Beruflich Dokumente
Kultur Dokumente
DOI: 10.1161/CIRCGENETICS.110.958116
270
June 2011
atria alters sodium and potassium channel expression, corroborating the electrophysiological findings. Thus, we provide evidence that Pitx2 is an upstream transcriptional regulator of distinct signaling pathways that provide cellular,
molecular, and electrophysiological substrates linked to atrial
arrhythmogenesis.
Statistical Analyses
Methods
Human Tissue and DNA Samples
Atrial myocardial tissue samples were obtained from patients undergoing cardiac surgery. The atrial samples were classified as patients
with (AF) and without (no AF) a recorded history of AF. Detailed
information regarding tissue processing is provided in the onlineonly Data Supplement. The study conforms to the principles outlined
in the Declaration of Helsinki.
Genomic DNA samples from 47 patients diagnosed of having AF
and 100 healthy donors with no cardiac structural and/or functional
diseases were obtained from the Spanish National DNA Bank
(BNADN, Salamanca). Polymerase chain reaction (PCR) amplification of both single nucleotide polymorphisms (SNPs) (rs2200733
and rs13143308) was carried out using flanking oligonucleotides, as
detailed in online-only Data Supplement Table 1, followed by direct
sequencing. This study was approved by the Ethics Committees of
the Spanish National DNA Bank (BNADN, Salamanca) and of the
University of Jaen, and the investigation conforms to the principles
outlined in the Declaration of Helsinki.
Results
rs2200733 and rs13143308 Correlate With AF
We performed a direct resequencing approach to study the
frequency of 2 SNPs previously associated with AF8 in a small
cohort of Caucasian patients with AF. A total 47 patients (25
men and 22 women) with paroxysmal or permanent AF were
recruited for the study (online-only Data Supplement Table 2).
Thirty patients presented isolated AF; 17 patients were also
diagnosed with cardiomyopathy and/or valvulopathy. Ages
ranged from 38 to 90 years (6811 years); 100 patients (44 men
and 56 women) without any cardiac structural or electrophysiological diagnosis were recruited as the control population.
Control ages ranged from 43 to 69 years (526 years) (onlineonly Data Supplement Table 3). rs2200733 (C/T or T/T) was
observed in 20 of 47 (42%) AF patients and 22 of 100 (22%)
control patients (odds ratio [OR], 2.607; 95% confidence interval [CI, 1.158 to 5.908; Fisher exact test; P0.01; Table 1).
rs13143308 (T/T or T/G) was observed in 26 of 47 (55%) AF
patients and was present in 10 of 100 (10%) control patients
(OR, 10.900; 95% CI, 4.325 to 29.594; Fisher exact test;
P0.001; Table 1) (online-only Data Supplement Figure 1).
Thus, the data demonstrate a highly significant prevalence of
rs2200733 (C/T or T/T) and rs13143308 (T/T or T/G) in patients
with AF compared with control subjects. No significant differences were obtained related to age for rs2200733 (C/T or T/T) or
for rs13143308 (T/T or T/G), respectively, using general linear
models. Furthermore, an increased frequency of rs2200733 (C/C
or C/T) but not of rs13143308 is obtained if patients are
subdivided into isolated AF (rs2200733, 16/30; 53%) and AF
patients with valvulopathy and/or cardiomyopathy (rs2200733,
4/17; 23%), although none of them reached statistical significance.
Chinchilla et al
Table 1.
271
Sex
Isolated
With CM/VM
Male
Female
Total
C/C
14/30 (47%)
13/17 (76%)
17/25 (68%)
10/22 (45%)
27/47 (57%)
C/T
14/30 (47%)
3/17 (18%)
9/25 (36%)
8/22 (17%)
17/47 (36%)
T/T
2/30 (6%)
1/17 (6%)
1/25 (4%)
2/22 (9%)
3/47 (5%)
C/C
NA
NA
35/44 (79%)
43/56 (77%)
88/100 (88%)
C/T
NA
NA
9/44 (21%)
13/56 (33%)
22/100 (22%)
T/T
NA
NA
0/44 (0%)
0/56 (0%)
0/100 (0%)
G/G
13/30 (42%)
8/17 (50%)
11/25 (44%)
10/22 (45%)
21/47 (45%)
G/T
13/30 (42%)
6/17 (37%)
10/25 (40%)
9/22 (41%)
19/47 (40%)
T/T
5/30 (16%)
2/17 (13%)
4/25 (16%)
3/22 (13%)
7/47 (15%)
G/G
NA
NA
42/44 (95%)
48/56 (86%)
90/100 (90%)
G/T
NA
NA
2/44 (5%)
8/56 (14%)
10/100 (10%)
T/T
NA
NA
0/44 (0%)
0/56 (0%)
0/100 (0%)
rs2200733
AF patients
Control subjects
rs13143308
AF patients
Control subjects
Isolated AF
Paroxysmal
Permanent
16/27 (59%)
1/3 (33%)
17/27 (62%)
1/3 (33%)
Age
5170 y
7190 y
11/16 (68%)
5/11 (45%)
13/16 (81%)
4/11 (36%)
272
June 2011
Because Pitx2 expression in the developing atria is confined to the left atrial chamber, we explored the expression
profile of several cardiac markers in the left atrial appendages
of NppaCrePitx2/ mutant embryos as compared with
age-matched control mice. In line with previous reports,15
Bmp10 expression was highly upregulated in the left atrial
chambers. In addition, a significant increase on Nkx2.5
expression was observed. On the contrary, Gata6, Mef2c, and
Nppa transcript levels were downregulated, whereas islet-1
and Gata4 displayed no significant differences (Figure 3J).
Chinchilla et al
273
Figure 2. Morphological remodeling of adult atrial-specific Pitx2 conditional mutants. Whole-mount ventral views (A) and isolated left
atria (B and C) corresponding to adult NppaCrePitx2flox/flox (A and B) and NppaCrePitx2/ (A and C) hearts, respectively. Observe
the increased heart size in NppaCrePitx2/ compared with control NppaCrePitx2flox/flox hearts. Left atria (la) size is significantly
enlarged in NppaCrePitx2/ (B) compared with control NppaCrePitx2flox/flox (C) hearts. Dashed lines in C represent the overlay of
the left atria dimensions illustrated in B. Four-chambered views of adult NppaCrePitx2flox/flox (D) and NppaCrePitx2/ (E) hearts are
shown. Note that atrial-specific Pitx2 mutants (E) display enlarged atrial and ventricular chambers and thickening of the interventricular
septum (IVS) (double arrows) compared with control (D) and right ventricular (rv) lumen is significantly dilated (asterisk, E). Transversal
histological sections of adult ventricular NppaCrePitx2floxed/floxed (F) and NppaCrePitx2/ (G) chambers illustrate a significant IVS
thickness (double arrows). Red sirius staining of atrial (H through K) and ventricular (L and M) histological sections of
NppaCrePitx2flox/flox (H, J, and L) and NppaCrePitx2/ (I, K, and M) adult hearts demonstrate increased fibrosis in the ventricular
(arrows, M) but not the atrial chambers in atrial-specific Pitx2 conditional mutants. qRT-PCR analyses of Col1a1 (K) and Col3a1 (L) expression in NppaCrePitx2flox/flox (black bars) and NppaCrePitx2/ (white bars) adult hearts are shown. *P0.05, **P0.01.
274
June 2011
We therefore overexpressed miR-1 in HL-1 atrial cardiomyocytes, which resulted in decreased Gja1 and Kcnj2 transcripts
levels (Figure 5B), in line with previous reports,16 but did not
modify Scn5a and/or Scn1b expression (Figure 5B). To
further investigate if Pitx2 directly regulates miR-1 expression and/or Scn5a expression, we transiently transfected
HL-1 atrial adult cardiomyocytes with Pitx2c. Overexpression of Pitx2c resulted in decreased miR-1 and increased
Scn5a and Scn1b expression (Figure 5C). Furthermore, Pitx2
silencing decreased Scn5a and Scn1b expression in HL-1
cells (Figure 5D).
Chinchilla et al
275
Figure 4. Electrophysiological and molecular remodeling of adult atrial-specific Pitx2 conditional mutants. Shown are representative
ECG recordings (A through D) of adult NppaCre (n5) (A), NppaCrePitx2flox/flox (n10) (C), and NppaCrePitx2/ (B and D) hearts,
respectively. Observe that in control mice (NppaCre and NppaCrePitx2flox/flox), conserved R-R intervals are recording, and in all
cases a p wave can be distinguished (arrows, A and C). In 40% (4/10) of the atrial-specific conditional mutant mice (NppaCrePitx2/), an
AV block ECG pattern can be observed, as delineated by arrowheads in B and B, whereas in the remaining atrial-specific conditional
mutant (6/10), in all but one, a p wave (arrow) was frequently missing (asterisks), as illustrated in D and D, and irregular R-R intervals
were also recorded (D, double arrows). E through I correspond to qRT-PCR expression analyses of Scn5a (E), Scn1b (F), Kcnj2 (J),
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June 2011
Preparation
Left atria (n5)
Pitx2 Conditional
RMP, mV
APA, mV
Vmax, V/s
APD20, ms
APD50, ms
APD90, ms
NppaCrePitx2flox/flox
87.02.7
114.11.8
174.08.7
7.31.4
23.33.5
72.28.9
89.710.0
83.84.2*
109.80.6*
170.014.8
7.30.8
24.32.6
NppaCrePitx2flox/flox
86.42.9
111.71.1
160.08.6
6.81.0
25.62.7
93.07.5
NppaCrePitx2/
85.41.7
111.52.0
164.013.4
9.12.2
31.75.6
102.38.8
Mlc2vCrePitx2flox/flox
85.32.1
116.41.7
190.014.9
8.81.7
37.05.8
145.114.6
86.23.0
114.02.5
186.08.7
14.62.5
40.23.8
124.87.8
NppaCre Pitx2
Right atria (n5)
Ventricular papillary
muscle (n5)
Mlc2vCre Pitx2
APA indicates action potential amplitude; APD, action potential duration; and Vmax, maximum upstroke velocity.
Discussion
AF is the most common cause of arrhythmogenesis in the
human population, yet, the genetic cause of AF remains
elusive.1719 Point mutations in potassium or sodium channel
genes have been associated with familial AF but account for
only a small AF fraction.19 23 Recent genome-wide association studies8 10 have reported several risk variants on chromosome 4q25, adjacent to PITX2 gene, which are associated
with AF. Although these studies do not provide any experimental evidence that links regulation of PITX2 expression/
activity to the risk variants, it has been suggested that PITX2
might be the causative link. In the present study, we demonstrate that 2 SNPs, rs2200733 and rs13143308, are highly
prevalent in a cohort of Spanish patients with AF, supporting
previous findings from other populations. In addition,
rs2200733 is more prevalent in patients with isolated AF as
compared with patients with AF and other cardiac structural
defects, providing a potential role for SNP genotyping as a
stratification tool as recently suggested.24 The mechanisms by
which these SNPs regulate PITX2 function, however, remain
unknown.
At the transcriptional level, we demonstrate for the first
time in this study that PITX2C is significantly decreased in
human patients with sustained AF, thus providing a molecular
link between PITX2 loss of function and AF. We have also
generated chamber-specific conditional Pitx2 mouse mutants,
which display a 60% reduction of Pitx2 expression in the
chamber myocardium, thus providing an experimental model
of Pitx2 insufficiency. Such incomplete Pitx2 deletion might
be attributed to incomplete and/or patchy Cre recombination
in the atrial chamber myocardium.11 Importantly, Cre recombination (NppaCre) is mainly restricted to the atrial appendage myocardium, with some weak and patchy expression in
the AV node (V. Christoffels, personal communication) but
excluding the sinoatrial and pulmonary veins myocardium.11
Lack of Pitx2 expression in the atrial myocardium leads to a
progressive enlargement of the atrial chambers, which is
consistent with the increased proliferation rate in the left
compared with the right atrium already observed from early
developmental stages.25 Furthermore, Bmp10 is highly up-
Figure 4 (Continued). Kcnj12 (H), and Kcnj4 (I) in right atrium (RA), left atrium (LA), and ventricular (V) chambers corresponding to
atrial-specific adult Pitx2 conditional hearts (white bars) as compared with control mice (black bars). Histological sections of the
AV conduction system of adult NppaCrePitx2flox/flox (J and L) and NppaCrePitx2/ (K and N) hearts are stained with picrosirius (J and K) and Mallory trichrome (L and M). Western blot analyses (N) of Nav1.5 and Kir2.1 expression correspond to adult
NppaCrePitx2flox/flox (wt) and NppaCrePitx2/ (Pitx2/) hearts. -Tubulin served as internal loading control. O, Semiquantitative
illustration of Nav1.5 protein expression normalized to -tubulin expression corresponding to control mice as compared with atrialspecific Pitx2 mutants. *P0.05, **P0.01.
Chinchilla et al
277
Figure 5. Pitx2-mediated microRNA molecular pathway. A, qRT-PCR analyses of microRNA miR-1 expression in adult
NppaCrePitx2flox/flox (control) and NppaCrePitx2/ (Pitx2/) hearts. Observe that miR-1 expression is increased approximately
2-fold in atrial-specific mutant mice as compared with control mice. B, qRT-PCR analyses of Scn5a, Scn1b, Gja1, Kcnj2, Kcnj12, and
Kncj4 expression corresponds to miR-1 overexpression in HL-1 atrial cardiomyocytes. Overexpression of miR-1 leads to a significant
decrease in the expression of Gja1, Kcnj2, and Kcnj4, in line with previous reports,14 whereas Scn5a, Scn1b, and Kcnj12 display no
significant differences. C, qRT-PCR analyses of Pitx2c, miR-1, Scn5a, and Scn1b expression in Pitx2c-transfected HL-1 atrial cardiomyocytes. Overexpression of Pitx2c leads to downregulation of miR-1 and enhanced expression of Scn5a and Scn1b. D, qRT-PCR
analyses of Pitx2b, Pitx2c, Scn5a, and Scn1b expression in Pitx2 siRNA-transfected HL-1 atrial cardiomyocytes. Silencing of Pitx2c
leads to downregulation of Scn5a and Scn1b. *P0.05, **P0.01.
278
June 2011
the Complutense University of Madrid to Dr Caballero; a translational CNIC grant (CNIC-13) to Drs Tamargo and Caballero; and the
Ministry of Science and Education (SAF2008-04903) to Dr Delpon.
This work was partially supported by a grant from the University of
Jaen (UJA2009/12/11) to Dr Dominguez.
Disclosures
None.
References
Acknowledgments
We thank Phil Gage (University of Michigan Medical School, Ann
Harbor, MI), Vincent Christoffels (Heart Failure Research Center,
Academic Medical Centre, Amsterdam, The Netherlands), and Kenneth Chien (University of California, San Diego, CA) for reagents,
Antonio Caruz and Francisco J. Esteban for expert counseling and
support on statistical analyses of genotype data, and Robert Kelly for
critical reading of the manuscript. We also thank the Spanish
National Bank of DNA (BNADN, Salamanca) for their valuable
supply of AF and control DNA samples (grant AL-09-0026). We
thank the Department of Surgery, Hospital de Sant Pau, for providing
tissue samples. Technical assistance of Berta Ballester and collaboration of the Cardiac Surgery Team at Hospital Sant Pau in providing
and handling human atrial samples is greatly appreciated.
Sources of Funding
This work was partially supported by the VI European Union
Integrated Project Heart Failure and Cardiac Repair, LSHM-CT2005-018630 to Dr Franco; a grant from the Junta de Andaluca
Regional Council to Dr Franco (CTS-1614); a grant from the Junta
de Andaluca Regional Council to Dr Aranega (CTS-03878); and
grants from the Ministry of Science and Innovation of the Spanish
Government to Dr Franco (MICINN BFU2009-11566) and to Dr
Aranega (MICINN BFU-2008-01217). This work was partially
supported by the Spanish national network REDINSCOR (RD006/
0003/0000) on heart failure, coordinated by Dr Cinca, and translational CNIC grant 2009/08 to Drs Franco, Caballero, and HoveMadsen. This work was partially supported by grants from Ministry
of Health and Consume (PI08/665 and HERACLES RD06/009
network) of the Spanish Government to Dr Tamargo; a grant from
Chinchilla et al
279
CLINICAL PERSPECTIVE
Atrial fibrillation (AF) is the most frequent cardiac arrhythmia, leading to a high risk of mortality and morbidity. Though
its prevalence is high, genetics of AF has remained rather elusive, with sporadic reports on point mutations in a wide variety
of ion channel encoding genes. Recently, genome-wide association studies have unraveled genetic variants (associated
with AF risk) that are located close to the homeobox transcription factor PITX2 in a large proportion of AF patients. In
the present investigation, we corroborated these findings in a small cohort of AF patients. We also provided evidence that
PITX2 is downregulated in AF patients and experimentally demonstrated that Pitx2 insufficiency results in cellular and
molecular changes leading to atrial electrical and cellular remodeling linked to atrial arrythmogenesis. Thus, these findings
provide insights into signaling pathways that are implicated in the pathogenesis of AF.
SUPPLEMENTALMATERIAL
crossed
with
homozygous
mice,
Pitx2floxed
and
NppaCre+Pitx2fl/-,
+
respectively)
-/-
and
Mouse genotyping
DNA for PCR screening was extracted from adult ear and/or tail
samples and from the yolk sac in embryos. Screening of Cre and
Pitx2 floxed alleles was routinely done using used specific primers as
detailed in Supplementary Table 1. Cycling conditions for Cre were as
follows; 5 min at 95C, 35 cycles of 30s at 95C, 30s at 60C and 90s
at 72C, and for Pitx2 as follows; 5 min at 95C. 40 cycles of 30s at
95C, 30s at 60C and 90s at 72C, followed by a final extension step
of 10 min at 72C, respectively. PCR products were separated in
standard agarose electrophoresis and classified according to the
expected band size.
solution
and
photographed.
Samples
processed
for
samples
processed
for
histochemistry
and
conditional
mutants.
Neonatal
(3
weeks)
and
adult
left atrial chambers, the right atrial chambers and the ventricular
chambers, and stored in liquid nitrogen. For MlcvCrePitx2 (Mlc2vCrePitx2flox/flox and Mlc2vCre+Pitx2-/-, respectively), only the ventricular
chambers were dissected and stored in liquid nitrogen.
RNA extraction was performed using six E17.5 pooled left, right
atrial or ventricular samples of embryonic NppaCrePitx2 conditional
mutants, respectively, corresponding on each case to either control
(NppaCre-Pitx2flox/flox) or homozygous (NppaCrePitx2-/-) mutants. For
Mlc2vCrePitx2 three pooled ventricular myocardium samples of
Mlc2vCre+Pitx2-/- and their corresponding controls (Mlc2vCre-Pitx2
flox/flox
qRT-PCR (mRNA)
RT-PCR was performed in Mx3005Tm QPCR System with a MxPro
QPCR Software 3.00 (Stratagene) and SYBR Green detection
system. Reactions were performed in 96-well plates with optical
sealing tape (Cultek) in 20 L total volume containing SYBR Green
Mix (Finnzymes) and the corresponding cDNA. Two internal controls,
mouse actin and GAPDH, were used in parallel for each run.
Amplification conditions were as follows: denaturisation step of 95C
for 10 min, followed by 40 cycles of 95C for 30s, 60C for 30s, 72C
for 30s; with final elongation step of 72C for 10 min. All primers were
designed to span exon-exon boundaries using online Primer3
software Primer3input (primer3 www. cgi v 0.2) as provided in Table
1. No amplifications were observed in PCR control reactions
containing only water as the template. Each PCR reaction was
performed at least three times to obtain representative averages. The
Livak method was used to analyze the relative quantification RT-PCR
data (37) and normalized in all cases taking as 100% the wild-type
(control) value, as previously described (38).
qRT-PCR (microRNA)
miR-1 microRNA qRT-PCR was performed using Exiqon LNA
microRNA qRT-PCR primers and detection kit according to
manufacturers guidelines. All reactions were always run in triplicates
using
5S
as
normalizing
control,
as
recommended
by
the
Q-Max
2005P
qRT-PCR
thermocycler.
Relative
Electrophysiological measurements
Transmembrane action potentials were recorded in isolated left and
right atria of male NppaCre-Pitx2flox/flox and NppaCre+Pitx2-/- mice
(n=5, per group), and in thin papillary muscles from male Mlcv2CrePitx2flox/flox and Mlc2vCre+Pitx2-/- mice through glass microelectrodes
filled with 3 M KCl (tip resistance, 8-15 M) using procedures
described previously (13,14). Multicellular preparations were perfused
with a modified Tyrodes solution of the following composition: NaCl
125, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaHCO3 24, NaH2PO4 0.42 and
glucose 11. The solution was bubbled with 95% O2 and 5% CO2
(pH=7.4)
and
maintained
at
temperature
of
35C.
The
ECG recordings
Mice were anesthetized with 2mg/Kg Ketamine (PARKE-DAVIS, S.L.)
intraperitoneally. Electrocardiogram (ECG) recordings were registered
and analyzed using a digital acquisition and analysis system (Power
Lab/4SP; www.adinstrument.com). Dual Bio Amplifier was connected
to the ECG Lead Switch Box to enable recording of standard lead
configurations. For routine screening, surface ECG (lead II) were
recorded from needle electrodes that were inserted subcutaneously in
the limbs and tape secured. The signal is acquired for about 10
minutes using Chart 4.2.3 software. When recordings were finished,
the limb electrodes are removed and mice were allowed to recover
and returned to their cage. The signal averaged ECG waveform and
rinsed in PBS and incubated in DRAQ-5TM (Red Fluorescent CellPermeable DNA probe, from Biostatus Limited, UK) diluted (1:1000)
in PBS for 10 min. The excess of DRAQ-5TM was removed by a wash
in PBS, briefly rinsed in water, dehydrated and mounted in DPX. The
specificity of the primary antibody was assessed by lack of primary
antibody incubation which resulted in all cases in no detectable signal.
The samples were conserved in total darkness until analysed. Images
were obtained using a Leica Laser Scanning Confocal Microscope
and further edited using Adobe Photoshop software (version 7.0).
Western blotting
Adult hearts from either wild type (NppaCre-Pitx2flox/flox) or homozygous
(NppaCre+Pitx2-/-) mutants, were collected, processed accordingly and
stored in liquid nitrogen. Total protein extraction of was done using single
hearts. These samples were lysated in a small volume of 1 ml RIPA buffer
(50mM Tris pH 8,2, 1mM EDTA, 0,1% p/v Triton X-100, 1mM PMSF,
cocktail protease) using sonication. Protein quantitation was performed
using standard Commassie Protein Assay (Pierce). 10mg of total protein
was loaded in homogeneous 12,5% SDS-PAGE gels. Gels were blotted
onto nitrocellulose and probed against Kir2.1 (ab-80969-500, Abcam) or
Nav1.5 (ASC-005, Alomone) while -tubuline was used as internal loading
control (T-5168, Sigma). Primary antibody incubation was performed at
1:200, 1:100 and 1:14000, respectively. Corresponding secondary antirabbit or anti-mouse antibodies (1:10000 dilution) were used to reveal
Kir2.1, Nav1.5 and -tubuline, respectively. Signal detection was performed
using ECL Plus (GE).
Statistical analyses
qRT-PCR data statistical analyses were performed using unpaired Student
t- test. p values <0.05 were considered statistically significant and are
stated on each corresponding figure legend. Deviation from the Hardy
Weinberg equilibrium was tested by a Fisher's exact test for testing the null
of independence of the group and genotype in a (2 by 2) contingency table
with fixed marginals (alternative hypothesis: true odds ratio is not equal to
References
37. Livak KJ. Schmittgen T. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-Delta Delta C(T))
Method. Methods. 2001;25(4):402-408.
38. Domnguez JN. Navarro F, Franco D, Thompson RP, Arnega
AE.Temporal and spatial expression pattern of beta1 sodium
channel subunit during heart development. Cardiovasc Res. 2005;
65:842-850.
39. Claycomb WC. Lanson NA Jr, Stallworth BS, Egeland DB,
Delcarpio JB, Bahinski A, Izzo NJ Jr. HL-1 cells: a cardiac muscle
cell line that contracts and retains phenotypic characteristics of the
adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998; 95:29792984.
A
250
200
150
100
50
**
**
**
NoAF
AF
1
2
NoAF
AF
3
4
NoAF
AF
5
6
NoAF
AF
7
8
NoAF
AF
9
10
NoAF
AF
11
12
B
**
250
200
150
100
50
**
1
2
NoAF
AF
**
3
4
NoAF
AF
5
6
NoAF
AF
**
7
8
NoAF
AF
9
10
NoAF
AF
11
12
NoAF
AF
rs2200733 (CT)
C/C
C/T
T/T
rs13143308 (GT)
G/G
G/T
T/T
B
NppaCrePitx2
Mlc2vCrePitx2
120
normalized
d units
100
80
60
40
20
0
1
Pitx2b
2
Pitx2c
4
Pitx2b
5
Pitx2c
Supplementary Table 1
Mouse qRT-PCR
Oligonucleotide sequence
Cre Forward
Cre Reverse
Pitx2 Forward
Pitx2 Reverse
Gapdh Forward
Gapdh Reverse
Gusb Forward
Gusb Reverse
Nkx2.5 Forward
Nkx2.5 Reverse
Pitx2b Forward
Pitx2b Reverse
Pitx2c Forward
Pitx2c Reverse
Nppa Forward
Nppa Reverse
Bmp10 Forward
Bmp10 Reverse
Kcnj2 Forward
Kcnj2 Reverse
Kcnj12 Forward
Kcnj12 Reverse
Kcnj4 Forward
Kcnj4 Reverse
Gja1 Forward
Gja1 Reverse
Scn1b Forward
Scn1b Reverse
Scn5a Forward
Scn5a Reverse
Mhl7 Forward
Mhl7 Reverse
Mef2c Forward
Mef2c Reverse
Mlc2a Forward
Mlc2a Reverse
Mlc2v Forward
Mlc2v Reverse
Col1a1 Forward
Col1a1 Reverse
Col3a1 Forward
Col3a1 Reverse
ATCTTCCAGGCGCACCATTGCCCCTGT
TGACGGTGGGAGAATGTTAATCCATATTGG
TCGTGTCTTAAAAGGATGTGTTTCTTC
TTCTGGAGGGTTTTCTTGTTCTAG
TCCTGGTATGACAATGAATACGGC
TCTTGCTCAGTGTCCTTGCTGG
ACGCATCAGAAGCCGATTAT
ACTCTCAGCGGTGACTGGTT
AGGTACCGCTGTTGCTTGAA
CAAGTGCTCTCCTGCTTTCC
GATAACCGGGAATGGAGACC
GTCTTTCTGGGGCAGAGTTG
GCCCACATCCTCATTCTTTC
CCTCACCCTTCTGTCACCAT
TCT CAG AGG TGG GTT GAC CT
CCT GTG TAC AGT GCG GTG TC
TCAAGACGCTGAACTTGTCG
GTTCAGCCATGACGACCTCT
GGTGTCAGCGCAAACAGTTGC
AGAGATGGATGCTTCCGAGA
GACAGAAACAGCATCCACCA
GTGTATGCACCTTGCCATTG
AGACCCTCCTCGGACCTTAC
AGACGTTACACTGGCCGTTC
ACAGCGAAAGACTGTT
TTTGACTTCACCAAGG
TGC TCA TTG TGG TGT TGA CC
CCT GGA CGC CTG TAC AGT TT
GGA GTA CGC CGA CAA GAT GT
ATC TCG GCA AAG CCT AAG GT
TGCTTTATTCCCACCT
AGTCCCAGGTAAGCTG
GGGGTGAGTGCATAAGAGGAG
AGAAGAAACACGGGGACTATGGG
AAGCCATCCTGAGTGCCTTCCG
GGTGTCAGCGCAAACAGTTGC
CCT CTC TGC TTG TGT GGT CA
AAA GAG GCT CCA GGT CCA AT
CACCTGGTCCACAAGGTTTC
ACCATCCAAACCACTGAAGC
AATGGCTCACACAAAG
CACCTGAAGGCGTGTT
Gata4 Forward
Gata4 Reverse
Gata6 Forward
Gata6 Reverse
islet-1 Forward
islet-1 Reverse
siRNA Pitx2 sense
siRNA Pitx2 antisense
GCAGCAGCAGTGAAGAGATG
GCGATGTCTGAGTGACAGGA
CTACACAAGCGACCACCTCA
CCAGAGCACACCAAGAATCC
TCCCATCCCTAAGCAC
ACCAATTGTCCACCAT
GUC CAU ACA AUC UCC GAU AdTdT
UAU CGG AGA UUG UAU GCA CdTdT
Oligonucleotide sequence
rs2200733 Forward
rs2200733 Reverse
rs13143308 Forward
rs13143308 Reverse
ACTAGCAAGCCCTCCAGGTT
GCAAACCACTGCCCTAAGAG
TGGGGGATGGACCAGTATAA
TTGCCAGAAGAGCTTCAGTATG
Human qRT-PCR
Oligonucleotide sequence
PITX2A Forward
PITX2A Reverse
PITX2B Forward
PITX2B Reverse
PITX2C Forward
PITX2C Reverse
GAPDH Forward
GAPDH Reverse
PPIA Forward
PPIA Reverse
ENEP Forward
ENEP Reverse
GGCGTGTGTGCAATTAGAGA
GGTCCACACAGCGATTTCTT
TCGAGTTCACGGACTCTCCT
GAGCTGCTGGCTGGTAAAGT
CTTTCCGTCTCCGGACTTTT
CGCGACGCTCTACTAGTCCT
AGCCACATCGCTCAGACAC
AACCATGTAGTTGAGGTCAATGAA
TCGAGTTGTCCACAGTCAGC
TTCATCTGCACTGCCAAGAC
TTTCTCCTGCTCCAGCTTGT
AGAAACCTTGGCCGAATTG
Supplementary Table 2
Sex
Age
1 Male
2 Male
3 Female
4 Female
5 Female
6 Male
7 Male
8 Female
9 Female
10 Female
11 Female
12 Male
13 Male
14 Male
15 Male
16 Female
17 Male
18 Female
19 Male
20 Female
21 Female
22 Male
23 Male
24 Male
25 Male
26 Male
27 Female
28 Female
29 Male
30 Male
31 Male
32 Male
33 Male
34 Male
35 Female
36 Female
37 Female
38 Female
39 Male
40 Female
41 Male
42 Male
43 Female
44 Female
45 Female
46 Male
47 Male
72
64
58
75
64
81
75
73
70
60
72
46
58
68
74
65
73
78
52
58
81
59
65
78
60
65
78
74
85
77
75
57
76
57
86
59
86
77
38
90
63
43
83
77
78
77
81
Diabetes
NO
NO
NO
NO
NO
NO
NO
YES
NO
YES
NO
NO
NO
YES
NO
YES
NO
YES
NO
NO
NO
YES
NO
NO
NO
NO
YES
NO
NO
NO
YES
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
NO
YES
NO
NO
NO
NO
Systole Dyastole
BP
BP
100
65
160
100
146
80
128
78
147
74
128
95
155
65
162
95
90
58
160
88
130
80
138
96
130
84
135
66
129
80
110
77
136
102
125
72
132
75
123
80
144
93
131
79
130
80
134
74
98
62
152
89
167
97
140
70
131
79
110
85
127
68
98
71
118
69
136
81
181
86
136
78
120
70
123
100
119
82
153
81
137
89
119
88
115
62
118
97
147
86
116
61
139
67
HTA
FA type
Isolated/CM
NO
NO
YES
NO
YES
YES
YES
YES
YES
YES
YES
NO
NO
YES
YES
NO
YES
YES
YES
NO
YES
YES
NO
YES
NO
YES
YES
YES
NO
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
NO
YES
YES
YES
NO
YES
NO
NO
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Permanent
Permanent
Permanent
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Permanent
Permanent
Permanent
Paroxysmal
Permanent
Paroxysmal
Permanent
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Paroxysmal
Permanent
Paroxysmal
Paroxysmal
Permanent
Permanent
CM
Isolated
Isolated
Isolated
Isolated
CM
Isolated
Isolated
Isolated
Isolated
Isolated
Isolated
Isolated
CM
Valvulopathy
Isolated
CM
Isolated
Isolated
Isolated
Isolated
Isolated
Isolated
CM
Isolated
Isolated
CM
CM
CM
CM
Isolated
CM
CM
Isolated
CM
Isolated
Isolated
CM
Isolated
Isolated
Isolated
Isolated
CM
Isolated
Isolated
CM
CM
rs2200733 rs13143308
T/T
C/T
C/C
C/T
C/T
C/C
C/C
T/T
C/C
C/T
C/C
C/C
C/T
C/C
C/T
C/C
C/C
C/C
C/C
C/T
C/T
C/T
C/T
C/C
C/C
C/T
C/C
C/T
C/C
C/C
C/T
C/C
C/C
C/T
C/C
C/T
C/C
C/C
C/C
T/T
C/T
C/C
C/T
C/C
C/C
C/C
C/C
G/G
T/G
T/T
T/G
T/G
T/G
T/T
G/G
G/T
G/T
G/G
G/G
G/T
G/G
G/T
T/T
G/T
G/G
G/T
G/G
G/T
G/T
G/T
G/G
G/G
G/T
G/G
G/T
G/G
G/G
G/G
T/T
G/G
T/T
G/T
G/G
G/G
T/T
T/T
G/G
G/T
G/G
G/G
G/T
G/G
G/T
G/G
Supplementary Table 3
Sex
1 Male
2 Male
3 Male
4 Female
5 Male
6 Female
7 Male
8 Female
9 Male
10 Male
11 Male
12 Female
13 Female
14 Female
15 Male
16 Female
17 Male
18 Female
19 Female
20 Female
21 Female
22 Female
23 Male
24 Female
25 Female
26 Male
27 Female
28 Female
29 Female
30 Female
31 Female
32 Female
33 Female
34 Female
35 Male
36 Male
37 Female
38 Male
39 Male
40 Female
41 Female
42 Female
43 Female
44 Male
45 Female
46 Male
47 Female
48 Male
49 Female
50 Female
51 Female
52 Female
53 Female
54 Male
55 Male
56 Female
57 Female
58 Male
59 Female
60 Female
Age
61
53
59
49
53
55
59
69
57
53
45
61
49
47
63
55
64
56
43
43
56
46
50
51
49
65
50
57
43
45
45
56
52
47
65
45
57
45
56
57
62
57
44
60
52
66
45
52
56
50
44
59
51
57
51
45
49
53
54
44
rs2200733 rs13143308
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/T
C/C
G/G
C/C
G/G
C/T
G/G
C/T
G/T
C/C
G/T
C/C
G/G
C/T
G/T
C/C
G/T
C/C
G/T
C/C
G/T
C/C
G/T
C/T
G/T
C/T
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/T
G/T
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/T
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/T
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/T
G/G
C/T
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/T
G/G
Sex
61 Male
62 Female
63 Male
64 Male
65 Male
66 Female
67 Male
68 Female
69 Male
70 Male
71 Female
72 Male
73 Male
74 Female
75 Male
76 Female
77 Female
78 Male
79 Male
80 Female
81 Female
82 Female
83 Male
84 Male
85 Male
86 Female
87 Female
88 Male
89 Female
90 Female
91 Male
92 Male
93 Male
94 Female
95 Male
96 Female
97 Male
98 Male
99 Male
100 Female
Age
52
53
51
54
52
56
54
44
58
53
52
48
58
44
51
54
55
53
63
51
48
43
56
52
52
45
54
58
62
45
58
51
64
58
49
44
53
64
55
53
rs2200733 rs13143308
C/T
G/G
C/C
G/G
C/C
G/G
C/T
G/G
C/T
G/G
C/T
G/G
C/C
G/G
C/T
G/G
C/C
G/G
C/T
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
C/C
G/G
Supplementary Table 4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Sex
Male
Male
Female
Female
Female
Male
Female
Male
Male
Male
Male
Male
Female
Female
Male
Male
Female
Female
Age
51
58
79
78
61
75
74
75
15
67
18
54
74
67
71
58
58
65
Diabetes
NO
NO
YES
NO
NO
NO
YES
YES
NO
NO
NO
NO
NO
NO
NO
YES
NO
NO
Hypertension
YES
YES
YES
NO
NO
YES
YES
NO
NO
NO
NO
NO
NO
YES
YES
NO
NO
NO
FA/ No AF
sinus rythmn
sinus rythmn
sinus rythmn
sinus rythmn
permanent AF
permanent AF
permanent AF
permanent AF
sinus rythmn
sinus rythmn
sinus rythmn
sinus rythmn
sinus rythmn
permanent AF
permanent AF
paroxysmal AF
permanent AF
permanent AF
Surgery
Biopsies
Valve replacement
LA
Heart transplatation
LA
Valve replacement
LA
Valve replacement
LA
Valve replacement
LA
Valve replacement
LA
Bypass
LA
Valve replacement
LA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Valve replacement
RA
Supplementary
Table 5
NoAF #1 RA
AF #1 RA
delta CT (PITX2C/PPIA)
right atrium
mean
SD
10,84
1,27
14,72
2,43
NoAF #2 RA
AF #2 RA
8,74
14,26
0,54
0,89
NoAF #3 RA
AF #3 RA
6,4
13,35
1,61
1,48
NoAF #4 RA
AF #4 RA
10,83
15,52
1,49
1,49
NoAF #5 RA
AF #45 RA
20,45
12,75
1,12
0,65
NoAF #1 LA
AF #1 LA
left atrium
mean
6,73
9,25
SD
0,81
0,44
NoAF #2 LA
AF #2 LA
8,51
16,35
1,13
0,49
NoAF #3 LA
AF #3 LA
7,28
20,83
0,27
0,01
NoAF #4 LA
AF #4 LA
17,58
7,92
0,92
0,86
Supplementary Table 1. Oligonucleotide sequences used for qRTPCR expression analyses, SNPs (rs2200733 and rs13143308)
genotyping and Pitx2 siRNA silencing.
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