Supporting InformationNadeau et al. 10.1073/pnas.0914888107SI Materials and MethodsECG.Mice were induced with 5% isoflurane with oxygen and thenmaintained with 2% isoflurane for the length of the acquisition.Lead I and II ECG recording from mice of all three genotypeswere obtained using s.c. electrodes. Ambulatory ECG recordingswere obtained by s.c. implantation of a TA10EA-F20 wirelessradiofrequency telemetry device (Data Sciences International).Mice were allowed a 72-h recovery period. Each mouse was thenplaced in an individual cage overlying a receiver and 48 h ofcontinuous ECG recording was acquired. Surface and ambulatoryrecordings were analyzed using the ECG Auto program (EMKATechnology). The following intervals were analyzed: PP, PQ,QRS, QT, QTc, and RR.

Physiology and Cardiac Function.Mice (n= 10 per group; five male,five female) were anesthetized with droperidol/fentanyl andechocardiography were performed as previously described. Tocompare the diastolic function, five 130-d-old littermates, malesfrom each group, were used to measure the LV pressure by usinga Millar pressure catheter (Millar Instruments). The pressurecatheter was introduced in the right jugular vein of anesthetizedmice and pushed in the LV. Millar recordings were analyzed withthe Chart program (ADInstruments). The τ and LV filling timewere analyzed.

Treadmill Evaluation. A total of 23 adult mice (eTbx5−/−, n = 12;eTbx5+/−, n = 5; eTbx5+/+:, n = 6) were trained for 3 d on thetreadmill. The treadmill was equipped with electrical bars at therear end. These bars are connected to an analogical counter.Mice were running for 10 min at 15, 20, 22.5, and 25 m/min fora total of 40 min. As several eTbx5−/− mice were unable tocomplete the exercise, the number of times the electrical barswere touched at each steps and the time spent on the treadmillfor each mouse was recorded.

Transfections. TC13 cells were maintained as previously described(2) and transfected using lipofection. NIH 3T3 cells were platedin DMEM with 10% fetal bovine serum. Cells were transfectedusing calcium phosphate with varying doses of Tbx5 and/orGATA-4 expression vectors. Luciferase reporter (500 ng) was

used. The Bcl-xL promoter plasmid was described previously.The −1.6-kbp NOS3 promoter was a gift of W. Sessa (YaleUniversity, New Haven, CT) and was previously described (3).Deletion and point mutants were obtained using PCR and wereall confirmed by sequencing. Total DNA was kept constant byaddition of empty backbone vector.

qPCR. qPCR analysis was performed on RNA from cells and hearttissues as described previously (4). Reverse transcription wasdone using Omniscript RT kit (Qiagen). qPCR was then ac-complished on 1:20 diluted cDNA using Qiagen qPCR kit. DNAtemplate and 300 nM oligonucleotides were used at an annealingtemperature of 58 °C using the Quantitect SYBR Green PCR kit(Qiagen) in an MX4000 real-time PCR machine (Stratagene).The primer sequences are available on request.

Immunohistochemistry. Immunohistochemistry was performed asdescribed previously. GATA4 and Tbx5 polyclonal antibodiesused were previously described (5, 6). The anti-NKX2-5 N-19antibody (SC-8697X; Santa Cruz Biotechnology) was used ata dilution of 1:5,000 to detect cardiomyocytes in the tissuesample. Ki67 antibody was purchased from Thermo Scientific.For BrdU detection, BrdU was injected 100 mg/kg i.p. in preg-nant mice 1 h before euthanasia. Tissue sections from litterswere stained with anti-BrdU monoclonal antibody (Develop-mental Studies Hybridoma Bank, University of Iowa). Anti-bodies used were revealed by an anti-rabbit biotinylated antibody(BA-1000; Dimension) or an anti-goat biotinylated antibody(BA-5000; Dimension) diluted at 1:250 and a streptavidin-HRPconjugate diluted at 1:500 (NEL 750; Dimension). TUNEL as-says were carried out using Apoptag kit (S7100; Chemicon) ac-cording to the Apoptag protocol.

Atrial Septum Analysis. Atria were removed from the hearts exceptfor the atrial septum and part of the atrial roof connected to theatrial septum.Then, under themicroscope, thephloxineBdyeflowthrough the atrial shunt in eTbx5+/− and eTbx5−/− mice was cap-tured on camera. The dye is injected in the right atria. InWTmice,the intact atrial septum prevents flow to the left atria, whereasPFOs and type II ASDs in mutant mice let the dye flow through.

1. Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M (2004) Essential role of GATA-4 incell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci USA 101:6975–6980.

2. Nemer G, Nemer M (2002) Cooperative interaction between GATA5 and NF-ATcregulates endothelial-endocardial differentiation of cardiogenic cells. Development129:4045–4055.

3. Zhang R, Min W, Sessa WC (1995) Functional analysis of the human endothelial nitricoxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription inendothelial cells. J Biol Chem 270:15320–15326.

4. Lavallée G, et al. (2006) The Kruppel-like transcription factor KLF13 is a novel regulatorof heart development. EMBO J 25:5201–5213.

5. Nemer G, Nemer M (2003) Transcriptional activation of BMP-4 and regulation ofmammalian organogenesis by GATA-4 and -6. Dev Biol 254:131–148.

6. Georges R, Nemer G, Morin M, Lefebvre C, Nemer M (2008) Distinct expression andfunction of alternatively spliced Tbx5 isoforms in cell growth and differentiation. MolCell Biol 28:4052–4067.

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α-TBX5 N

50 μm E14

RA

LA

EC

AS I

50 μm E9

A BC

V

E

50 μm E12

EC

50 μm E12

LA RA

Ca p

α-G AT A4

50 μm

50 μm

50 μm

50 μm

α-TBX5 C

50 μm

50 μm

50 μm

50 μm

α-NKX2. 5

50 μm

50 μm

50 μm

50 μm

Fig. S1. TBX5 expression in endocardial cells in late embryonic stages. Immunohistochemical staining with homemade polyclonal antibody directed at the N-terminal of TBX5 (α-TBX5 N) and a monoclonal TBX5 antibody directed at the C-terminal (α-TBX5C) on staged mouse embryos. Staining with anti-GATA4 (whichmarks myocardial and endocardial cells) and anti-NKX2.5 (which marks only myocardial cells) are used as control. Note the modest TBX5 staining in endocardialcells (negative for α-NKX2.5) in E9 hearts; TBX5 staining is present in endocardial cells of the atrial septum primum (AS I) and the endocardial cushion (EC) ofthe atrioventricular canal starting on embryonic day 12 and is clearly evident in embryonic day 14 hearts.

B

eTbx5 +/+

eTbx5 +/-

eTbx5 -/-

Genotype n

5

5

5

PR

40.50 ± 0.744

41.12 ± 2.551

42.15 ± 1.717

QRS

13.32 ± 0.487

13.66 ± 0.557

15.3 ± 1.325

QT

49.25 ± 2.121

41.89 ± 0.694

46.44 ± 2.934

QT cf 3

43.19 ± 1.405

37.93 ± 1.302

42.02 ± 1.922

RR

130.1 ± 5.496

119.6 ± 8.425124.1 ± 4.057

A

eTbx5 -/-

P P

0.5

0.0

mV

eTbx5 -/- P

0.0

0.5

mV

eTbx5 +/+

PR

QRS

QT

P

0.5

0.0

mV

50 ms

Fig. S2. ECG recording on aged (460 d) mice: two typical patterns are shown for the eTbx5−/− mice. The middle panel corresponds to mice with arrhythmiasand the lower panel is characteristic of the majority of eTbx5−/− and control WT mice.

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A T ie2 Cre- Tbx5 Wt/Wt

a -Gal

Hoescht

a -Sarcomeric Actin

a -Dig

E14.5

eTbx5 +/+

a -Gal

Hoescht

a -Sarcomeric Actin

a -Dig

eTbx5 +/+

IVSTV

MV

ASI

E14.5

eTbx5 +/+

a -Gala -Sarcomeric Actin

T ie2 Cre- Tbx5 Flox/Flox

IVS

TVMV

ASI

E14.5

eTbx5 -/-

a -Gala -Sarcomeric Actin

eTbx5 -/-

a -Gal

Hoescht

a -Sarcomeric Actin

a -Dig

E14.5

eTbx5 -/-

a -Gal

Hoescht

a -Sarcomeric Actin

a -Dig

B T ie2 Cre- Tbx5 Flox/Flox

E14.5

eTbx5 -/-

eTbx5 -/-

E14.5

eTbx5 -/-

T ie2 Cre-Tbx5 Wt/Wt

eTbx5 +/+

E14.5

eTbx5 +/+

E14.5

eTbx5 +/+

Fig. S3. Excessive apoptosis of the septum primum in eTbx5−/− mice. (A) Immunofluorescence TUNEL assays of eTbx5−/− heart sections in the Rosa26 back-ground reveals excessive apoptosis. Apoptotic cells are in green; nuclei are stained with Hoescht (blue); endocardial cells are positive for α-β-Gal (red) andmyocardial cells are positive for α-sarcomeric actin (yellow). (B) BrdU immunofluorescence staining of consecutive tissue sections.

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Movie S1. Phloxine B dye was injected in the right atrium and prevented to flow to the left atrium by the intact interatrial septum of an eTbx5+/+ mouse(control heart).

Movie S1

Movie S2. Phloxine B dye flow through the atrial shunt caused by a PFO in the interatrial septum of an eTbx5+/− mouse.

Movie S2

Movie S3. Phloxine B dye flow through the atrial shunt caused by the subtotal absence of the VFO and the superior limbus of the septum secundum in theinteratrial septum of an eTbx5−/− heart.

Movie S3

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