Pfister et al.
SUPPLEMENTAL MATERIAL
Flt3 activation improves post-myocardial infarction remodeling involving a
cytoprotective effect on cardiomyocytes
Otmar Pfister1, 2, MD; Vera Lorenz1, MSc; Angelos Oikonomopoulos3, PhD; Lifen Xu1,
PhD; Stéphanie P. Häuselmann1, MSc; Christopher Mbah3, MD, MA; Beat A.
Kaufmann1, 2, MD; Ronglih Liao3, PhD; Aleksandra Wodnar-Filipowicz1, PhD; and
Gabriela M. Kuster1, 2, MD
From the 1Department of Biomedicine, University Hospital Basel and University of Basel, the 2Division of
Cardiology, University Hospital Basel, Switzerland, and the 3Cardiovascular Division, Department of
Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA
Correspondence:
Gabriela M. Kuster, M.D.
Myocardial Research, Department of Biomedicine, and Division of Cardiology
University Hospital Basel
Hebelstrasse 20
4031 Basel, Switzerland
Tel: ++41 61 328 77 36
Fax: ++41 61 265 23 50
Email: Gabriela.Kuster@usb.ch
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Pfister et al.
Supplemental Methods
Cell culture. Hearts were digested in 0.05% Trypsin-EDTA (Gibco) followed by serial digestions
in 0.07%(w/v) collagenase type II (Worthington) and pre-plated twice in T75 culture flasks (Sarstedt) to
remove fibroblasts. Cardiomyocytes were seeded at a density of 1000 cells/mm2 on plastic culture dishes
(BD Falcon) and kept at 37˚C in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with
7%(v/v) heat-inactivated fetal calf serum (FCS), penicillin (P, 100U/mL, Gibco), streptomycin (S,
100µg/mL, Gibco) and 5-bromo-2-deoxyuridine (BrdU, 100μmol/L, Sigma) for 24hrs. Prior to treatments,
the medium was changed to serum-free DMEM for 16hrs.
Cell treatments. Cardiomyocytes were treated with recombinant mouse FL (FL, R&D, 427-FL025/CF) or vehicle (phosphate buffered saline (PBS) with 0.1% (w/v) bovine serum albumin) at various
concentrations and for the times indicated, as well as 30min prior to H2O2 treatment (200µmol/L, Sigma).
The FL concentration producing maximal signal (Akt-phosphorylation, Figure 3E) was chosen for all other
experiments. Akt inhibitor VIII (Akti-1/2, 2µmol/L, Calbiochem) was added 30min prior to FL. Cells were
cultured at 20%O2/5%CO2 except for hypoxia experiments, which were conducted at 1%O 2/5%CO2.
RNA interference. Freshly isolated cardiomyocytes were suspended in 100µL Nucleofector
solution and either transfected with Flt3 siRNA (Qiagen, SI02953433) or non-targeting siRNA (Qiagen,
1027310) (2µg/106 cells) according to Amaxa’s Neonatal Rat Cardiomyocyte Nucleofector kit (VPE-1002).
Cells were transferred to 400µL 7% (v/v) FCS RPMI medium 1640 (Gibco) supplemented with P/S and
incubated for 10min at 37°C. Cells were then plated at a density of 2000 cells/mm2 on laminin (10µg/mL;
Sigma)-pre-coated 6-well plates (BD Falcon) in 1.5mL 7% (v/v) FCS DMEM (P/S and BrdU), kept at 37°C
for 24hrs and changed to fresh FCS or serum-free DMEM.
RT-PCR and quantitative, real-time RT-PCR. Whole hearts were cut into small pieces and
homogenized with a polytron PT-DA1212/2 EC-E homogenizer (Kinematica). RNA was extracted from
whole heart homogenates and cultured cardiomyocytes using TRI Reagent (Sigma) and 2µg RNA were
reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
according to the manufacturer’s instructions. PCR was performed on a Thermocycler (Biometra) and
quantitative real-time PCR on an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems)
using Power SYBR Green PCR Master Mix (Applied Biosystems) and mRNA levels were calculated using
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the comparative CT method with 18SrRNA as endogenous control. Primer sequences (all from Microsynth)
were as follows: for PCR, Flt3 forward 5’-TGGTGACCTGCTCAACTACCTA-3’ and reverse 5’AGTCACAGATCTTCACCACCT-3’, and for quantitative real-time PCR, Flt3 forward 5’AGGTCTCAGGGTTCAATGGGA-3’ and reverse 5’-ACACGACTTGAACTCCAGGAACTC-3’ and
18SrRNA forward 5’-CCATTCGAACGTCTGCCCTAT-3’ and reverse 5’-GTCACCCGTGGTCACCATG-3’.
Flow cytometer analysis of Flt3. Flt3 protein expression was measured in trypsinized, 70% (v/v)
ethanol-fixed cardiomyocytes using a PE-conjugated rat anti-mouse CD135 antibody (BD Bioscience,
553842) and a PE-conjugated rat IgG2a,  as isotype control on a DakoCytomation CyAn ADP
Flowcytometer (excitation 488nm; emission 519nm) and analyzed using FlowJo software.
Western blotting. Western Blotting was performed according to standard technique. In brief,
cultured cardiomyocytes were homogenized in lysis buffer (Cell Signaling) containing 80mmol/L Pefabloc
SC plus (Roche). Protein concentrations were measured using the Micro BCA (bicinchoninic acid) protein
assay kit (Pierce). Equal protein amounts were resolved on SDS-PAGE under reducing conditions and
transferred to Polyvinylidene fluoride (PVDF) membranes (Amersham). The following antibodies were
used: anti-phospho-MEK1/2 (Ser217/221), anti-total MEK1/2, anti-phospho-ERK1/2 (Thr202/Tyr204),
anti-total ERK1/2, anti-phospho-Akt (Ser473), anti-total Akt, anti-Bax, anti-Bcl-2 and anti-caspase-3 (all
Cell Signaling), and anti--tubulin (Sigma). Blots were subsequently incubated with horseradish
peroxidase-conjugated secondary antibodies (Jackson Immuno Research) and band intensities were
detected by enhanced chemiluminescence (Western Lightening Plus; Perkin Elmer) and quantitated using
NIH ImageSXM software (http://rsbweb.nih.gov/ij/docs/guide/146-2.html).
Cytochrome c release. Cardiomyocytes were lysed in digitonin buffer (KCl 75mmol/L, NaH2PO4
1mmol/L, Na2HPO4 8mmol/L, sucrose 250mmol/L and digitonin 190µg/mL, Sigma) to extract the cytosolic
fraction. The lysates were centrifuged at 500g for 5min at 4°C and 80mmol/L Pefabloc SC plus (Roche)
was added. Samples were prepared for Western Blotting as described and a mouse monoclonal anticytochrome c antibody (BD Pharmingen) was used.
Apoptotic nuclei by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling of DNA strand breaks (TUNEL). TUNEL staining was performed on cardiomyocytes plated on
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plastic coverslips and in deparaffinized formalin-fixed tissue slides using the in situ cell death detection kit
fluorescein (Roche) according to the manufacturer’s instruction. A total of five random fields (equalizing
env. 3500 nuclei) were quantified using an Olympus BX61 microscope and a 20x/0.50 Ph1 objective
controlled by Cell^P software, and the amount of apoptosis is given in % TUNEL-positive nuclei.
Mouse surgery. Mice were anesthetized using 65mg/kg pentobarbital and ventilated on a standard
rodent ventilator. An incision was performed between the fourth and the fifth intercostal space. After
opening of the pericardium, the LAD was ligated with a silk suture. A total of 12µl of recombinant mouse
FL (10µg) or vehicle (PBS) were injected at three different sites (4µl per injection) of the infarct border
zone as demarcated immediately after LAD ligation. The dose of FL was chosen based on a single daily
dose of 10µg/mouse for s.c. or i.p. application used in previous studies (1). Sham-operated mice (N=4)
underwent an identical procedure except for LAD occlusion. All mice received buprenorphine (0.03-0.06
mg/kg) s.c. for up to 48 hours after surgery.
Echocardiography. Prior to echocardiography, the chest was shaved and the mice were place on a
heated platform in the supine position. Echocardiography was performed in conscious mice at a
physiological heart rate (Tables S1 and 1). Left parasternal long axis views of the left ventricle were used to
measure left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension
(LVESD) as well as interventricular septal and posterior wall thickness. Left ventricular ejection fraction
(LVEF) was calculated using the cubed method as follows: LVEF = (LVEDD3 –LVESD3)/LVEDD3 x
100. All parameters were measured in three consecutive cardiac cycles and values were averaged. All data
were acquired and analyzed in a blinded fashion.
Heart fixation and histology. Mice were euthanized using pentobarbital and 100IU/ml heparin
was injected intraperitoneally. Hearts were rapidly excised and washed in Krebs buffer (NaCl 137mmol/L,
KCl 4.0 mmol/L, CaCl2 1.8mol/L, KH2PO4 1.2mmol/L, MgSO4 1.2mmol/L, NaHCO3 24.9mmol/L, and
dextrose 11.2mmol/L; pH 7.4). The isolated hearts were mounted on a Langendorff apparatus, paced and
retrogradely perfused via a short cannula inserted into the aortic root. Hearts were arrested in diastole at an
end-diastolic pressure of 5mmHg with KCl and perfused with 10% buffered formalin solution at a
hydrostatic pressure of 40-50mmHg for 15min and kept overnight at 4°C. Excess formalin was then
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removed and the hearts were imbedded in paraffin, cut in three parts (apex, middle, base) and analyzed by
immunohistochemistry.
Infarct size. Infarct size was calculated using histological imaging (Image J,
http://rsb.info.nih.gov/ij/) on a composite obtained from four images acquired on an Olympus BX61
microscope with a 2x/0.08 objective after staining with Masson trichrome (Sigma) as the mean percentage
of epicardial and endocardial circumference occupied by scar tissue averaged for all three ventricular levels
(base, midpapillary and apex).
Immunohistochemistry. Sections of 5µm in thickness were obtained using a Hyrax M55
microtome. Tissue slides were deparaffinized through sequential five minutes incubations in UltraClear
(Biosystems), ethanol (100%, 96%, 70%) and water. Antigen retrieval was performed by Proteinase K
(Fermentas) incubation for 30min at room temperature. After blocking with tris-buffered saline containing
4%(w/v) milk, slides were incubated with primary antibody against -sarcomeric actinin (Sigma) overnight
at 4°C, followed by incubation with TRITC-labeled secondary antibody (Sigma) for 1hr and with Hoechst
33342 (Sigma) for 30min at room temperature. Slides were subsequently washed and mounted with
antifade medium (SlowFade®Antifade Kit, Invitrogen).
Reference
1.
Brasel K, McKenna HJ, Morrissey PJ et al. Hematologic effects of flt3 ligand in vivo in mice.
Blood 1996;88:2004-12.
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Supplemental Table S1. Echocardiographic measurements at baseline
Sham (N=4)
MI-V (N=13)
MI-FL (N=12)
Heart Rate (min-1)
674±9
632±27
652±12
IVSd (mm)
0.88±0.08
0.77±0.03
0.75±0.02
LVPWd (mm)
0.74±0.04
0.75±0.03
0.77±0.03
LVEDD (mm)
2.67±0.08
2.88±0.07
2.91±0.06
IVSs (mm)
1.30±0.12
1.17±0.04
1.19±0.03
LVPWs (mm)
1.00±0.05
0.99±0.04
1.01±0.04
LVESD (mm)
1.57±0.07
1.78±0.06
1.79±0.05
LVEF (%)
79.5±1.6
76.0±1.4
76.6±1.1
Values are given as mean±SEM. IVS: interventricular septum; d: diastole; LVPW: left-ventricular posterior
wall; LVEDD: left-ventricular end-diastolic diameter; s: systole; LVESD: left-ventricular end-systolic
diameter; LVEF: left-ventricular ejection fraction.
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Supplemental Figure S1.
A
fold expression
(ddCt Flt3R/18SrRNA)
1.5
1.0
**
0.5
0.0
siCtr
siFlt3
B
p-AktSer473
60 kD
6
Akt
siCtr
mFL (2µg/ml)
-
siFlt3
-
siCtr siFlt3
+
+
Figure S1. FL-induced signaling in cardiomyocytes is Flt3-dependent. (A) Flt3 mRNA expression was
measured by quantitative real-time RT-PCR in cardiomyocytes transfected with control (siCtr) or Flt3targeting (siFlt3) siRNA (N=3, **p<0.01). (B) Cardiomyocytes were transfected with Flt3-targeting
(siFlt3) or non-targeting (siCtr) siRNA and treated with mouse Flt3 ligand (mFL) (2µg/mL, 15min) to
induce Akt-phosphorylation. Representative blot from N=3 independent experiments.
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