrial adipose tissue from the mitral valve plane up to the mid part of the pulmonary
artery (Supplemental figure 2B).
Validation of adipose tissue volume assessment by cardiac MRI was performed by correlating
the mass of paracardial fat at autopsy with total MRI fat (i.e. ventricular and atrial fat) (Supplemental
figure 2C). Total MRI fat volume was converted to mass by multiplying it with a density of 0.9g/ml.
Paracardial adipose tissue was collected at autopsy by removing all parietal pericardium with its
associated paracardial adipose tissue.
In order to determine adipose tissue coverage of the left atrium, we reconstructed the left
atrium and atrial adipose tissue (Supplemental Figure 3). Contours obtained from the LA endocardium
and the adipose tissue were reconstructed into 3D triangulated surface mesh by Poisson surface
reconstruction algorithm. To determine adipose tissue coverage of the LA, proximity of the two
meshes was observed. For each face in the LA endocardium mesh we have found the closest face in
the adipose tissue mesh, and if the distance between centers of those triangles was less than 5 mm
coverage was assigned.
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Histological study of human and sheep samples
In total samples from 135 patients were collected, 12 from the LA and 123 from the RA. Of
these 10 LA and 92 RA samples with sufficient histological quality and at least 5mm in epicardial
length were included for analysis. Right atrial samples were collected during cannulation of the right
atrium, at the level of the appendage. These RA samples could be obtained during routine cardiac
surgery from a large number of patients and samples were taken from a fixed area of the right atria and
were of regular anatomic morphology allowing a comparison between samples. LA samples were
collected in a limited number of patients, in which a LA incision was necessary for the procedure
mainly for mitral valve replacement. The AF burden was classified according to the ESC guidelines.7
The classification was performed by the treating physician. In accordance with the recommendation of
2014 HRS/ACC, the term “permanent AF” was used when patients remained in AF on ECG
performed at 6 months interval and when no further attempts to restore and/or maintain sinus rhythm
was done.
Atrial appendage samples, fixed in 4% paraformaldehyde and embedded in paraffin, were
used for the quantification of fibrosis, inflammation and fatty infiltrates. After deparaffinization,
sections were stained with Sirius Red and Haematoxylin Eosin (HE). Modified Sirius Red staining
was performed on sheep atrial sections (i.e. additional staining with Haematoxylin), allowing an
assessment of inflammatory cells. All analyses were performed in a blinded fashion. Adipose tissue
was quantified on all HE stained sections, using manual delineation, and expressed as a percentage of
total area.
In humans, the fibrotic extent of fatty infiltrates was assessed on Sirius Red stained sections.
The extensiveness of fibrotic remodeled epicardium was visually assessed and expressed as a
percentage of total epicardial length using Image J (ImageJ 1.48v, National Institutes of Health, USA).
These visual assessments were validated in a subset of samples using semi-automated digital
processing (ImageJ). Total myocardial fibrosis was quantified on Sirius Red stained sections, using the
method described in supplemental Figure 4.
4
In sheep, analysis of fatty infiltrations was performed on left atrial appendage samples. AF
sheep were sacrificed at 24±8 weeks of rapid atrial pacing (14±12 weeks of persistent AF).
Subepicardial fatty infiltrations were assessed on Sirius Red stained sections. The less extensive
remodeling of the epicardial region, compared to the human samples, allowed segmentation of
individual fatty infiltrations (Supplemental figure 5A). A histological scoring system was developed to
visually grade the fatty infiltrations according to a four grade system. Grading was performed twice by
the primary investigator and by a second investigator to determine the intra- and inter-observer
agreement. A quantitative validation of the grading system was performed in a subset of fatty
infiltrates (Supplemental Figure 5B).
Inflammatory cells were assessed on HE stained sections using a magnification of 100x and
400x in both human and sheep samples. For human samples, at least one cluster of inflammatory cells
in the subepicardial adipose tissue was necessary to designate a patient as ‘inflammation positive’.
Immunohistochemistry
For immunohistochemistry, six µm-thick sections were deparafinized in toluene and hydrated
in ethanol. After blocking in 5% BSA, sections were incubated with primary antibodies (rabbit antihuman CD3, polyclonal; mouse anti-human CD20, L-26; mouse anti-human CD8, C8/144B, Dako;
rabbit anti-human CD4, sp35, Fisher; mouse anti-human CD15, HI98, BioLegend; rabbit anti-human
CD14, EPR3653; rabbit anti-human Granzyme, polyclonal, Abcam) overnight at 4°C. After several
washes in PBS, slides were incubated with the appropriate secondary antibody (anti-rabbit DL649;
anti-mouse A488, Jackson Immunoresearch). TUNEL staining was performed following the
manufacturer’s instructions (Roche). Nuclei were stained with Hoechst 53542 and slides were covermounted with Prolong Gold Antifade Reagent® (Invitrogen). Images were captured with a
Nanozoomer 2.0 RS (Hamamatsu) and with a Zeiss axiovert 200 M microscope equipped with the
AxioCam Mrm vers.3 camera, the ApoTome® system and the AxioVision® image capture software.
Chemotaxis assay
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Epicardial adipose tissue of sheep was collected at the predefined locations described in supplemental
figure 7(A). The collected adipose tissue was cultured as previously described (0,1g of adipose tissue
per 1 ml of culture media).8 After 24 hours the conditioned medium was collected. Peripheral blood
mononuclear cells (PBMC) were prepared from fresh blood of a single healthy sheep using cell
preparation tubes with sodium citrate (BD Vacutainer CPTTM). A total of 500 000 PBMCs were added
to the upper chamber of the Transwell (3 µm pores) (Greiner Bio-one, Vilvoorde, Belgium). Control
medium or conditioned medium from AF or non-AF sheep was added to the lower chamber
(Supplemental Figure 6A). The migration test was performed for 120 minutes in a cell culture
incubator (37°C, 5% CO2). Subsequently, the concentration of PBMCs in the lower chamber was
counted. The chemotactic capacity of conditioned medium was expressed as fold increase in
comparison with control medium.
Results
Cardiac adipose tissue volume in sheep
Sixteen weeks of rapid atrial pacing resulted in persistent AF in nine of eleven sheep, with a
minimal duration of 14 hours. Two sheep were not in AF when stopping RAP. Four sheep remained in
AF for the complete duration of the monitoring week and needed electrical reconversion before
cardiac MRI at 16 weeks. The weight of the sheep slightly increased in the sham group (baseline:
46±4kg vs 16weeks: 50±2 kg, p=0.0327) and remained stable in the AF group (baseline: 48±2kg vs
16weeks: 50±3 kg p=0.0683). The total cardiac MRI fat mass (i.e. ventricular and atrial adipose tissue)
correlated strongly with autopsy measured paracardial fat (r=0.8599; p<0.0001). However, cardiac
MRI systematically over-estimated autopsy fat mass by 53±18g. This difference can be at least
partially explained by the fact that the epicardial adipose tissue was not collected at autopsy due to
methodological considerations.
6
The total LA surface area significantly increased (LA surface area: 81.2 ± 11.3 cm² vs
16weeks: 109.0 ± 21.4 cm², p=0.003). However, the fraction of the LA surface area covered by
adipose tissue did not change (0 week: 50.8 ± 9.9% vs 16weeks: 47.6 ± 12.8 %, p=0.576). These
results suggest an increase of preexisting areas as an explanation for the increase of atrial adipose
tissue.
Increased chemotactic capacity in AF sheep
The chemotactic capacity of atrial epicardial adipose tissue on peripheral blood mononuclear
cells was significant higher in the AF sheep in comparison to non AF sheep (Supplemental Figure 8).
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Supplemental Figures
Supplemental figure 1: Illustrative examples of a transition zone between non-fibrotic and
fibrotic remodeled fatty infiltrations. (human samples)
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Supplemental Figure 2: Assessment of the cardiac adipose tissue volume by cardiac magnetic
resonance imaging. (A) A region of interest including all cardiac adipose tissue was delineated in all
short axis images (yellow line). Within this region of interest adipose tissue was segmented by
adjusting the gray scale threshold. (B) Ventricular adipose tissue (AT) was measured from the apex up
to the mitral valve plane and atrial adipose tissue from the mitral valve plane up to the mid part of the
pulmonary artery. (C) Good correlation between total cardiac MRI fat mass (i.e. ventricular and atrial
adipose tissue) and autopsy measured paracardial fat.
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Supplemental figure 3: Illustrative example of adipose tissue coverage (green) of the left atrium
(grey). (Postero-lateral view; 51.1% fat coverage in example)
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Supplemental figure 4: Quantification of myocardial fibrosis on Sirius Red stained sections. (A)
The myocardial area was manually selected, with exclusion of the epicardium and associated adipose
tissue. (B) Total myocardial area. (C) Calculation of fibrosis by threshold based selection of red areas.
Myocardial fibrosis was expressed as a percentage of total myocardial area. (Image processing
performed with ImageJ).
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Supplemental figure 5: Selection of fatty infiltrates and validation of the histological scoring
system in atrial sheep samples. (A) In sheep, the epicardial area was less remodeled in comparison
with the human samples. This allowed segmentation of individual subepicardial fatty infiltrates (dotted
boxes), which were photographed (using a magnification of x100) and digitalized for further analysis.
Grading according to the new histological scoring system was performed offline in a blinded fashion.
(B) A subset of fatty infiltrates (five infiltrates in every grade) were quantitatively assessed for
fibrosis, epicardial thickness and adipocyte area (ImageJ). Fibrosis content was quantified by
calculating the ratio of area occupied by fibrosis to the total fatty infiltration area. Epicardial thickness
was measured at 3 locations above the fatty infiltration. Adipocyte area was determined by averaging
the area of ten adjacent adipocytes covering the full thickness of the fatty infiltration. Fibrotic content
of the fatty infiltrate and epicardial thickness increased with increasing grade. Whereas, the size of
adipocytes decreased with increasing grade (adipocyte area was not assessed in grade 3, as only few
adipocytes remained). (C) Results of the second observer scoring the fatty infiltrations demonstrates
similar results as in figure 2.
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Supplemental figure 6: Histological characteristics of cellular inflammation in the subepicardial
fatty infiltrations. (A, B) Most often the cellular inflammation was located epicardially (arrow). (B)
At several of these epicardial sites of cellular inflammation, a retraction of the epicardium could be
observed (arrows). (C, D) Inflammation (arrows) was also seen adjacent to extensive fibrotic
remodeled areas (asterisk) with no or limited remaining adipocytes. (E) Crown-like structures, i.e.
accumulation of inflammatory cells around an adipocyte (arrowheads, also in B and D). (F) On
Haematoxylin Eosin staining, the major component of these inflammatory infiltrates were
lymphomononuclear cells. (Human samples, Haematoxylin Eosin staining)
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Supplemental figure 7: Adipose tissue is abundant at atrial level and in close proximity with the
myocardial wall. (A) Macroscopic view of the sheep heart demonstrates epicardial adipose tissue at
both the right and left atrium, including the appendages, Bachmann bundle (*1), posterior wall (*2)
and coronary sinus. (B) The right atrial appendage in AF sheep demonstrates multiple fatty
infiltrations. In this AF sheep the fatty infiltrates show clear fibrotic transformation and cellular
inflammation. (C) The left atrial appendage in a control sheep with several fatty infiltrations.
However, here the fibrotic content is low and none infiltrating, also the adipocytes are larger and no
inflammation can be seen. Although both the right and left atrial appendage contains fatty infiltrations,
a difference in the extent of fatty infiltrates could be observed (right atrium: 7.4 ± 5.5% vs left atrium:
2.0 ± 3.2%; p=0.0234; AF (n=3), control sheep (n=5). (D) The pulmonary veins are covered by a
deposition of adipose tissue, which is located between the inner layer of the visceral pericardium and
the myocardium, and thus in direct contact with the myocardium. (E) Large fat depositions can also be
seen at the left atrial posterior wall, again in direct contact with the cardiomyocytes. (Composite
images recorded at a magnification of x50; LAPW= left atrial posterior wall; PV= pulmonary vein;
asterisks = larger depositions of adipose tissue around the atria; arrows = subepicardial fatty
infiltrations)
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Supplemental figure 8: Chemotaxis assay. (A) In vitro functional test of leucocyte migration, using
Transwells with 3 µm pores. Peripheral blood mononuclear cells (500 000 cells) were placed in the
upper chamber, and control medium or conditioned medium from either AF sheep or non AF sheep (6
control sheep and 5 sham sheep) in the lower chamber. The concentration of cells in the lower
chamber was counted after 120 minutes. Migration capacity of conditioned medium was expressed as
fold increase in comparison with control medium. (B) The chemotactic capacity of atrial epicardial
adipose tissue was significant higher in the AF sheep in comparison to non AF sheep.
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