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SUPPLEMENTARY MATERIAL
Detailed Methods
Mice.
The FoxO3-CA cDNA (T32A, S256A, S308A) kindly provided by Marc Schmidt (then
Netherlands Cancer Institute, Amsterdam) was subcloned into a bidirectional vector
(Clontech pBI-5) with luciferase and the FoxO3-CA transgene under the control of a Tetoperon (tetO.FoxO3-CA; Sup. Fig. 1A). After microinjection of pronuclei, founder mice were
identified by PCR of genomic tail DNA. These transgenic mice (CD-1 background) were
crossed with mice expressing the tetracycline transactivator under the control of the myosin heavy chain promoter (-MyHC-tTA, C57Bl/6) to obtain double-transgenic animals
(FoxO3MyHC) with cardiomyocyte-specific expression.
Mice were kept under specific pathogen free conditions at the animal facilities of the
University of Ulm and, for MRI analyses, the Max-Planck-Institut for Heart and Lung
Research, Bad Nauheim. Breedings were done in the presence of doxycycline in the drinking
water (1mg/ml, Sigma). Offspring was kept under doxycycline until the age of 6-10 weeks.
Wild type and single transgenic animals were used as controls. All experiments were in
accordance with German animal protection law and approved by the responsible government
agency (Regierungspräsidium Tübingen, Versuch-Nr. 1018).
In vivo bioluminescence imaging.
Mice were anesthetized by continuous inhalation of 2% vol/vol isoflurane. 5 minutes after i.p.
injection of 3.5 mg luciferin, luminescence was detected using an IVIS 200 System (Caliper).
An exposure time of 10 seconds was used for quantitative analysis.
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Cardiac magnetic resonance imaging.
For magnetic resonance imaging (MRI), mice were anaesthetized with isoflurane (1.7 2.0%), and body temperature was maintained throughout the measurements. The scan was
performed on a Bruker Pharmascan 7.0 T, equipped with a 300 mT/ m gradient system, using
a custom-built circularly polarized birdcage resonator and the Early Access Package for selfgated cardiac Imaging (Intragate, Bruker). Measurements were based on the gradient echo
method (repetition time= 6.3 ms; echo time= 1.5 ms; flip angle = 15°; field of view = 22x22
mm; slice thickness = 1.0 mm; matrix =128x128; repetitions = 100; resolution = 0.172 mm/
pixel). The imaging plane was localized using scout images showing the 4- and 2-chamber
view of the heart, followed by acquisition in short axis view, orthogonal on the septum in
both scouts. Multiple contiguous short-axis slices consisting of 6 to 8 slices were acquired for
complete coverage of the left ventricle. All MRI data were analyzed using Qmass digital
imaging software (Medis).
Adult murine cardiomyocyte isolation.
60 IU heparin (Ratiopharm) and 20 mg xylazin/ 125 mg ketamin per kg body weight were
injected intraperitoneally for anti-coagulation and narcosis. Hearts were excised and a 23G
hypodermic needle was inserted in the aorta. Digestion was achieved by retrograde
Langendorff perfusion with a calcium-free buffer supplemented with 6.5 mg/mL Liberase
DH (Roche) and 3.5 mg/mL Trypsin (Sigma). Digestion was stopped with FCS. Cells were
isolated with a 25 ml wide mouth pipette, filtered through a 100 µm mesh. After step-by-step
reintroduction of calcium, cells were seeded on laminin-coated 6 well culture dishes at a
density of 1 x 105 cells / well. Purity of the culture was >95% as determined by staining for
sarcomeric actinin.
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Histological analysis of transgenic hearts and immunofluorescence on tissue sections.
Organs were washed in PBS and snap-frozen in liquid nitrogen for cryosections, or incubated
in PBS-buffered formalin, dehydrated and embedded in paraffin. Cryosections were cut in 5
µm sections with a Cryotome (Leica), warmed and immediately fixed with PBS/ 4%
paraformaldehyde for 15 min. Paraffin sections were cut in 3 µm sections using a rotation
microtome (Thermo Scientific), deparaffinized and rehydrated. Sections were blocked with
10% goat serum containing 0.5% BSA in TBS for one hour at room temperature. First
antibody incubation with antibodies against FoxO3 (Santa Cruz) and sarcomeric actinin
(Sigma) was performed over night at 4°C with a first antibody diluent (Dako). Co-staining
was performed with 4',6-diamidino-2-phenylindole (DAPI) and wheat germ agglutinin
(WGA, Molecular Probes). Secondary antibodies coupled with Alexa Fluor 488 or 596
(Molecular Probes) were incubated for one hour at room temperature. TUNEL staining was
performed with an in situ TUNEL detection kit (Roche) following the manufacturer's
instructions. All fluorescence-labeled samples were analyzed with a fluorescence microscope
(Axiovert 200M; Zeiss) equipped with a digital camera (AxioCam MR3, Zeiss) and
Axiovision software. Leica DM5500B light microscope equipped with a DFC420C camera
(Leica) was used for visualization of hematoxyline/ eosin sections. Whole heart sections
stained with hematoxyline/ eosin were photographed with a Mirax scanner (Zeiss). Heart
photographs were taken with a Leica MZ7.5 stereomicroscope equipped with a Jenoptik
camera (ProgRes C14) and OpenLab software.
Protein extraction.
Mice were killed by cervical dislocation and organs were rapidly excised, washed with PBS
and snap-frozen in liquid nitrogen. Tissue was ground and aliquoted for protein and RNA
isolation. Tissue for SDS-PAGE was resuspended in RIPA buffer (0.01 mol/l Tris-HCl; 0.15
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mol/l NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% Triton X100), incubated for 15 min
on ice, followed by a 15 min centrifugation step with 13000 rpm at 4°C. To detect luciferase
activity, tissue or cells were resuspended in TNT buffer (0.1mol/l Tris-Hcl, 0.15mol/l NaCl,
and 0.05% Triton X100), sonicated (Diagenode Bioruptor), and centrifuged (13000 rpm, 15
min, 4°C). Luciferase activity measurements were performed on a Lumat (Berthold) with a
luciferin-containing buffer. Protein concentration was measured using the BCA detection kit
(Pierce).
Western blotting.
SDS Page was performed with 10 to 50 µg of protein. Separation was carried out on 4- 12%
gradient gels or 10% NuPage Bis/ Tris precast gels (Invitrogen) according to the
manufacturer's instructions. Protein transfer was performed with a semi-dry blotting device
(Biorad) onto a PVDF membrane (Millipore). Membranes were blocked for 1 h in TBS /5%
low-fat milk powder and incubated with the primary antibody over night in TBS/ 5% BSA or
5% low fat milk. The secondary antibody, coupled with horseradish peroxidase (HRP), was
applied for one hour at room temperature. Chemiluminescence detection was done with HRP
Juice (PJK) and a CCD camera (Fuji LAS 1000). For stripping, membranes were incubated
for 30 min at 37°C with a commercial buffer (Pierce, Restore plus western blot stripping
buffer). Specific antibodies used were purchased from Cell Signaling/ New England Biolabs:
mTor, phospho-mTor, Akt, phospho-Akt, Bnip3, phospho-GSK3, 4E-BP, phospho-4E-BP,
phospho-S6RP; from Santa Cruz Biotechnology: GSK3, GAPDH, FoxO3; and from Sigma:
destrin, -smooth muscle actin.
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RNA extraction, cDNA synthesis, and quantitative PCR.
Total RNA was isolated using the mirVana isolation kit (Ambion) following the
manufacturer's instructions. RNA concentration was measured using the Nanodrop (Peqlab)
device. cDNA synthesis was performed with the transcriptor high fidelity kit (Roche)
according to the manufacturer's instructions. A LightCycler 480 and the Universal Probe
Library (Roche) were used for quantitative RT-PCR; primer sequences are shown in
supplementary table III. PCRs were performed in triplicates according to the manufacturer's
instructions, with Rpl13 used as a reference for relative quantification. Data were corrected
for primer efficiency, which was determined by serial dilution of cDNA.
For quantification of mitochondrial DNA, complete DNA was extracted with a commercially
available kit (Qiagen). Quantitative PCR was performed on 40 µg of DNA with primers for a
mitochondrial gene (F-ATPase) and a genomic gene (Rpl13).
Gene expression profiling.
Total RNA was isolated using the mirVana isolation kit (Ambion) following the
manufacturer's instructions. Microarray analyses were performed with 200ng total RNA as
starting material and 5.5µg ssDNA per hybridization (GeneChip Fluidics Station 450;
Affymetrix). Total RNA was amplified and labeled with the Whole Transcript Sense Target
Labeling Assay (Affymetrix). Labeled ssDNA was hybridized to Mouse Gene 1.0 ST
Affymetrix GeneChip arrays. Chips were scanned with an Affymetrix GeneChip Scanner
3000 and analyzed using Affymetrix Expression Console Software. Probe level data were
obtained using the robust multichip average (RMA) normalization algorithm. Analysis of
differentially expressed genes was done on a gene-by-gene basis using annotation supplied by
the GeneSifter microarray data analysis system (www.genesifter.net, VizX Laboratories). For
both genotypes, four biological replicas were analyzed.
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Transmission electron microscopy.
For electron microscopy, left ventricular tissue of control or FoxO3MyHC mice was fixed over
night in 2.5% glutaraldehyde in 0.1mol/l cacodylate buffer (pH 7.4). Specimens were washed
in pure cacodylate buffer, postfixed for 1h in 2% OsO4 in cacodylate buffer, dehydrated in
ascending series of 2-propanol and propylene oxide, bloc-stained in 2% uranyl acetate in
ethanol for 30min and flat-embedded in Epon. Using an ultramicrotome (Ultracut, Leica),
semi- (0.5µm) and ultrathin (70 nm) sections were cut. Ultrathin sections were stained with
lead citrate, mounted on copper grids and finally analyzed with a Phillips EM400 electron
microscope.
Adult murine cardiomyocytes for transmission electron microscopy based on high pressure
freezing were grown on laminin coated and glow-discharged sapphire discs. Cardiomyocytes
were cultured for 36 h in the absence of doxycycline, and subsequently high-pressure frozen
with a HPF Compact 01 (Wohlwend) without prefixation, freeze-substituted and embedded in
Epon. Ultrathin sections were analyzed with a Phillips EM400 electron microscope.
For tomograms, sections of cardiomyocytes, about 400 nm thick, were collected on bare
copper grids with grid bars in one direction to prevent the grid bars from hiding the biological
structures at high tilt angles (3 mm grids for tomography, Plano GmbH, Wetzlar, Germany).
Gold particles (15 nm, Aurion, The Netherlands) on both section sides served as fiducial
markers for the calculation of the tomograms. Finally, the mounted sections were coated with
5 nm carbon from both sides by electron beam evaporation to increase electrical conductivity
and mechanical stability. The tilt series (-72° to +72°) were recorded with a 300 kV field
emission STEM (Titan 80-300 TEM, FEI, Eindhoven) with a high angle annular dark field
detector1 (Fischione, Export, PA, USA). Tomograms were reconstructed by SIRT using the
IMOD software package2.
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Measurement of autophagic flux.
For in vitro measurements, isolated adult cardiomyocytes were cultured for 32h and treated
for 4h with a mixture of lysosomal inhibitors (E64D (5µg/ml), bafilomycin A1 (100nM) and
pepstatin A methyl ester (5µg/ml). Cells were harvested and proteins were analyzed by
western blot. For in vivo measurements, mice were injected with either saline or chloroquine
(30 mg/kg body weight). 4h after injection, hearts were harvested and proteins analyzed by
immunoblot.
Transverse aortic constriction (TAC).
Mice were kept without doxycycline to allow the development of heart atrophy in FoxO3MyHC
mice and examined in a 1st MRI. Mice were then anaesthetized with isoflurane and
endotracheally intubated with a 20-gauge catheter (Braun, Germany). Mice were placed in a
supine position on a heating pad maintained at 37ºC. Subsequently, under the general
anaesthesia with 2 % isoflurane supplemented with 100 % oxygen, mice were ventilated
(MiniVent, HSE, Germany) with 220 strokes/min and 300 µl stroke volume. The transverse
aortic arch was surgically accessed and ligated (Prolene 7-0) between the right innominate
and left common carotid arteries with an overlying 24-gauge needle. The needle was then
removed, leaving a stenosis. Sham-operated mice underwent the same surgical procedure
without the actual ligation. Mice were analyzed 4 weeks after surgery in a 2nd MRI and killed
for further analysis.
Statistical analysis.
All parameters are expressed as arithmetic mean, with the standard deviation (SD) as a
measure of dispersion. Statistical differences were assessed with the Student’s t test
(*p<0.05; **p<0.01; ***p<0.001).
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References for Detailed Methods
1.
Höhn K, Sailer M, Wang L, Lorenz L, Schneider EM, Walther P. Preparation of
cryofixed cells for improved 3D ultrastructure with scanning transmission electron
tomography. Histochem Cell Biol. 2011 135, 1-9.
2.
Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of threedimensional image data using IMOD. J Struct Biol. 1996; 116:71-76.
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Legends to Supporting Figures
Fig. S1
Transgenic mouse model for conditional expression of constitutively active FoxO3 (FoxO3CA) in cardiomyocytes. (A) The tetracycline transactivator (tTA) is expressed under the
control of the -myosin heavy chain (-MyHC) promoter. In the absence of doxycycline,
tTA protein can bind to a bidirectional promoter (tetO) and activate the transcription of
luciferase and FoxO3-CA (right). Doxycycline prevents binding of tTA to tetO, thereby
shutting off transgene expression (left). (B) In vivo imaging of luciferase activity. Shown are
two pairs of a double-transgenic (FoxO3MyHC) and a control animal under the influence of
(left, Transgene OFF) or without doxycycline (right, Transgene ON). (C) Kinetics of
luciferase activation after removal of doxycycline from the drinking water (quantification of
in vivo imaging data). (D) Extracts of FoxO3MyHC and control mice 6 weeks after
doxycycline removal were analyzed in Western blots for FoxO3 and luciferase; GAPDH
shown as a loading control.
Fig. S2
Body weight of FoxO3MyHC animals. Shown are the mean body weights +/- SD of FoxO3
MyHC
mice in absolute values (upper diagram) and relative to age-matched control animals
(lower diagram; N=5-8 per group).
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Fig. S3
Hypertrophy pathways in FoxO3MyHC hearts and cardiomyocytes. (A) Quantification of
Western blot data from Fig. 2D, relative to the non-phosphorylated protein or GAPDH.
(B) Cardiomyocytes were isolated from mice kept under doxycycline. After isolation,
doxycycline was removed, allowing the transgene to be activated. Left panels: Immunoblot
against phosphorylated AKT (Thr-308) and ERK (loading control), 24 hours after
doxycycline removal. Right panel: Immunoblot against phosphorylated S6RP (Ser-235/236)
and GAPDH (loading control), 36 hours after doxycycline removal.
Fig. S4
Electron microscopy of FoxO3MyHC hearts and cardiomyocytes. (A-B) Transmission electron
microscopy (TEM) of glutaraldehyde-fixed left ventricular tissue of control (A) and
FoxO3MyHC (B) mice, 10 weeks after transgene activation. Scale bars: 3 µm. (C-D) TEM of
primary adult cardiomyocytes of FoxO3MyHC and control mice. Cells were cultivated for 36 h
without doxycycline and cryo-fixed under high pressure. In the control cell (C), the typical
assembly of contractile structures and mitochondria can be seen, whereas the transgenic cell
(D) manifests a disrupted architecture with degenerating contractile structures and
mitochondria engulfed by membranes. M, mitochondrium; asterisk denotes the M line of the
sarcomere. Scale bars: 1 µm. (E-F) Transmission electron microscopy (TEM) of
glutaraldehyde-fixed left ventricular tissue of control and FoxO3MyHC mice, showing the
preinuclear area. Scale bars: 1 µm.
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Fig. S5
Mechanisms of FoxO3-induced cardiac atrophy. Cardiomyocytes were isolated from mice
kept under doxycycline and cultivated without doxycycline, allowing ex vivo transgene
activation.
(A) Cells were cultivated without doxycycline for the indicated time, and
luciferase as a surrogate marker for transgene expression was measured in protein extracts.
(B) Validation of genes significantly up-regulated in the gene array analysis (see Table S2).
Quantitative RT-PCR of extracts from cardiomyocytes (N≥7 per genotype) 36 hours after ex
vivo transgene activation. Expression levels were normalized to endogenous Rpl13
expression and shown as fold change over controls, measured in triplicates. (C) Immunoblots
for LC3-I and LC3-II. After 6 weeks of doxycycline removal, mice were treated with the
lysosomal inhibitor chloroquine (30 mg/kg body weight) for 4 hours, or with saline. Heart
extracts were probed with an antibody detecting LC3-I and LC3-II. GAPDH: loading control.
(D) Western blot for the lysosomal marker protein LAMP1 with heart extracts of FoxO3MyHC
and control mice. GAPDH: loading control. (E) Analysis of cathepsin D activity. Upper
panel: Cardiomyocytes of FoxO3MyHC (black bars) and control mice (white bars) cultivated
36h without doxycycline were treated or not treated with E64D (5mg/ml) for 4 hours. Then,
cathepsin D activity was determined with a commercially available kit (Abcam). Lower
panel: The same measurement was done in vivo, in hearts of FoxO3MyHC (black bars) and
control mice (white bars). N=5 per genotype.
Fig. S6
Expression of genes involved in mitochondrial biogenesis and metabolism. Quantitative RTPCR of extracts from FoxO3MyHC mice, 6 weeks without doxycycline (N≥5 per genotype).
(A) Mitochondrial transcription factor A. (B) Acyl- CoA dehydrogenase. Expression levels
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were normalized to endogenous Rpl13 expression and are shown as fold change over
controls.
Fig. S7
Quantification by MRI of heart wall thickness in FoxO3MyHC and control mice before (1st
MRI) and after (2nd MRI) transverse aortic constriction. (A) Anterior septum thickness
(diastolic) (B) Posterior wall thickness thickness (diastolic). N≥4/ group.
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Legends to Supporting Tables
Table S1
Microarray analysis of ventricular tissue of FoxO3MyHC versus control mice (10 weeks after
doxycycline removal). Ratio: Ratio in expression between FoxO3MyHC and control tissue,
values >1 denote up-regulation. Cut-off: Regulation 1.8-fold (or 1 / 1.8), p<0.05, adjusted by
Benjamini-Hochberg method.
Table S2
Microarray analysis of FoxO3MyHC versus control cardiomyocytes (36 hours after doxycycline removal in cell culture). Ratio: Ratio in expression between FoxO3MyHC and control
cardiomyocytes, values >1 denote up-regulation. Cut-off: Regulation 1.8-fold (or 1 / 1.8),
p<0.05, adjusted by Benjamini-Hochberg method.
Table S3
List of primers used for quantitative RT-PCR, with the specific probe used for the primer
pairs (UPL, Universal Probe Library, Roche).
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Legend to Supporting Video Files
The video files "Tomogram_Control.mov" and "Tomogram_Transgenic.mov" show
tomograms of a depth of approximately 400 nm through a control and a FoxO3MyHC
cardiomyocyte, reconstructed as described in the Detailed Methods section. The control
movie shows the aspect of normal mitochondria and sarcomere architecture. In the transgenic
cell, several degenerated mitochondria with dilated cristae can be seen (left), as well as a
mitochondrium surrounded by autophagic membranes (right).
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