Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
SUPPLEMENTAL MATERIALS AND METHODS
Human Adipose and Cardiac Tissue
Between 2009 and 2011, we obtained paired samples of EAT and SAT from 39 patients
undergoing routine cardiac bypass surgery. SAT biopsies were taken from the parasternal
region, while EAT samples from the left interventricular groove before the cardiopulmonary
bypass (paracardial fat was not collected). The clinical parameters of patients are indicated in
Table 1. The LVEF was assessed in 4 chambers apical view of the echocardiography using
Simpson measurement. Each patient underwent two echocardiographies performed by
independent operators, one in the cardiology department and one in the surgical department. It
is <45% the criteria used for low LVEF. 12 paired samples were dedicated to transcriptomic
study (Group 1), 13 to the organo-culture experiments (Group 2) and 14 to the proteomic
analysis of the conditioned media (Group 3). In 18 patients, a sample of the right (n=15) or
left (n=3) atrial appendage was obtained for histological study. Ventricle human myocardium
samples (n=5) were obtained from autopsy. The ethical committee of Pitié-Salpêtrière
Hospital approved the clinical investigations.
Organo-culture of Adult Rat Atria
Animals were handled in accordance with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health. Adult male Wistar rats (200 to 250
g) were anesthetized using an intraperitoneal injection of sodium pentobarbital (0.1 mL/100
mg body weight) 15 min after intraperitoneal heparin injection. Whole hearts were rapidly
excised and atria (right and left) were isolated from the whole heart and transferred into cold
CO2-independent medium (Gibco, Invitrogen). Left and right atria were dissected out from
the aperture of the mitral valve or the superior vena cava, respectively, under a binocular
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
microscope. The septal parts were discarded, and only the trabeculated myocardium walls
were conserved. Inserts containing polyester porous membranes (pore size, 0.4 µm;
Transwell, Corning) were filled with M199 culture medium (Gibco, Invitrogen) containing
ITS (Insulin, Transferrin and Sodium selenite, 1/1000, Sigma), 10% fetal calf serum (FCS;
Invitrogen), 10 mM glucose, 1 nM isoproterenol, and 100 U/mL penicillin/streptomycin. The
trabeculated myocardium walls were then placed on the porous membranes in a drop of
culture medium, with the endothelial face toward the porous membrane. Next, atria were
flattened by removal of the drop by capillary action, and incubated for 15 min at 37°C (5%
CO2). After adhesion, 20 µL of culture medium were dropped onto the epicardial face, and
atria were kept for 7 days in standard incubation conditions (37°C, 5% CO2). The culture
medium was changed daily. Each adipose tissue-conditioned media (EAT, OAT, SAT) was
mixed in a proportion of 1:4 with culture medium. One drop of the mix was poured directly
onto the specimen to keep it hydrated. In a different set of three experiments, rat atria were
treated with culture medium containing 5 ng/mL of recombinant human/rat Activin A (R&D
Systems). The effects of monoclonal transforming growth factor  (TGF-β)neutralizing
antibody (1 µg/mL; R&D Systems), mouse monoclonal Inhibin βA-neutralizing antibody (1
µg/mL; Abcam), or non-immune IgG preincubated for 30 min with adipose tissue-conditioned
media and culture medium alone were also tested. At the end of the experiment, atria were
washed with PBS and fixed with 4% formaldehyde for 15 min. Fixed atria were embedded in
cryo-conservation medium (Shandon Cryomatrix, Thermo Scientific), frozen with liquid
nitrogen, and stored until use at 80°C (Fig.1 supplement File).
Rat Cardiac Fibroblasts Culture
Atria from three-month-old rats were used to isolate cardiac fibroblast as previously described
20
. Next, the cells were cultured in DMEM 4.5 g/L glucose (Gibco) supplemented with 10%
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
SVF, 100 U/mL penicillin, and 100 mg/mL streptomycin, in standard conditions (37°C, 5%
CO2).
Immunohistological Analysis
Frozen sections of rat atria (10 μm) were stained with Masson’s trichrome and picrosirius red
for the study of fibrosis which was quantified by histomorphometry using an Alphelys
platform (Histolab Software, Plaisir, France) and expressed as the ratio of fibrous tissue area
to total tissue surface area 8. Procedure used for the immunofluorescence has been already
described
20
; the following primary antibodies were used: anti-collagen I (1/200, rabbit
polyclonal, Novus Biological), anti-collagen III (1/100, rabbit polyclonal, Abcam), anticollagen VI (1/100, mouse monoclonal, Abcam) and anti-sarcomeric α-actinin (1/400, mouse
monoclonal, Sigma); the following secondary antibodies Alexa Fluor® 488 goat anti-rabbit
IgG, Alexa Fluor® 546 goat anti-mouse IgG (1/400, Invitrogen), and Alexa Fluor® 594 goat
anti-mouse IgG (1/400, Molecular Probes, Invitrogen). Nuclei were stained with DAPI
(Invitrogen).
Deconvolution Microscopy
Images were acquired with an epifluorescent microscope (60X, UPlanSApo, 0.17) equipped
with a cooled CoolSnap camera (Roper-Scientific). 26 images of 0.2 µm-thick were collected
in the z-axis for each sample using a piezoelectric system and deconvoluted with the
Metamorph software (Molecular Devices) using acquired PSF. For each sample, 3
consecutive z-images were thresholded to similar levels and z-projections were carried on
using the Image J software. Phase contrast images were also acquired to provide information
on cell shape and cell membranes.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
Multiplexed Proteomics and Enzyme-Linked Immunosorbent Assay
Cytometric bead arrays were used to analyze protein composition of EAT and SAT
conditioned-media. Multiplex assays were performed according to the manufacturer
instructions (R&D Systems, France). Multianalyte profiling was performed on the Luminex200 system and the Xmap Platform (Luminex Corporation, Austin, TX). Fluorescence data
were analyzed with Xponent software by using standard curves obtained from serial dilutions
of standard cytokines mixtures. Concentrations of Activin A and Follistatin were measured by
Enzyme-Linked Immunosorbent Assay (ELISA) according to the manufacturer instructions
(R&D System, France).
Second Harmonic Generation Imaging of Rat Auricular Fibrosis
Second-harmonic generation (SHG) is an optically nonlinear coherent process used to image
the collagen network, based on 2-photon excitation. Multiphoton imaging was performed
using 10-μm thick slices covered with coverslips without any staining.
Statistical Analysis
To study the relationship between clinical parameters and the composition of the EAT
secretome a multiple factor analysis was used (Multiple Factor Analysis (AFMULT
package)). This analysis is analogous to a Principal Component Analysis (PCA) approach but
it allowed us to combine quantitative (Age, Body Mass Index (BMI), Activin A, MMP8) and
qualitative (diabetes, hypertension (HTA), dyslipidemia, Left Ventricular Ejection Fraction
(LVEF)) analysis of parameters.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
SUPPLEMENTAL RESULTS
Supplemental Figure 1
A
B
Adipose tissue
conditioned medium
+
Cardiomyocyte
culture medium
Sarcomeric -actinin
Connexin 43
Permeable polycarbonate Transwell ®
Caveolin 3
Dissection of atria
Legend Figure S1. Adult rat atrial interface culture model. (A) Left and right atria were
dissected out and cultured on permeable polycarbonate membrane in either control
cardiomyocyte culture medium or with adipose tissue conditioned medium. (B) One weekcultured
atria
were
assessed
for
survival
and
architecture
integrity
using
immunohistochemichal approach (right panel). (Top) -actinin staining showing the wellconserved striation of the myocytes, (middle) connexin 43 staining showing the intercalated
discs location and, (bottom) caveolin 3 staining showing the integrity of the sarcolemma.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
Supplemental Figure 2
DAPI+Collagen type VI
DAPI+Collagen type III
DAPI+Collagen type I
Second Harmonic
Ctrl
EAT
SAT
A
B
C
D
E
F
G
H
I
J
K
L
Figure S2. Epicardial adipose tissue promotes atrial collagen deposition. Second
Harmonic Imaging (A-C; green, collagen fibers) and immunostaining of collagen types I (DF), III (G-I), and VI (J-L) (D-L; green, collagen; blue, DAPI) were performed in rat atrial
sections (10 μm) after incubation with epicardial (EAT), or subcutaneous (SAT) adipose
tissue-conditioned media, or with control medium (Ctrl) [63× (A-D) and 10× magnification].
Data are representative of four separate experiments.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
Supplemental Figure 3
A
EAT
SAT
DAPI+Collagen I
Ctrl
Relative mRNA levels
B
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
*
Ctrl
EAT
SAT
* *
Collagen I
α-SMA
Figure S3. The EAT secretome has pro-fibrotic and pro-differentiating effects on
cardiac fibroblasts. Adult cardiac rat fibroblasts were incubated for four days with epicardial
(EAT) or subcutaneous (SAT) adipose tissue-conditioned medium, or with control medium
(Ctrl). (A) Collagen I immunostaining (green, collagen; blue, DAPI) (60× magnification). (B)
Collagen I and alpha-smooth muscle actin (α-SMA) expressions were measured using real
time PCR. Data are representative of three separate experiments. The nonparametric MannWhitney test was used *P<0.01.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
Supplemental Figure 4
Ctrl
Activin A 2 ng/mL
Activin A 5 ng/mL
Collagen 1
Red picrosirius
A
B
Ctrl
Activin A
(2ng/mL)
Activin A
(5ng/mL)
Figure S4: Activin A promotes atrial remodelling. (A) Picrosirius red was used to visualize
collagen deposition in rat atrial sections (10 μm) after one-week incubation with human
recombinant Activin A (2ng/mL and 5ng/mL) or control medium (Ctrl) (Upper panel)
Immuno-staining of collagen types 1 (green, collagen) in rat atrial sections (10 μm) after
incubation with Activin A (2ng/mL and 5ng/mL) or control medium (Ctrl) (20×
magnification) (Lower panel). (B) Total fibrosis in atrial sections: data depict the amount of
fibrotic area as percentage of the total tissue area.
Vanteclef & Guglielmi ref. EURHEARTJ-D-12-03207
Supplemental Figure 5
Coll3
Overlay
Ctrl
Phase contrast
10 µm
Coll3
Overlay
Activin A
Phase contrast
10 µm
Figure S5: Distribution of fibroblasts and collagen fibers in rat atrial explants treated
with Activin A. Immunostaining of collagen types 3 (Coll3) (green, collagen) in rat atrial
sections (10 μm) after incubation with Activin A or control medium (Ctrl) (20×
magnification). Data are representative of four separate experiments.
Supplemental Figure 6
Activin A 0.1 ng/ml
Activin A 1 ng/ml
Activin A 2 ng/ml
Collagen 3
Ctrl
Figure S6: Distribution of fibroblasts and collagen fibers in rat atrial explants treated
with Activin A (D) Distribution of fibroblasts and collagen fibers in rat atrial explants treated
with Activin A at 0.1, 1 and 2ng/mL. Immunostaining of collagen types 3 (Collagen 3) (green,
collagen) in rat atrial sections (10 μm) after incubation with Activin A or control medium
(Ctrl) (20× magnification). Data are representative of four separate experiments.