Pitx2c increases in atrial myocytes from chronic atrial fibrillation

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Cardiovascular Research (2016) 109, 431–441
doi:10.1093/cvr/cvv280
Pitx2c increases in atrial myocytes from chronic
atrial fibrillation patients enhancing IKs and
decreasing ICa,L
Marta Pérez-Hernández 1,2†, Marcos Matamoros 1,2†, Adriana Barana 1,2, Irene Amorós 1,2,
Ricardo Gómez1,2, Mercedes Núñez1,2, Sandra Sacristán1,2, Ángel Pinto 2,3,
Francisco Fernández-Avilés 2,3, Juan Tamargo 1,2, Eva Delpón1,2*, and Ricardo Caballero1,2
1
Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid 28040, Spain; 2Instituto de Investigación Sanitaria Gregorio Marañón, School of Medicine,
Universidad Complutense, Madrid 28040, Spain; and 3Cardiology and Cardiovascular Surgery Services, Hospital General Universitario Gregorio Marañón, Madrid 28007, Spain
Received 31 July 2015; revised 23 November 2015; accepted 15 December 2015; online publish-ahead-of-print 28 December 2015
Time for primary review: 35 days
Aims
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Pitx2c † Chronic atrial fibrillation † Human atrial myocytes † IKs † ICa,L
1. Introduction
Atrial fibrillation (AF) is the most frequent arrhythmia and the main risk
factor associated with myocardial-related cerebrovascular events.1
Nowadays, treatment of AF is clearly suboptimal,2 mainly due to rapid
changes in the electrical properties of the atria (electrical remodelling)
induced by the arrhythmia itself.1 This electrical remodelling promotes
the maintenance and recurrence of AF3 and is characterized by a
marked shortening of the atrial action potential duration (APD) and
refractoriness as a consequence of alterations in the expression and
function of L-type Ca2+ and inward rectifier and voltage-dependent
K+ channels.4 – 6 In chronic AF (CAF) patients, the L-type Ca2+ current
(ICa,L) decrease4 and the slow delayed rectifier K+ current (IKs) increase5
critically contribute to the APD shortening.7
Pitx2 is a transcription factor that plays a critical role in directing
cardiac asymmetric morphogenesis.8 In humans, the PITX2 gene generates three main isoforms (Pitx2a, Pitx2b, and Pitx2c), the latter being
the predominant isoform in the heart.8 Increasing evidence pointed
to a role of Pitx2c in the pathophysiology of AF. Indeed, it has been described that single nucleotide variants on human chromosome 4q25,
≈170 kb distal to the PITX2 gene, are associated with an increased
risk of familial AF.9 Pitx2c expression can be increased10 or decreased11
in atrial appendage samples obtained from AF patients, compared with
those from patients in sinus rhythm (SR). However, the putative role of
* Corresponding author. Tel: +34 913941474; fax: +34 913941470, Email: [email protected]
†
The first two authors contributed equally.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected]
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Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that
promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K+ current
(IKs) and down-regulation of the voltage-gated Ca2+ current (ICa,L) are hallmarks of electrical remodelling and critically
contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that
Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis,
could play a role in atrial remodelling. However, its effects on IKs and ICa,L are unknown.
.....................................................................................................................................................................................
Methods
Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher
and results
in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was
positively and negatively correlated with IKs and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased
IKs density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of
KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide.
.....................................................................................................................................................................................
Conclusion
Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and
suggested that this transcription factor could contribute to the CAF-induced IKs increase and ICa,L reduction observed in
humans.
432
Pitx2c in electrical remodelling in humans has not been explored as
data available thus far have been obtained in mouse models.11 – 15
Therefore, this study was undertaken to measure Pitx2c expression
in human atrial myocytes from SR and CAF patients and to determine
the effects of Pitx2c on IKs and ICa,L. Importantly, the results obtained
demonstrated that Pitx2c expression is augmented in atrial myocytes
from CAF patients. Furthermore, Pitx2c increases IKs by enhancing expression of Ks channel encoding genes, whereas it decreases ICa,L by a
post-translational effect.
2. Methods
This study was approved by the Investigation Committee of the Hospital
Universitario Gregorio Marañón (CNIC-13) and conforms to the principles
outlined in the Declaration of Helsinki. Each patient gave written informed
consent. Clinical data of the patients are included in Supplementary material online, Table S1.
2.1 Analysis of the mRNA expression in human
atrial myocytes
2.2 Patch clamping
Outward K+ currents and ICa,L were recorded in human atrial myocytes
and HL-1 cells at room temperature using the whole-cell patch-clamp technique (micropipette resistance ,3.5 MV).5,7,16 Human atrial myocytes
were isolated from right atrial appendages obtained from SR (n ¼ 10)
and CAF (n ¼ 10) patients as described.5,7,16,17 HL-1 cells were cultured
as described previously16,18 and transfected with CMV-Pitx2c (2 mg) by
using Lipofectamine 2000. In HL-1 cells, ICa,L was recorded by using Ba2+
as charge carrier to increase current density and to eliminate Ca2+dependent inactivation.16 Dofetilide (1 mM) and HMR-1556 (1 mM) (Sigma,
UK and Thermo Fisher, USA) were used to inhibit the rapid delayed rectifier current (IKr) and IKs,19 respectively. Series resistance was compensated
manually and usually ≥80% compensation was achieved. Thus, no significant voltage errors (,5 mV) due to series resistance were expected
with the micropipettes used.
2.3 Luciferase gene expression reporter assays
Luciferase activity assays were conducted in CHO cells transfected with the
corresponding promoters20 cotransfected with an empty vector or with
Pitx2c (100 ng). Luciferase activity was measured 48 h after transfection
and normalized to sample protein concentration. All reporter assays
were performed in triplicate.
2.4 Western blot analysis
Western blot analysis17 was conducted to measure Kv7.1, minK, and
Cav1.2 protein expressions in HL-1 cells transfected or not with Pitx2c
by using anti-Kv7.1 (1:250; Sigma, USA), anti-minK (1:250; Abcam, UK),
and anti-Cav1.2 (1:1000; clone L57/46; Neuromab, USA) antibodies.
2.5 Statistical analysis
Results are expressed as mean + SEM. Unpaired t-test or one-way analysis
of variance (ANOVA) followed by Newman – Keuls test was used where
appropriate. In small-sized samples (n , 15), statistical significance
was confirmed by using non-parametric tests. Comparisons between categorical variables were performed using Z-test. To take into account
repeated sample assessments, data were analysed with multilevel
mixed-effects models. A value of P , 0.05 was considered significant.
Additional details are presented in Supplementary material.
3. Results
3.1 Pitx2c expression increases in atrial
myocytes from CAF patients
The expression of Pitx2c was measured by qPCR in myocytes enzymatically isolated from atrial appendages obtained from SR (n ¼ 10) and
CAF (n ¼ 10) patients. Comparison of DCt values demonstrated that
Pitx2c expression was significantly larger in CAF myocytes (Figure 1A).
Indeed, transformation of DCt values to fold differences demonstrated
that Pitx2c expression was approximately two times larger in CAF than
in SR myocytes (Figure 1B).
3.2 Pitx2c expression levels correlate
with IKs and ICa,L densities in SR and
CAF myocytes
An increase in IKs and a decrease in ICa,L densities are hallmarks of
CAF-induced electrical remodelling.4,5,7,16 Therefore, we analysed a
putative correlation between Pitx2c levels and IKs and ICa,L densities.
In these experiments, myocytes enzymatically isolated from each atrial
appendage were separated in two fractions: one for Pitx2c expression
assay and another for ICa,L or IKs recordings. Interestingly, association
studies demonstrated significant correlations of Pitx2c expression with IKs
density at +60 mV (Figure 1C) and ICa,L density at +10 mV (Figure 1D),
showing that the larger the Pitx2c expression the higher IKs and the lower
ICa,L densities. To test whether these correlations were driven by differences between SR and CAF groups, a partial correlation was calculated.
Pitx2c expression and IKs or ICa,L densities are correlated in the absence of
the influence of the group effect (Figure 1E and F). Moreover, a positive
correlation was observed between Pitx2c mRNA levels and the expression of KCNQ1 and KCNE1 mRNA. However, Pitx2c mRNA expression was not correlated with CACNA1C mRNA levels (Supplementary
material online, Figure S1).
The existence of such correlations suggested that Pitx2c may modulate
the expression of the channels that generate these currents. To test this
hypothesis, we first analysed the properties of IKs and ICa,L recorded in SR
and CAF myocytes. As demonstrated previously,5,7 cell capacitance of
CAF myocytes was greater than that of SR myocytes (96.9 + 6.2 vs.
56.7 + 2.2 pF, P , 0.0001; n ¼ 194). In the presence of 4-aminopyridine
(2 mM), used to inhibit the transient outward (Ito1) and the ultrarapid
component of the delayed rectifier (IKur) K+ currents, a current whose
time- and voltage-dependent properties are consistent with those of IKs
was recorded.5,7 Figure 2A and B shows current traces and current density–voltage curves for IKs recorded in SR and CAF myocytes. IKs was measured as the difference between the amplitudes at the end and beginning
of 4 s pulses and normalized by cell capacitance to obtain current density.
Confirming previous results,5,7 in myocytes obtained from CAF patients,
IKs density increased approximately two-fold compared with myocytes
from SR patients (2.3 + 0.2 vs. 1.2 + 0.2 pA/pF at +60 mV, P , 0.01;
n ¼ 40 and 86, respectively) and accordingly, KCNQ1 and KCNE1
mRNA expressions were also increased (Supplementary material online,
Figure S1). Moreover, the time course of current activation was significantly faster, and the conductance curve was shifted to more hyperpolarized
potentials in CAF myocytes relative to SR myocytes (Table 1). ICa,L was
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Real-time quantitative polymerase chain reaction (qPCR) was performed
using TaqMan gene expression assays (Life Technologies, USA). The cycle
to threshold (Ct) values were normalized to 18S rRNA. To compare CAF
vs. SR expression differences, the respective data were transformed from
DCt values to equivalent fold differences using the following equation: Fold
Difference (mean DCtSR – mean DCtCAF) ¼ 2(mean DCtSR – mean DCtCAF).7,16
M. Pérez-Hernández et al.
Pitx2c and human atrial currents
433
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Figure 1 Pitx2c increases in human atrial myocytes from CAF patients. (A) DCt values of Pitx2c mRNA measured by qPCR in isolated right atrial
myocytes obtained from SR (n ¼ 10) and CAF (n ¼ 10) patients. Each bar represents mean + SEM. (B) Relative expression levels of Pitx2c in SR and
CAF samples. Correlation between Pitx2c expression (represented as 1/DCt) and IKs density at +60 mV (C) and ICa,L density at +10 mV (D) recorded in
right atrial myocytes from SR (black circles) and CAF (white circles) patients. In (C ) and (D), each point corresponds to the mean values of 1/DCt and
current density obtained for each patient. (E and F) Partial correlation calculated to suppress the influence of the group effect. The corresponding residuals (labelled with ‘) were calculated after regressing IKs or ICa,L density (Y ) and Pitx2c expression (X1) to variable X2 (to be in the SR or CAF group).
Solid lines represent the linear regression to the data, and dashed lines represent the SEM of the fit. In (A) and (B), *P , 0.05 vs. SR. Unpaired t-test.
measured as the difference between the peak amplitude and the amplitude at the end of the pulses and normalized by cell capacitance to obtain
the current density. As expected,4,7,16 CACNA1C mRNA expression
(Supplementary material online, Figure S1) and ICa,L density in CAF myocytes (22.0 + 0.3 vs. 23.8 + 0.3 pA/pF at +10 mV, P , 0.01; n ¼ 27
and 41, respectively) (Figure 2C and D) were significantly smaller than in
SR myocytes. The midpoint of the ICa,L inactivation curve in CAF was significantly shifted to more positive potentials compared with SR myocytes
(Table 1). However, no differences were observed in the activation
(tact ¼ 1.2 + 0.1 vs. 1.1 + 0.1 ms at +10 mV) and inactivation kinetics
and voltage dependence of activation (Table 1).
3.3 Pitx2c increases IKs density in HL-1 cells
The results suggested that Pitx2c could be involved in the CAF-induced
IKs increase and ICa,L decrease. Next, we analysed the molecular mechanisms underlying the putative Pitx2c-induced IKs increase and ICa,L
decrease in the mouse atrial-derived HL-1 cardiomyocyte-like cell
line. Cells non-transfected with Pitx2c randomly patched (n ¼ 32)
could be separated into three groups, depending on the main outward
K+ current recorded by applying 4 s pulses from 280 mV to potentials
ranging from 280 to +40 mV. About 36% of the cells (Figure 3A) exhibited an outward current with time- and voltage-dependent properties concordant with those of IKr and sensitive to dofetilide (1 mM)
434
M. Pérez-Hernández et al.
Table 1 Electrophysiological properties of IKs and ICa,L recorded in isolated myocytes from atrial appendages obtained from
SR and CAF patients
IKs
...............................................
ICa,L
.........................................................................................................................
tact (s)
Vhact (mV)
kact
Erev (mV)
Vhact (mV)
kact
tinactf (ms)
tinacts (ms)
Vhinact (mV)
kinact
1.8 + 0.1
1.3 + 0.1*
34.8 + 1.8
13.3 + 2.5*
10.7 + 0.8
12.8 + 0.9
56.1 + 1.5
54.8 + 2.1
24.6 + 0.6
24.0 + 0.9
6.4 + 0.1
6.6 + 0.2
21.3 + 6.5
21.7 + 2.3
188.7 + 19.8
162.6 + 15.4
222.2 + 1.6
217.1 + 1.0*
7.2 + 0.5
6.1 + 0.4
.......................................................................................................................................................................................
SR
CAF
Each value represents mean + SEM of more than 27 experiments conducted in isolated myocytes obtained from samples from 10 SR patients and 10 CAF patients.
CAF ¼ chronic atrial fibrillation; Erev ¼ reversal potential; SR ¼ sinus rhythm; tact ¼ time constant of IKs activation measured at +60 mV; tinactf and tinacts ¼ fast and slow time
constants of ICa,L inactivation measured at +10 mV; Vhact and kact ¼ midpoint and slope values of the conductance-voltage curves; Vhinact and kinact ¼ midpoint and slope values of the
ICa,L inactivation curves.
*P , 0.05 vs. SR.
(IKr-predominant) (Supplementary material online, Figure S2). IKr was
completely absent in ≈23% of the cells, in which a slow-activating noninactivating dofetilide-resistant current with biophysical properties
similar to those of IKs was recorded (IKs-predominant). In the rest of
the cells (≈41%), both dofetilide-sensitive and -resistant currents
could be recorded (IKr + IKs). Transfection of Pitx2c did not modify
cell capacitance (50.6 + 8.7 pF, P . 0.05) but significantly changed celltype distribution (n ¼ 41). Pitx2c decreased the percentage of the cells
that exhibited the IKr-predominant pattern, whereas it increased the
percentage of the cells with the IKs-predominant pattern (P , 0.05)
(Figure 3B). IKr was recorded in IKr-predominant and IKr + IKs cells.
The results demonstrated that Pitx2c transfection did not significantly
modify IKr density, the voltage dependence of activation, or the
time dependence of current activation or deactivation (Table 2 and
Supplementary material online, Figure S2). In cells with IKs-predominant
and IKr + IKs patterns perfused with dofetilide, IKs was recorded by
applying 4 s pulses from 280 mV to potentials ranging between 280
and +60 mV, followed by repolarizing pulses to 230 mV to record the
tail currents (Figure 3C and D). Under these conditions, the timedependent current was completely inhibited by HMR-1556 (1 mM)
(inset of Figure 3D). Importantly, Pitx2c significantly increased
the current density (from 2.6 + 0.4 to 6.1 + 1.1 pA/pF at +60 mV;
P , 0.05) (Figure 3D and E) and accelerated the time course of activation (Table 2). It also significantly increased the tail current density
(Figure 3F) and shifted the activation curves to more hyperpolarized
potentials (Table 2). Densitometric analysis from western blots conducted in HL-1 cells (Figure 4A) demonstrated that Pitx2c increased
by approximately two-fold Kv7.1 and minK protein expression
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Figure 2 Pitx2c increases IKs and decreases ICa,L in human atrial myocytes. Current traces recorded by applying the protocol shown at the top (A) and
current density – voltage curves (B) for IKs recorded in SR and CAF myocytes. Current traces recorded by applying the protocol shown at the top (C) and
current density– voltage curves (D) for ICa,L recorded in SR and CAF myocytes. Each point represents mean + SEM of more than 27 experiments conducted in isolated myocytes obtained from samples from 10 SR patients and 10 CAF patients. *P , 0.05 vs. SR. ANOVA followed by Newman–Keuls test
and multilevel mixed-effects model (B and D).
435
Pitx2c and human atrial currents
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Figure 3 Pitx2c increases IKs in HL-1 cells. Percentage of HL-1 cells with IKr-predominant, IKs-predominant, and IKr + IKs patterns in non-transfected
cells (A) or in cells transfected with Pitx2c (B). Outward K+ current traces recorded in two IKs-predominant cells transfected (D) or not (C) with Pitx2c.
The inset shows an outward K+ current recorded at +60 mV and tail current recorded at 230 mV in the absence and presence of HMR-1556 (1 mM).
Current density– voltage (E) and tail current density– voltage (F ) relationships for dofetilide-resistant current (Dofe-insensitive) recorded in cells transfected or not with Pitx2c. In (F), continuous lines represent the fit of a Boltzmann function to the data. In (E) and (F ), each point represents mean + SEM
of more than 15 cells from at least 4 independent batches in each group. In (B), (E), and (F ), *P , 0.05 vs. cells non-transfected with Pitx2c. Z test (B) and
ANOVA followed by Newman – Keuls test and multilevel mixed-effects model (E and F ).
Table 2 Electrophysiological properties of IKr and IKs recorded in HL-1 cells in the absence and presence of Pitx2c
IKr
...............................................................................
tact (ms)
tdeactf (ms)
tdeacts (ms)
Vhact (mV)
IKs
....................................................................................
kact
tact (ms)
2800 + 824
tdeactf (ms)
tdeacts (ms)
Vhact (mV)
kact
.......................................................................................................................................................................................
Pitx2c (2)
866 + 197
140 + 25.2
905 + 206
27.7 + 2.0
8.0 + 0.6
Pitx2c (+)
780 + 216
211 + 54.5
1037 + 197
28.6 + 3.2
9.3 + 2.2
856 + 164*
341 + 74
2582 + 595
36.5 + 4.8
12.0 + 2.5
117 + 23*
1522 + 359*
25.6 + 2.6*
13.1 + 2.0
Each value represents mean + SEM of more than 15 cells from at least 4 independent batches in each group.
tact, time constants of IKr and IKs activation measured at 0 and +60 mV, respectively; tdeactf and tdeacts, fast and slow time constants of IKr and IKs deactivation measured in the tail currents
recorded at 260 and 230 mV, respectively, after pulses to +60 mV; Vhact and kact, midpoint and slope values of the activation curves for IKr and IKs.
*P , 0.05 vs. cells non-transfected with Pitx2c.
436
M. Pérez-Hernández et al.
(Figure 4B and C ). These results suggested that Pitx2c regulates the expression of the ion channels responsible for IKs.
It has been previously demonstrated that Pitx2c binds the DNA
sequence TAATCC by means of a lysine residue located in position
9 of helix 3 in its homeodomain.21 Therefore, the consensus sequence
TAATCC was searched near the transcription start site (TSS) of
KCNQ1 and KCNE1 genes. In KCNQ1, a single consensus sequence
was found at a distance of 2 kb upstream of the TSS (Figure 5A). In
KCNE1, two consensus sequences were found at 1.4 and 1.2 kb
upstream of the TSS (Figure 5C). To determine whether Pitx2c may increase KCNQ1 and KCNE1 transcription, luciferase activity assays were
conducted in CHO cells. A construction carrying the KCNQ1 proximal
promoter and including the Pitx2c-binding site (22186/0) was cloned
in the pLightSwitch_Prom luciferase expression reporter vector
(KCNQ1_WT) and transiently transfected. The same construction
but with the Pitx2c-binding site mutated to AAAAAA (KCNQ1_mut)
was also tested (Figure 5A). Cotransfection of Pitx2c significantly increased luciferase activity in cells transfected with KCNQ1_WT
(Figure 5B), indicating that Pitx2c increased KCNQ1 transcription. However, Pitx2c did not increase luciferase activity in cells transfected with
KCNQ1_mut, revealing that the presence of the consensus sequence
TAATCC was critical for the Pitx2c effect on KCNQ1 transcription
(Figure 5B). To determine the putative interaction of Pitx2c with
KCNE1, a construction carrying the KCNE1 proximal promoter and including both Pitx2c-binding sites (21429/+16) was cloned in the
PGL3-Basic luciferase expression reporter vector (F2)20 and transfected in CHO cells. A shorter construction (F7) without the Pitx2cbinding sites (2311/+16) was also tested (Figure 5C). Pitx2c produced
a 1.8-fold increase in luciferase activity in cells transfected with F2, demonstrating that Pitx2c was able to increase KCNE1 transcription.
Conversely, it did not increase luciferase activity in cells transfected
with F7, indicating that the presence of the TAATCC sequence is critical for the Pitx2c effects on KCNE1 transcription. It is known that atrial
natriuretic peptide (ANP) encoded by the NPPA gene is one of the main
targets of Pitx2c and is frequently used to evaluate transcriptional activity of Pitx2c as the gold standard.21 Pitx2c increased NPPA transcription by interacting with several Pitx2c-binding sites within its promoter
region.22,23 For this reason, CHO cells expressing the pLightSwitch_
Prom luciferase expression reporter vector carrying NPPA minimal
promoter were used as a positive control. As expected, in both groups
of experiments, transfection of Pitx2c significantly increased luciferase
activity generated by the NPPA promoter (Figure 5B and D).
3.4 Pitx2c decreases ICa,L density in HL-1
cells
In HL-1 cells, both ICa,L and ICa,T can be recorded.16 To determine the
effects of Pitx2c on ICa,L, currents were recorded using Ba2+ as the
charge carrier (IBa) and the holding potential was fixed to 230 mV
to fully inactivate ICa,T. Under these conditions, 80% of the cells exhibited a measurable IBa that reached its maximum value at +20 mV
when 500 ms pulses to potentials ranging between 240 and
+50 mV were applied (n ¼ 49) (Figure 6A). Transfection of Pitx2c
decreased the proportion of cells with measurable current to 57%
(P , 0.01, n ¼ 53), without modifying cell capacitance (40.7 + 8.3 pF;
P . 0.05). Pitx2c markedly decreased IBa density at potentials between
0 and +50 mV (n ¼ 23, P , 0.05) (Figure 6A and B). Moreover, it
shifted the midpoint of the inactivation curve to more positive potentials (Figure 6C) and slowed recovery from inactivation (Figure 6D and
Table 3). Conversely, it did not modify the voltage dependence of
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Figure 4 Pitx2c increases Kv7.1 and minK expressions in HL-1 cells. (A) Western blot images and their corresponding stain-free gels showing Kv7.1
and minK expression in HL-1 cells transfected (right lanes) or not (left lanes) with Pitx2c. Mean densitometric analysis of Kv7.1 (B) and minK (C) levels
normalized to total protein in cells transfected or not with Pitx2c. Results are presented as mean + SEM of four independent batches of cells for each
group. *P , 0.05 vs. non-transfected cells. Unpaired t-test (B and C).
Pitx2c and human atrial currents
437
Figure 5 Pitx2c increased KCNQ1 and KCNE1 transcription. (A) Schematic diagram of the human KCNQ1 region proximal to the TSS (22186/0).
Figure 6 Pitx2c decreases IBa in HL-1 cells. Current traces recorded by applying the protocol shown at the top (A) and current density – voltage relationships (B) for IBa recorded in HL-1 cells transfected or not with Pitx2c. (C) Normalized steady-state IBa activation and inactivation curves. In (C ), solid
lines represent the fit of a Boltzmann function to the data. (D) Recovery from inactivation data for IBa recorded by applying the protocol shown in the
inset in HL-1 cells transfected or not with Pitx2c. The solid lines represent the fit of a monoexponential function to the data. Each point represents
mean + SEM of more than 15 cells from at least 5 independent batches in each group. In (B), *P , 0.05 vs. non-transfected cells. ANOVA followed
by Newman – Keuls test and multilevel mixed-effects model (B – D).
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(B) Normalized luciferase activity in CHO cells expressing the pLightSwitch_Prom luciferase expression reporter vector carrying KCNQ1_WT,
KCNQ1_mut, or the NPPA minimal promoter cotransfected or not with Pitx2c. (C) Schematic diagram of the human KCNE1 region proximal to the
TSS. In (A) and (C ), the TAATCC sequences are represented by black boxes. (D) Normalized luciferase activity in CHO cells expressing the PGL3-Basic
luciferase expression reporter vector carrying F2 or F7 cotransfected or not with Pitx2c. In (B) and (D), each bar represents the mean + SEM of five
independent batches of cells for each group. **P , 0.01 vs. non-transfected cells. One-way ANOVA followed by Newman – Keuls test (B and D).
438
M. Pérez-Hernández et al.
Table 3 Electrophysiological properties of IBa recorded in HL-1 cells in the absence and presence of Pitx2c
IBa
..................................................................................................................................................................
Erev (mV)
tact (ms)
Vhact (mV)
kact
tinact (ms)
Vhinact (mV)
215.7 + 6.0
kinact
trecu (ms)
.......................................................................................................................................................................................
Pitx2c (2)
75.3 + 1.8
1.7 + 0.1
9.2 + 0.9
5.9 + 0.3
167 + 8.8
Pitx2c (+)
74.0 + 2.2
2.1 + 0.1
10.6 + 1.0
5.8 + 0.3
175 + 14.5
26.6 + 0.9*
10.6 + 1.9
314 + 14
10.0 + 0.8
480 + 44*
Each value represents mean + SEM of more than 15 experiments in each group.
Erev, reversal potential; tact, time constant of IBa activation measured at +20 mV; tinact, time constant of IBa inactivation measured at +20 mV; trecu, time constant of IBa reactivation
measured at 230 mV; Vhact and kact, midpoint and slope values of the conductance – voltage curves; Vhinact and kinact, midpoint and slope values of the inactivation curves.
*P , 0.05 vs. cells non-transfected with Pitx2c.
4. Discussion
To our knowledge, this is the first study comparing Pitx2c expression in
isolated atrial myocytes from SR and CAF patients and analysing the effects of Pitx2c on IKs and ICa,L. The results demonstrate that expression
of Pitx2c increased in CAF myocytes and that this increase directly and
inversely correlated with the densities of IKs and ICa,L, respectively.
4.1 Pitx2c and AF
In 2007, Gudbjartsson et al. 9 published the first genome-wide association study, describing that non-coding single nucleotide polymorphisms on chromosome 4q25, 170 kb distal to the PITX2 gene, were
strongly associated with AF. This association has been further
confirmed in populations of distinct ethnic backgrounds26 and has
also been reported for post-cardiac surgery AF27 and left atrial dilatation28 and responsiveness to anti-arrhythmic drug therapy.29 The proposed mechanisms underlying this relationship are mainly based on its
role in cardiac development, including alterations in the sinoatrial node
genetic programme13 and in the development of myocardial sleeves of
the pulmonary veins.14 However, recent evidences in mouse models
suggest a role of Pitx2c in adult atria and, specifically, in the events
that form part of the remodelling process associated with AF.11,12,15
Chinchilla et al. 11 showed that Pitx2c mRNA expression was markedly
decreased in right and left atrial appendages obtained from patients
with AF when compared with those obtained from SR patients.
More recently, it was demonstrated that Pitx2c expression increased
in atrial tissue from patients in AF at the time of sample collection.10
These discrepancies could be attributed to patient characteristics and
AF progression. AF patients included in these two studies were not
specifically classified as CAF patients, and the disease duration was
not mentioned. More importantly, both studies were conducted using
whole atrial appendage samples. It is known that the presence of fibroblasts and other non-myocyte cells in whole atrial samples may complicate the interpretation of gene expression analysis in myocytes.16 To
overcome these issues, we conducted qPCR experiments in isolated
myocytes from atrial appendages obtained from patients in SR and patients diagnosed with CAF (.6 month at the time of surgery). Under
these conditions, our qPCR experiments showed that Pitx2c mRNA
expression was approximately two-fold greater in CAF than in SR
myocytes.
4.2 Pitx2c modulates IKs and ICa,L
Up-regulation of IKs and down-regulation of ICa,L are hallmarks of
CAF-induced electrical remodelling in humans and critically contribute
to the abbreviation of APD and atrial refractory period.3 – 5,7,16 Our results confirmed previous observations5,7 and showed that in atrial myocytes from CAF patients, IKs density was approximately two-fold higher,
the activation kinetics was significantly faster, and the conductance
curve was shifted to more hyperpolarized potentials when compared
with SR myocytes. In contrast, in CAF myocytes, ICa,L density was reduced and the inactivation curves were shifted to more positive potentials in comparison to SR myocytes. Importantly, in human atrial
myocytes, increase in Pitx2c expression was positively and inversely
correlated with the IKs increase and ICa,L decrease, respectively, suggesting that Pitx2c could be involved in the changes in these currents
that characterize CAF-induced electrical remodelling. On the basis of
our results, Pitx2c deletion should lead to APD prolongation. However, in mice heterozygous for Pitx2c 15 and in atrial-specific Pitx2c-
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activation (Figure 6C), kinetics of activation and inactivation, and the reversal potential (Table 3). Additionally, western blot analyses demonstrated that Pitx2c did not significantly modify Cav1.2 expression
(Figure 7A and B), suggesting that Pitx2c does not regulate CACNA1C
transcription, despite the presence of a TAATCC sequence 100 bp
downstream of the TSS of CACNA1C. In contrast, neither CACNB2 nor
CACNA2D minimal promoters exhibit the consensus sequence for
Pitx2c binding. From this result, we surmised that the inhibition could
be due to post-translational modifications. As shown earlier, Pitx2c activates the NPPA promoter. Moreover, it was previously described that
ANP inhibits ICa,L in atrial myocytes through a mechanism involving an
increase in cyclic GMP levels.24 Furthermore, Pitx2c significantly increased ANP concentration in the culture medium of HL-1 cells
(n ¼ 6, P , 0.05) (Figure 7C). Therefore, we hypothesized that the effects of Pitx2c on ICa,L could be mediated by ANP. To preliminarily answer this question, the effects of Pitx2c transfection on IBa were
analysed in HL-1 cells incubated or not with the ANP type A receptor
antagonist A71915 (0.5 mM) for 24 h.25 As shown in Figure 7D, incubation with the antagonist completely prevented the inhibitory action induced by Pitx2c on IBa, suggesting that ANP was responsible for this
effect. It was described that isoproterenol-induced ICa,L potentiation
is larger in atrial myocytes from rats with heart failure than in control
myocytes. This result was associated to higher levels of ANP in heart
failure animals and due to an ANP-induced effect on cGMP-dependent
phosphodiesterases.24 We tested whether the Pitx2c-induced increase
in ANP expression would modulate isoproterenol effects on IBa recorded in HL-1 cells. In cells non-transfected with Pitx2c, isoproterenol
(50 nM) increased IBa recorded at +20 mV by 26.8 + 7.7% (n ¼ 7),
whereas in Pitx2c-transfected cells, the IBa increase induced by isoproterenol was significantly higher (65.0 + 11.3% at +20 mV, P ,
0.05, n ¼ 7) (Figure 7E and F, inset).
Pitx2c and human atrial currents
439
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Figure 7 Pitx2c does not modify Cav1.2 expression in HL-1 cells. (A) Western blot images showing Cav1.2 expression in HL-1 cells transfected (right
lane) or not (left lane) with Pitx2c. GAPDH (bottom) was used as loading control. (B) Densitometric analysis of the western blots. Results are presented
as mean + SEM of four batches of cells for each group. (C) ANP concentration measured in the culture medium of HL-1 cells transfected or not with
Pitx2c. Bars represent the mean + SEM of three batches of cells for each group. (D) Current density – voltage relationships for IBa recorded in HL-1 cells
transfected with Pitx2c in the absence or presence of the ANP type A receptor antagonist A71915 (0.5 mM). Each point represents the mean + SEM of
more than 12 cells from at least 3 independent batches in each group. In (C ) and (D), *P , 0.05 vs. cells non-transfected with Pitx2c. IBa density – voltage
relationships for currents recorded in HL-1 cells transfected (F ) or not (E) with Pitx2c in the presence or absence of 50 nM isoproterenol. The inset
shows IBa density at +20 mV. Each point/bar represents the mean + SEM of seven cells from at least two independent batches in each group. In (E) and
(F), *P , 0.05 vs. control and #P , 0.05 vs. cells non-transfected with Pitx2c. Unpaired t-test (B and C ), ANOVA followed by Newman–Keuls test (D – F,
and inset), and multilevel mixed-effects model (D).
deficient mice,11 atrial APD was shortened and unmodified, respectively, compared with wild-type animals. It is known that atrial action potential repolarization of mouse and human is different.30 Indeed,
mouse atrial myocytes lack functional Kv11.1 + miRP1 and Kv7.1 +
minK channels and, thus, IKr and IKs, respectively, cannot be recorded.
Therefore, changes in APD secondary to modulation of Kv7.1 + minK
channels would not be expected in mouse. In contrast, repolarizing sustained K+ currents in mice are generated by Kv1.5 and Kv2.1 channels.
Therefore, it is possible that Pitx2c-deficient mice display altered
expression of Kv2.1 channels or other unknown factors, which may
compensate the effects on APD secondary to the ICa,L increase upon
Pitx2c deletion.
To elucidate the molecular mechanism underlying Pitx2c-induced effects, HL-1 cells were used as experimental model. These cells are
mouse atrial-derived cells that can be maintained in culture and have
been previously used to test Pitx2c actions.11,31 Depending on the
main outward K+ current recorded, three groups of HL-1 cells were
established: IKr-predominant, IKs-predominant, and intermediate (with
440
4.3 Study limitations
All samples came from right atrial appendages, which could not be
representative of the rest of the atria. Furthermore, Pitx2c expression is higher in left than in right atria,8 and thus it cannot be ruled
out that the increase in Pitx2c is more relevant for left atrial myocytes. In this case, it is possible that Pitx2c would play a role in the
exacerbation of electrophysiological heterogeneity that characterizes CAF.5 Pitx2c expression and ion channel function could be influenced by age, sex, pharmacological treatment, and/or underlying
cardiac diseases of the patients. Interestingly, ICa,L density and plasma
ANP levels are influenced by the clinical history of the donors, in
such a way that the ANP levels are higher and ICa,L density is smaller
in patients with mitral valve disease or decreased left ventricular
function.34 In our sample, the proportion of patients with ischaemic
heart disease alone and combined with valvular cardiomyopathy was
equally distributed in both groups. However, more patients were in
decompensated heart failure [New York Heart Association (NYHA)
III and IV] in the CAF group than in the SR group. Interestingly, multiple linear regression analysis confirmed that CAF influenced both
I Ca,L and I Ks densities, whereas the NYHA class and the presence
of mitral valve disease did not (Supplementary material online, Tables
S2 and S3).
5. Conclusions
We demonstrate that Pitx2c expression increases in human atrial
myocytes from CAF patients, and this increase correlates with the IKs
increase and ICa,L decrease that characterize CAF-induced electrical remodelling. We propose that through this mechanism Pitx2c is involved
in the APD and refractory period shortening that enhances arrhythmia
recurrence and maintenance.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Acknowledgement
We thank Paloma Vaquero for her technical assistance and Dr Diego
Franco for providing us with Pitx2c plasmids.
Conflict of interest: none declared.
Funding
This work was supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC-08-2009), Ministerio de Ciencia e Innovación
(SAF2014-58769-P), Instituto de Salud Carlos III (Red HERACLES RD06/
0009, Red Investigación Cardiovascular RD12/0042/0011 and PI11/
01030), Comunidad Autónoma de Madrid (S2010/BMD-2374), and Fundaciones Mutua Madrileña and BBVA Grants.
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