Supplemental Digital Content
Title
Connection between cardiac vascular permeability, myocardial oedema and inflammation during sepsis: role of the α1AMPK isoform
Authors
Diego Castanares-Zapatero, MD, Claire Bouleti, MD, PhD, Caroline Sommereyns, PhD1, Bernhard Gerber, MD, PhD, Christelle Lecut, PhD, Thomas Mathivet, PhD, Michael Horckmans, PhD, Didier Communi, PhD, Marc Foretz, PhD, Jean-Louis Vanoverschelde, MD, PhD, Stéphane Germain, PhD, Luc Bertrand, PhD, Pierre- François Laterre, MD, Cecile Oury, PhD, Benoit Viollet, PhD, Sandrine Horman, PhD* and Christophe Beauloye, MD, PhD*
*These authors contributed equally to this work and should be considered as joint last authors.
Supplemental material online
Materials and reagents
The following materials and reagents were obtained from the sources indicated:LPS from E. Coli O55B5 and Evans blue dye (EBD, Sigma, Bornem, Belgium); 5- aminoimidazole-4-carboxamide riboside (AICAr, Toronto Research Chemicals Inc., Toronto, Ontario, Canada); 24-well and 24-Transwell plates, streptavidin-horserarish peroxidase with DAB (BD Biosciences, Erembodegem, Belgium) or AEC chromogen (Dako, Everlee, Belgium), cytometric bead array-based immunoassays, anti-CD45 (BD Biosciences) and anti-F4/80 (AbDSerotec, Düsseldorf, Germany); blocking reagent (Perkin Elmer, Zaventem, Belgium); fluorescent mounting medium (Dako);
Alexafluor 488, anti-ZO1 and pCR4-Topo (Invitrogen, Gent, Belgium);OCT (optimal cutting temperature) compound (VWR, Leuven, Belgium), RNeasy mini kit, proteinase K and DNAse (Qiagen, Venlo, Netherlands); iScriptTMcDNA synthesis kit (Bio-Rad, Nazareth Eke, Belgium),qPCR core kit for Sybergreen (Eurogentec, Seraing, Belgium), PRKAA1 Silencer Validated siRNA (Ambion, Invitrogen, Gent, Belgium), Lipofectamine (Invitrogen, Gent, Belgium), anti-AMPK (Cell Signaling, Leiden, Netherlands), anti-eEF2 (Santa Cruz Biotechnology, Heidelberg, Germany).
Supplemental figure legends
Supplemental Figure S1.Survival, left ventricular function and cardiac permeability in C57BL/6J mice subjected toendotoxaemia.
C57BL/6J mice were administered saline vehicle (NaCl 0.9%) or increasing doses of LPS. (A) Mortality 24 h after the indicated LPSdose, n=4 to 8 mice/group(y=- 7.87+1.82x+0.037(x-14)2; r2:0.99; p<0.05). (B)EF was measured by echocardiography 6 and 24 h after 10 mg.kg-1LPS. The data are means ± SEM, at least n=5. *indicates values statistically different from the saline group.Time-course (C) and dose-response curve (D) of the LPS-induced effect on cardiac vascular permeability. EBD was administered simultaneously with LPS or saline vehicle.
Mouse hearts were excised at different times. The bars represent a scale of 200µm.
The data are means ± SEM, n=5. *denotes values statistically different from the saline group.
Supplemental Figure S2.Absence of α1AMPK in macrophages: a potential mechanism of LPS-induced vascular hyperpermeability?
Vascular permeability 24 h after injection of 10 mg.kg-1 LPS (black bars) or saline vehicle (white bars) in mice with constitutive α1AMPK deletion in macrophages and corresponding wild type animals. The data are means ± SEM, n=5 to 8/group.
*indicates values statistically different from the saline group.
Supplemental Figure S3. Cytokine and chemokine expression in the myocardium of α1AMPK-/-mice.
Cytokine expression was measured in heart extractsfrom α1AMPK+/+and α1AMPK-/- mice by RT-qPCR24 h after injection of 10 mg.kg-1LPS(black bars)or saline vehicle(white bars). The data are means± SEM, n=4 to 12/group. The results were log10-transformed for analysis. *indicates values statistically different from the saline group. $denotes values statistically different from the α1AMPK+/+ group.
Supplemental Figure S4. AMPK and endothelialtight junctions
(A, C) ZO-1immunostaining in HCAEC (A) and HDMEC (C) monolayers.Cells were incubated in the presence or absence of 1mMAICAr1 h before being treated with 50 µg/mlLPS or vehicle for 24 h. White arrows indicate ZO-1 linear staining. Pictures are representative of 3 different experiments. (B) Gap areaswereassessed and expressed as percentages of microscopic fieldson serial pictures. The data are means ± SEM, n=3/group.*indicates values statistically different from the saline group. $denotes values statistically different from LPS without AICAr pre-treatment.
Supplemental Table S1
Gene amplified
Sense(s) Antisense
(as) Sequence temp. (°C) Annealing
RPL32 (s) ggcaccagt cag acc gat at (as) cag gat ctg gcc ctt gaa c 60
Il-6 (s) aagagttgtgcaatggcaattct 60
(as) aaattttcaataggcaaatttcct gat Il-1β (s) caaccaacaagt gat attctc cat g
64.4 (as) gatccacactctccagctgca RANTES (s) act ccc tgctgctttgcc ta
64.4 (as) ccc act tcttctctgggttgg TNF-α (as) agggtctgggccatagaact (s) gaactggcagaa gag gcact 64.4
CXCL1 (s) gcgcctatcgccaat gag c 62.5
(as) gcaagcctcgcgaccatt c CXCL2 (s) gat act gcccaaaggcaaggctaa c
64.4 (as) gcaggcacatcaggtacgatc MPO (s) acctac ccc agtacc gat ct
62.5 (as) actctc cag ctggcaaaa a
Tcf7 (s) atcctt gatgctgggatctg 64.4
(as) gcaatgaccttggctctcat
Cd11b (s) gactcagtgagc ccc atc at 62.5
(as) agatcgtcttgg cag atgct
A B
C D
Figure S1
mortality (%)
LPS dose (mg.kg-1)
0 1 2 3
0 20 40 60 80 100
10 20 30 40
baseline 1 hour
6 hours 24 hours
Permeability (relative fluorescence)
0 10 20 30
*
*
LPS dose (mg.kg-1) time (hours)
EF (%)
0 6 24
0 20 40 60
*
time (hours)
EF (%)
0 6 24
0 20 40 60
*
saline vehicle LPSLPS
saline
30 10
1 0
0 10 20 30 40 50
* *
NS
macro
α1AMPK +/+
macro
α1AMPK -/-
LPS
- + - +
Permeability (relative fluorescence)
Figure S2
c-DNA copies/RPL32 0.0 0.1 0.2 0.3
IL -1
β*
*
0.00 0.05 0.10 0.15
*
*
IL-6
TNF-α
c-DNA copies/RPL32
0.000 0.001 0.002 0.003 0.004 0.005
*
$
*
0 200 400 600 800
CXCL1
*
*
c-DNA copies/RPL32
0.0 0.2 0.4 0.6 0.8
*
*
CXCL2
0.0 0.2 0.4 0.6 0.8
* *
RANTES
$
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
NS
$
$
$
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
α1
AMPK +/+
α1AMPK -/-
LPS
- + - +
Figure S3
A B
C
area of gap (%)
0 10 20 30 40
*
$
vehicle AICAr
LPS LPS+AICAr
vehicle LPS LPS+AICAr
LPS
- - +
AICAr
