PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers

Abstract

Background

Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA).

Objectives

After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs.

Methods

LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na⁺)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials.

Results

Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c^+/–). Resting membrane potential was more depolarized in Pitx2c^+/– atria, and TWIK-related acid-sensitive K⁺ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide’s effects between wild-type and Pitx2c^+/– atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Na_v1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized.

Conclusions

PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.

Central Illustration

Atrial fibrillation (AF) causes cardiovascular death, frequent hospitalization, and cognitive decline even in patients treated according to guidelines ^{1, 2, 3}. Antiarrhythmic drug (AAD) therapy remains the most commonly used treatment to maintain sinus rhythm in AF patients, but AAD effectiveness remains limited (3). Unfortunately, we lack a basic understanding of why AADs prevent AF over long periods in some patients but not in others ^{4, 5}. Identifying factors that modify the effects of AADs would allow the selection of responsive patients and could help guide development of novel AADs (6).

Paired like homeodomain-2 transcription factor (PITX2) is a transcription factor that regulates the development of the left atrium (LA) and thoracic organs. Its c isoform is expressed in the adult LA and regulates the expression of LA ion channels ^{7, 8, 9}. Low atrial Pitx2 expression renders mice susceptible to AF and shortens the LA action potential ^{8, 10, 11}. In this study, we investigated how atrial PITX2 modifies the effects of AADs.

We detected variable LA PITX2 messenger ribonucleic acid (mRNA) expression in AF patients requiring rhythm control therapy. After finding that low Pitx2c enhanced the effect of flecainide, mediated by a more positive resting membrane potential (RMP), we identified reduced TWIK-related acid-sensitive K⁺ channel 2 (TASK-2) expression as a possible driver of this effect and replicated these effects in cells expressing human sodium (Na) channels and in a human atrial action potential model.

Methods

All experiments were conducted under the Animals (Scientific Procedures) Act 1986, and approved by the home office (PPL number 30/2967) and the institutional review board at the University of Birmingham. Analyses of human atrial tissue were approved by the institutional review board of Academic Medical Center, Amsterdam, the Netherlands. All patients provided written informed consent.

Left atrial appendages (LAAs) were excised from 95 patients undergoing bilateral thoracoscopic AF ablation either in the AFACT (Atrial Fibrillation Ablation and Autonomic Modulation via Thoracoscopic Surgery) trial (12) or undergoing similar procedures in the same centers using an endoscopic stapling device, snap frozen in liquid nitrogen and stored at –80°C (13). Deoxyribonucleic acid and ribonucleic acid were extracted using DNeasy and RNeasy kits (Qiagen Ltd., Manchester, United Kingdom), respectively. PITX2 mRNA content was quantified by quantitative polymerase chain reaction. Single nucleotide polymorphisms (SNPs) rs2200733, rs6838973, and rs1448818 (14) were identified using TaqMan assays (Thermo Fisher Scientific Inc., Waltham, Massachusetts).

Adult mice (age 12 to 16 weeks) on an MF1 background with normal or reduced (Pitx2c^+/−) atrial Pitx2c expression were studied (8).

LA epicardial monophasic action potentials were recorded from Langendorff-perfused murine hearts ^{8, 15}. Programmed stimulation was performed at baseline and with flecainide 1 μmol/l or d,l-sotalol 10 μmol/l. Arrhythmia inducibility and effective refractory period (ERP) were measured by using single right atrial extrastimuli after steady-state pacing in 1-ms decrements ^{15, 16, 17, 18}. Transmembrane action potentials were recorded using borosilicate glass microelectrodes from superfused murine LAs (17), RMP, action potential duration (APD), upstroke velocity, and activation times were analyzed ^{15, 17, 18}.

The human atrial cell model of Courtemanche et al. (19) was used. Pitx2c^+/– deficiency was modeled by reducing I_K1 conductance by 25% and doubling I_Kr conductance. Simulations were run in strands of 100 atrial cells (cell length 100 μm). The 5 leftmost cells of the strand were paced (S1) for 2 min at 1,000- and 500-ms basic cycle lengths. Premature stimulation (S2) was applied to determine the ERP and conduction velocity as measured from cells 25 to 75. Values for all other parameters were measured from the 50th cell. For the modeling, post-repolarization refractoriness (PRR) was calculated as the difference between APD at –60 mV repolarization and ERP.

LA cell isolation was performed as previously reported (20). Standard I_Na and I_K1 currents were recorded as previously published ^{18, 19, 20}. Background K⁺ (TASK-like) currents sensitive to high Ba²⁺ (10 mM) were measured ^{21, 22, 23}. Human embryonic kidney (HEK) 293 cells stably expressing the human Na_v1.5 channel were obtained (SB Ion Channels, Glasgow, UK).

Ribonucleic acid and complementary deoxyribonucleic acid were synthesized from murine LA, (SuperScript VILO, Thermo Fisher Scientific Inc.) to quantify expression of 20 atrial ion channels and genes with suspected PITX2-dependent regulation (9) using custom-designed Taqman low density array plates (Thermo Fisher Scientific Inc.). Western immunoblotting was performed on murine LA tissue lysates with antibodies detecting TASK-2, K_v1.6, Na/K ATPase alpha-1, Na/K ATPase alpha-2, Na/Ca exchanger 1, Serca2a, Na_v1.5, or calnexin, using standard methods.

Optical action potentials and calcium ion (Ca²⁺) transients were recorded in murine LA and analyzed using custom-made MATLAB algorithms (MathWorks, Natick, Massachusetts) as previously described (17).

Statistical analysis

All experiments were performed and analyzed in a blinded fashion. Murine studies were performed and analyzed blinded to genotype in littermate pairs. Categorical data were compared using the Fisher exact test. Numerical data were compared by 2-sided paired parametric Student t tests (e.g., measurements before and after perfusion of flecainide or sotalol) and Wilcoxon signed rank tests. Multiple measurements were assessed by repeated measures of analysis of variance followed by correction for multiple comparison (Bonferroni test) if the overall test was significant. Two-sided p < 0.05 were considered significant. Box plots depict individual measurements (points), mean, and SEM. Statistics and figures were created using Prism 5 (GraphPad Software, San Diego, California).

Results

PITX2 mRNA varied markedly in human LAA (Central Illustration) harvested from AF patients (Table 1) (13), suggesting that a 50% lowered PITX2 expression defines a large, potentially clinically relevant group of AF patients. This did not directly correlate with SNP haplotype (Table 2), although we found numerically lower PITX2c levels in patients with 5 risk alleles.

Central Illustration.

PITX2 Deficiency Potentiates Flecainide's Antiarrhythmic Effects

Paired like homeodomain 2 (PITX2) might play an important role in regulating gene expression and electrical function of the left atrium. (Left)Pitx2 messenger ribonucleic acid (mRNA) levels are variable in left atrial appendages of patients undergoing thoracoscopic atrial fibrillation (AF) ablation therapy. The differentiation between “low” (orange) and “high” (blue)PITX2 levels is somewhat arbitrary. (Right) A low left atrial PITX2c mRNA expression slightly depolarizes left atrial resting membrane potential (RMP). A depolarized RMP, in turn, enhances the antiarrhythmic effect of sodium-channel blockers such as flecainide. PRR = post-repolarization refractoriness.

Table 1.

Baseline Characteristics (N= 101)^∗

Age, yrs	59.7 ± 8.4 (40–76)
Male	79
Congestive heart failure	6
Hypertension	34
Age ≥75 yrs	1
Diabetes	9
Stroke/transient ischemic attack/embolus	10
Vascular disease	10
Female	22
Age ≥65 yrs	31
CHA2DS2-VASc score
0	60
1	24
≥2	17
Previous catheter ablation for AF	20
Type of AF
Paroxysmal	44
Persistent	56
Longstanding persistent	1
AF duration, yrs	6.0 (1–35)
Antiarrhythmic drugs and rate control agents
Quinidine or disopyramide	4
Flecainide or propafenone	33
Amiodarone, dronedarone, or sotalol	41
Beta blockers	53
Verapamil or diltiazem	17
Digoxin	15
Anticoagulant agents (before PVI procedure)
Vitamin K antagonists	89
Antiplatelets	6

Values are mean ± SD (range), n, or mean (range).

PVI = pulmonary vein isolation.

^∗Left atrial appendages were collected from these patients with atrial fibrillation (AF).

Table 2.

PITX2 mRNA Expression in Left Atrial Appendages From AF Ablation Patients^∗

Risk Alleles	25% IQR	Median	75% IQR	Mean	SEM	No. of Patients
0	3.22	3.69	5.22	4.04	0.6	3
1	2.96	4.25	6.25	4.54	0.5	13
2	2.65	3.78	4.75	3.94	0.3	22
3	2.74	3.72	4.92	3.83	0.4	17
4	3.00	4.29	5.41	4.39	0.5	10
5	1.96	2.66	4.66	3.10	0.7	4
6	4.95	4.95	4.95	4.95	0.0	1

IQR = interquartile range; LA = left atrium; other abbreviations as in Table 1.

^∗This dataset was grouped according to the number of risk single nucleotide polymorphism (SNP) alleles for AF on chromosome 4q25 (rs2200733, SNP2 rs6838973, rs1448818 [13]). Although PITX2 mRNA is numerically lower in patients with 5 or 6 risk alleles, we did not find a PITX2 mRNA gradient according to AF risk.

Flecainide suppressed atrial arrhythmias in murine Pitx2c^+/– hearts. Flecainide abolished induced atrial arrhythmias in hearts with reduced Pitx2c expression (0 of 17 hearts with atrial arrhythmias) but not in hearts with normal Pitx2c expression (atrial arrhythmias remained in 3 of 12 hearts) (Figures 1A to 1C). Flecainide prolonged ERPs and refractoriness beyond the end of repolarization (PRR) calculated as the difference between ERP and APD₉₀ (ms). Flecainide prolonged PRR more in hearts with reduced Pitx2c expression (Figures 1D and 1E, Table 3). PITX2c^+/– hearts had shorter atrial action potentials (8). Flecainide abolished APD differences between Pitx2c^+/– and wild-type LA by prolonging early repolarization (APD₃₀, APD₅₀, and APD₇₀) (Table 3). Murine atrial PITX2 expression did not modulate the effects of sotalol on atrial APD or ERP (Table 4).

Figure 1.

Atrial Arrhythmia Inducibility in Pitx2c^+/– Murine Whole Hearts

(A) Image and schematic representation of the Langendorff-perfused heart. (B) Atrial arrhythmia inducibility in isolated, beating hearts from wild-type (WT) and reduced paired like homeodomain 2 messenger ribonucleic acid (Pitx2c^+/–) mice. Flecainide abolished atrial arrhythmia inducibility in Pitx2c^+/– hearts only. *p < 0.05 flecainide versus baseline. (C) Representative trace of atrial fibrillation (AF) induced during programmed stimulation at baseline, showing reduced severity of arrhythmias with 1 μmol/l flecainide in Pitx2c^+/– atria. (D) Effects of flecainide on atrial effective refractory period (ERP) in wild-type and Pitx2c^+/– isolated, beating hearts. Shown is the difference in atrial ERP between baseline and 1 μmol/l flecainide at 80- to 120-ms paced cycle length following a single extrastimulus (S2) in WT and Pitx2c^+/– isolated, beating hearts. *p < 0.05 between genotypes across all cycle lengths. (E) Whereas flecainide prolonged ERP in both genotypes, this effect was more pronounced in Pitx2c^+/– atria. Flecainide caused post-repolarization refractoriness (PRR), the difference between ERP (orange and grey lines) and APD₉₀(blue lines), in WT and Pitx2c^+/– atria. Flecainide-induced PRR in Pitx2c^+/– is almost 3 times that of WT atria. *p < 0.05 WT versus Pitx2c^+/–. ^#p < 0.05 baseline versus 1 μmol/l flecainide. APD = action potential duration; EG = intracardiac electrogram; EP = electrophysiology; LA = left atrium; LV = left ventricle; MAP = monophasic action potential; RA = right atrium; RV = right ventricle.

Table 3.

Effect of Flecainide on Refractoriness and Repolarization in Mouse Hearts

Paced CL, ms	Wild-Type						Pitx2c+/–
	120		100		80		120		100		80
	Baseline	Flecainide	Baseline	Flecainide	Baseline	Flecainide	Baseline	Flecainide	Baseline	Flecainide	Baseline	Flecainide
LA ERP, ms
	23.5 ± 2.3 (11)	29.8 ± 3.0 (11)	22.2 ± 2.1 (11)	29.6 ± 3.3 (11)	21.9 ± 2.4 (10)	28.7 ± 3.5∗ (10)	30.5 ± 2.4 (11)	38.5 ± 3.3∗ (11)	28.0 ± 2.3 (13)	40.2 ± 2.8∗ (13)	27.5 ± 2.5 (13)	41.2 ± 3.0∗† (13)
LA monophasic APD, ms
APD50	10.2 ± 1.3 (8)	14.5 ± 1.7 (8)	10.8 ± 1 (8)	11.9 ± 1.6 (8)	10.4 ± 0.7 (7)	12.0 ± 1.1 (7)	12.4 ± 1.1 (15)	14.4 ± 1.3 (15)	11.5 ± 1.0 (15)	12.4 ± 1.1 (15)	10.6 ± 0.9 (11)	10.3 ± 1.0 (11)
APD70	17.8 ± 2.2 (9)	23 ± 2.1 (9)	18.4 ± 1.6 (9)	18.7 ± 2.2 (9)	18.1 ± 1.2 (8)	18.1 ± 1.9 (8)	18.0 ± 1.6 (15)	19.2 ± 1.8 (15)	16.0 ± 1.4 (13)	16.2 ± 1.4 (13)	14.9 ± 1.0† (10)	13.1 ± 0.7 (10)
APD90	31.3 ± 3.0 (8)	37.4 ± 2.8 (8)	31.5 ± 2.5 (9)	29.9 ± 2.9 (9)	31.0 ± 1.4 (8)	28.4 ± 2.7 (8)	28.3 ± 2.2 (13)	29.9 ± 2.2 (13)	27.4 ± 2.2 (13)	26.6 ± 1.5 (13)	26.8 ± 1.7 (10)	23.1 ± 1.4 (10)
LA transmembrane APD, ms
APD30	4.5 ± 0.1 (30)	5.5 ± 0.3 (22)	4.5 ± 0.1 (30)	5.4 ± 0.3 (22)	4.4 ± 0.1 (30)	5.2 ± 0.3 (22)	4.0 ± 0.1 (31)	4.7 ± 0.2 (24)	3.9 ± 0.1 (31)	4.6 ± 0.2 (24)	3.8 ± 0.1† (31)	4.4 ± 0.2 (24)
APD50	6.7 ± 0.2 (30)	8.2 ± 0.5 (22)	6.6 ± 0.2 (30)	8.0 ± 0.4 (22)	6.4 ± 0.2 (30)	7.8 ± 0.5 (22)	5.9 ± 0.2 (31)	7.1 ± 0.4 (24)	5.7 ± 0.2 (31)	7.0 ± 0.4 (24)	5.6 ± 0.2† (31)	6.7 ± 0.3 (24)
APD70	10.5 ± 0.4 (30)	12.7 ± 0.8 (22)	10.1 ± 0.4 (30)	12.1 ± 0.7 (22)	9.6 ± 0.4 (30)	11.8 ± 0.7 (22)	8.9 ± 0.4 (31)	10.7 ± 0.6 (24)	8.6 ± 0.4 (31)	10.3 ± 0.6 (24)	8.3 ± 0.3† (31)	9.8 ± 0.5 (24)
APD90	20.9 ± 1.0 (30)	23.4 ± 1.5 (22)	19.9 ± 0.9 (30)	22.2 ± 1.4 (22)	18.4 ± 0.8 (30)	21.6 ± 1.3 (22)	17.6 ± 0.9 (31)	20.3 ± 1.1 (24)	16.5 ± 0.8 (31)	19.2 ± 1.0 (24)	15.7 ± 0.8† (31)	17.9 ± 0.9 (24)
LA optical APD, ms
APD30	6.1 ± 0.3 (10)	7.3 ± 0.6 (6)	6.4 ± 0.8 (10)	5.9 ± 1.0 (6)	6.1 ± 0.4 (10)	6.9 ± 1.3 (6)	4.9 ± 0.4 (10)	7.7 ± 0.9 (8)	4.6 ± 0.3 (10)	5.4 ± 0.7 (8)	4.3 ± 0.4† (10)	5.7 ± 0.7 (8)
APD50	8.5 ± 0.6 (10)	10.7 ± 1.2 (6)	8.9 ± 1.1 (10)	8.5 ± 1.2 (6)	8.3 ± 0.7 (10)	10.3 ± 1.8 (6)	6.9 ± 0.4 (10)	10.0 ± 1.0 (8)	6.6 ± 0.4 (10)	8.1 ± 0.9 (8)	6.1 ± 0.4† (10)	8.0 ± 0.9 (8)
APD70	11.7 ± 1.2 (10)	15.0 ± 2.1 (6)	12.5 ± 1.5 (10)	12.8 ± 1.7 (6)	11.5 ± 1.1 (10)	14.4 ± 2.5 (6)	9.4 ± 0.0 (10)	13.3 ± 1.2 (8)	9.4 ± 0.6 (10)	11.6 ± 1.5 (8)	9.1 ± 0.5† (10)	11.2 ± 1.2 (8)

Values are mean ± SEM (number of atria).

APD = action potential duration; CL = cycle length; ERP = effective refractory period; Pitx2c^+/– = PITX2 deficient; other abbreviations as in Tables 1 and 2.

^∗p < 0.05 vs. baseline.

^†p < 0.05 vs. wild-type.

Table 4.

Electrophysiological Effects of Sotalol

Paced CL, ms	Wild-Type				Pitx2c+/–
	120		100		120		100
	Baseline	Sotalol	Baseline	Sotalol	Baseline	Sotalol	Baseline	Sotalol
LA ERP, ms
	38.7 ± 7.8 (7)	33.9 ± 6.3 (7)	32.2 ± 6.1 (6)	29.2 ± 5.3 (6)	39.3 ± 4.0 (4)	26.8 ± 3.5 (4)	37.0 ± 5.7 (4)	24.0 ± 3.7 (4)
LA APD, ms
APD50	11.5 ± 1.2 (9)	13.4 ± 1.2 (9)	10.9 ± 2.0 (7)	12.2 ± 1.3 (7)	10.8 ± 1.1 (7)	11.2 ± 1.0 (7)	8.3 ± 0.9 (4)	11.1 ± 1.7 (4)
APD70	17.6 ± 2.2 (9)	20.0 ± 1.9 (9)	16.0 ± 1.3 (7)	18.2 ± 2.3 (7)	16.5 ± 1.6 (7)	17.3 ± 1.2 (7)	13.0 ± 1.5 (4)	17.0 ± 1.9 (4)
APD90	30.7 ± 3.2 (9)	33.5 ± 2.7 (9)	29.0 ± 1.9 (7)	30.9 ± 3.2 (7)	29.7 ± 2.7 (7)	31.2 ± 2.0 (7)	23.8 ± 2.8 (4)	29.6 ± 2.9 (4)

Values are mean ± SEM (number of atria).

Abbreviations as in Tables 2 and 3.

RMP was slightly depolarized in LA murine cells with reduced Pitx2c expression (range of mean depolarization 1.2 to 2.4 mV over 5 cycle lengths; all p < 0.05) (Figures 2A and 2B). Atrial Pitx2c levels did not significantly affect dV/dt_max (100-ms paced cycle length: wild-type: 104.4 ± 4.3 V/s; Pitx2c^+/–: 93.7 ± 4.5 V/s) (Figure 2C). Flecainide did not modify atrial RMP (Figure 2B) but reduced action potential amplitude consistent with its Na-channel blocking effect, specifically at 100-ms cycle length: wild-type baseline: 77.5 ± 1.2 mV (n = 30); wild-type flecainide: 71.3 ± 1.2 mV (n = 31); Pitx2c^+/– baseline: 73.4 ± 1.3 mV (n = 22); and Pitx2c^+/– flecainide: 65.1 ± 1.45 mV (n = 24).

Figure 2.

Resting Membrane Potential and Maximum Upstroke Velocity

(A) Schematic representation of sharp microelectrode electrode recordings from the superfused whole LA. (B) Resting membrane potential (RMP) in WT and Pitx2c^+/– LA at baseline and with 1 μmol/l flecainide. Pitx2c^+/– LA have depolarized RMP. The difference between WT and Pitx2c^+/– is exaggerated with flecainide. *p < 0.05; ***p < 0.001 across all cycle lengths. (C) Flecainide unmasked a lower maximum upstroke velocity (dV/dt_max) in Pitx2c^+/– LA compared to WT. **p < 0.01 versus WT. Abbreviations as in Figure 1.

Because the Courtemanche–Ramirez–Nattel model does not incorporate background K⁺ currents (19), we simulated a depolarized RMP in this model by a 25% reduction in I_K1. This reduced the RMP at 500-ms paced cycle length by 2 mV from 79.9 mV (“normal PITX2”) to –77.9 mV (“low PITX2”). Na channels recovered from inactivation more slowly upon partial I_Na block (50% or 60%) (Figure 3A). Furthermore, PRR was enhanced in the PITX2 deficiency model (Figure 3B and Table 5). Inhibition of I_Na reduced upstroke velocity (dV/dt_max) and conduction velocity in both models, and reproduced the prolongation of PRR (Figure 3B).

Figure 3.

Modeling of the Electrophysiological Consequences of Pitx2c Deficiency

(A) Propagated action potentials (top) simulated with the Courtemanche–Ramirez–Nattel model modified to reflect the more positive RMP of murine Pitx2c^+/– atria and the effect of 60% sodium current (I_Na) block at pacing cycle lengths of 500 and 1,000 ms, with 2-min pre-pacing, and corresponding time courses of the product of the 2 inactivation gates h and j of I_Na(bottom), reflecting I_Na availability. (B) Reduced sodium conductance (g_Na) increased post-repolarization refractoriness (PRR) in the reference model and the Pitx2c deficiency model. After reducing g_Na, PRR was greater in the Pitx2c deficiency model than in the reference model (grey lines). Lines denote APD₆₀(blue) and ERP (orange: with 100% gNa; grey: with 40% gNa). Abbreviations as in Figures 1 and 2.

Table 5.

Electrophysiological Effects of Reduced Sodium Conductance in a Human Atrial Model

Paced CL, ms	Wild-Type Model		Pitx2c Deficiency Model
	500	1,000	500	1,000
RMP, mV
gNa, %
100	-79.92	-81.28	-77.90	-79.61
50	-79.60	-81.12	-77.33	-79.37
40	-79.43	-81.01	-76.99	-79.23
APD at repolarization to -60 mV, ms
gNa, %
100	217	253	206	226
50	239	266	233	239
40	248	273	245	245
ERP, ms
gNa, %
100	266	301	261	280
50	308	335	316	320
40	327	352	342	339
PRR, ms
gNa, %
100	49	48	55	54
50	69	69	83	81
40	79	79	97	94
Conduction velocity, cm/s
gNa, %
100	49.5	50.0	50.3	50.5
50	36.9	37.0	37.5	37.5
40	32.9	32.7	33.0	33.1

g_Na = reduced sodium conductance; PRR = post-repolarization refractoriness; RMP = resting membrane potential; other abbreviations as in Table 3.

Kcna6 and Kcnk5 mRNA expression were reduced in Pitx2c^+/– murine LA (Figure 4A, Online Table 1), whereas mRNA concentrations of 20 other ion channels or related genes were not altered. Kv1.6 protein concentration was unaltered, whereas TASK-2 protein concentration was reduced in murine atria with reduced Pitx2c expression (Figure 4B). Na_v1.5 mRNA and protein expression were not changed (Figures 4A and 4B).

Figure 4.

mRNA and Protein Expression in Murine LA

(A) Relative quantity (RQ) of messenger ribonucleic acid (mRNA) of selected ion channels in LA from Pitx2c^+/– and WT mice. Expression levels were measured relative to WT sample 1. Kcnk5 encodes TWIK-related acid-sensitive K⁺ channel 2 (TASK-2), Kcna6 encodes K_v1.6, and Scn5a encodes Na_v1.5. *p < 0.05. (B) Concentration of TASK-2, K_V1.6, and Na_v1.5 proteins relative to calnexin (arbitrary units). Representative immunoblots are displayed below the corresponding dot plot. *p < 0.05. Abbreviations as in Figures 1 and 2.

Atrial Pitx2c expression did not modify peak Na⁺ currents (I_Na) recorded from isolated murine cardiomyocytes at holding potentials ranging from –100 to –65 mV (Figures 5A to 5C). Peak I_Na was reduced at more depolarized holding potentials (Figure 5). Flecainide inhibited I_Na better at more positive holding potentials (inhibition at –70 mV: 68 ± 5%; inhibition at –65 mV: 75 ± 5%; n = 86 cells from n = 17 atria) in cells from murine atria with normal or reduced Pitx2c expression, suggesting that the greater efficiency of flecainide in atria with reduced Pitx2c expression is secondary to RMP depolarization (Figure 5C). Consistent with this, flecainide inhibited human Na_v1.5 channels expressed in HEK cells more potently at more depolarized test potentials (–65 to –75 mV) (Figures 5D and 5E).

Figure 5.

Effect of Membrane Potential on Sodium-Channel Inhibition

(A) Schematic representation of patch clamp experiments carried out in isolated atrial cardiomyocytes. Human embryonic kidney (HEK) cells were patch clamped using the same setup. (B) Representative traces showing I_Na measured by patch clamping in isolated atrial cardiomyocytes, at baseline and with flecainide, at increasing holding potentials (–100 to –65 mV). (C) Reduction in peak I_Na was enhanced with increased holding potentials, irrespective of cardiomyocyte origin. (D) Representative traces showing I_Na in Na_v1.5-transfected HEK cells at baseline and with flecainide at increasing holding potentials (–100 to –65 mV). (E) Reduction in peak I_Na with flecainide in Na_v1.5-transfected HEK cells was enhanced with increased holding potentials. The greatest difference in percent reduction was between –70 and –65 mV. Abbreviations as in Figures 1, 2, and 3.

Background K⁺ currents, which include TASK currents, were reduced in Pitx2c^+/– murine atria, whereas I_K1 did not differ between genotypes (Figure 6).

Figure 6.

Currents Responsible for RMP

(A) Representative raw traces showing I_K1 currents (50 μmol/l Ba²⁺ sensitive) in isolated WT and Pitx2c^+/– LA cardiomyocytes at test potentials ranging from –120 to 50mV. (B) No difference is seen in the I_k1 current/voltage relationship for LA cardiomyocytes between WT and Pitx2c^+/– LA at test potentials ranging from –120 to 50 mV. (C) Representative raw traces show background current response to 10 mmol/l Ba²⁺ in isolated WT and Pitx2c^+/– LA cardiomyocytes. (D)Pitx2c^+/– cardiomyocytes had significantly reduced background K⁺ (10 mmol/l Ba²⁺ sensitive) currents than WT cardiomyocytes. *p < 0.05. Abbreviations as in Figures 1, 2, and 3.

Reduced Pitx2c expression did not alter atrial conduction velocities or activation patterns (Online Figures 1A to 1C, Table 6), consistent with published data (8). We found that 1 μmol/l flecainide decreased atrial conduction velocities without differences between wild-type and Pitx2c^+/– mice (Online Figures 1B and 1C). Calcium transient relaxation times at 50% relaxation were not different between wild-type and Pitx2c^+/– (Online Figures 1D and 1E). Flecainide 1 μmol/l shortened 50% Ca²⁺ relaxation times by approximately 10% and decreased Ca²⁺ transient amplitude by approximately 50% in murine atria with normal and reduced Pitx2c expression (Online Figures 1E and 1F). Additionally, expression of the Na/Ca exchanger Serca2a and Na/K ATPase alpha-1 and alpha-2 subunit protein did not differ between wild-type and Pitx2c^+/– atria (Online Figure 2).

Table 6.

Electrical Activation Time and Conduction Velocity in Isolated Atria in the Presence of Flecainide (1 μmol/l)

	Wild-Type					Pitx2c+/–
Paced CL, ms	1,000	300	120	100	80	1,000	300	120	100	80
Activation time (isolated left atrium), ms	6 ± 0.3 (22)	6 ± 0.3 (22)	9 ± 0.5 (22)	12 ± 1.0 (22)	16 ± 1.4 (22)	6 ± 0.2 (24)	7 ± 0.3 (24)	12 ± 0.9 (24)	13 ± 0.9 (24)	18 ± 1.2 (24)
Conduction velocity (optical mapping), cm/s	—	30 ± 1.8 (8)	25 ± 2.4 (8)	25 ± 1.9 (8)	23 ± 1.9 (8)	—	29 ± 1.5 (8)	26 ± 1.6 (8)	25 ± 1.6 (8)	23 ± 1.6 (8)

Values are mean ± SEM (number of atria).

— = not applicable; other abbreviations as in Table 3.

Discussion

This study demonstrated that LA PITX2 mRNA concentrations vary in patients with AF requiring rhythm control therapy (Central Illustration). Furthermore, flecainide increases PRR and suppresses arrhythmias more effectively in atria with halved Pitx2c expression, mediated by a more depolarized RMP (Central Illustration). Drug-induced PRR is thought to prevent arrhythmias, as reactivation can then occur only after full recovery of excitability, avoiding slow propagation during the vulnerable period ^{16, 24, 25}. We found similar effects in cells expressing human Na channels and in the Courtemanche–Ramirez–Nattel model of human atrial action potentials.

Thus, this study highlighted modulation of the atrial RMP by PITX2, possibly mediated by background currents such as TASK-2, as a target for AAD therapy, including atrial-selective therapy. Furthermore, the results suggested that markers for atrial PITX2 expression may identify AF patients who benefit from Na-channel blocker therapy (Central Illustration).

Low atrial PITX2 expression was identified as an important determinant of the antiarrhythmic effects of Na channel blockers. Low LA Pitx2c mRNA depolarized atrial RMP (Figure 2), consistent with a previous report (11). A depolarized RMP increased flecainide-induced PRR (Figure 1) ^{26, 27, 28, 29, 30}. The conduction-slowing effect of flecainide was not modulated by reduced atrial Pitx2c (Online Figure 1), an important surrogate for drug safety. Both the modeling experiments (Figure 3) and the experiments in HEK cells expressing human Na channels (Figure 5) confirmed that small changes in RMP can markedly modulate Na-channel inhibition.

Resting membrane potential

Open-state Na-channel blockers such as flecainide and propafenone bind preferentially to Na channels integrated in membranes with slightly depolarized resting potentials, where more channels are in the open or inactivated state ^{31, 32}. Our data can be interpreted as suggesting that AAD combinations that include a Na-channel blocker with a membrane potential modifying substance, such as amiodarone ^{16, 33} or the combination of dronedarone and ranolazine ^{29, 34, 35}, may have synergistic antiarrhythmic effects because they modulate atrial RMP and thereby enhance the effect of Na-channel blockade. Further studies of such drug combinations and the relationship between their effectiveness and the patient’s atrial PITX2 mRNA levels are warranted. Our data also suggested that such combined effects may be of special relevance in patients who have a depolarized RMP, such as secondary to low LA PITX2. Because PITX2 expression is confined to the LA in the heart, AAD therapy that leverages modifications in RMP may achieve “atrial-specific” AAD therapy.

RMP is maintained by an intricate balance of different transmembrane currents and is closely related to the potassium equilibrium potential. We identified that PITX2 modifies expression of the genes encoding K_v1.6 and TASK-2 (Figure 4). Complete deletion of PITX2 regulates other potassium and Na channels such as Kcnj2 ^{8, 36}, which alter the RMP, but these were not responsible for the depolarized RMP observed in our study. Two-pore domain potassium channels, such as TASK-2, contribute to RMP in various cells, including skeletal and cardiac muscle ^{37, 38}. To date, an altered function of the TASK-1 channel and of I_K1 has been implicated in atrial remodeling and AF ^{39, 40}. This study demonstrated that TASK-2 is expressed in atrial myocardium (Figure 4B), suggesting that a reduced function of TASK-2 could depolarize RMP (Figures 1 and 5) ^{8, 11}, analogous to the effect of TASK-2 in neuronal and cartilage tissue ^{41, 42}.

Developing clinical markers for patients with depolarized RMP

It will be challenging to directly assess LA RMP in AF patients, but our data suggested that differences in atrial RMP could explain the effectiveness of Na-channel blockers in carriers of common gene variants on chromosome 4q25 (43), although LA PITX2 levels are modulated by factors other than SNP status (Table 2) (44). It seems desirable to develop and validate drivers that modify RMP and clinical markers for patients prone to a depolarized atrial RMP to select appropriate AADs for individual patients in the future, thus enabling personalized AAD selection ^{6, 45}.

Study limitations

This study provided robust evidence that LA PITX2 expression varies in AF patients and that reduced PITX2c expression enhances the antiarrhythmic effects of Na-channel blockers by modulating atrial RMP. The study was partly motivated by the assumption that gene variants on chromosome 4q25 modify PITX2 expression, an assumption that has not been definitively proven ^{9, 11, 44, 46}. Our analysis (Table 2) and that of others indicate that SNP status does not always correlate with PITX2 levels ^{47, 48}. Our findings are relevant to AAD therapy even if the presumed link between PITX2 expression and genetic variants on chromosome 4q25 proves elusive. The mechanisms by which reduced PITX2 mRNA concentrations shorten the LA action potential at high heart rates remain to be fully elucidated ^{8, 20}. Validating our findings in patients is desirable but will be challenging because access to fresh LA cardiomyocytes and LA tissue is limited.

Due to the novelty of our findings, we could not perform a priori power calculations for our mechanistic experiments, and we analyzed several functional parameters to identify potential mechanisms conveying the antiarrhythmic effects of flecainide in atria with low Pitx2c concentrations. Our findings thus require independent validation.

Conclusions

This study shows that low LA PITX2 mRNA levels increase atrial RMP and thereby increase the effectiveness of flecainide (Central Illustration). This finding calls for appropriately designed clinical studies to assess whether AF patients with low atrial PITX2 levels respond favorably to Na-channel blockade. Further studies exploring the relevance of TASK channels to atrial RMP also are warranted.

Perspectives.

COMPETENCY IN MEDICAL KNOWLEDGE: PITX2, a transcription factor linked to left–right asymmetry in the chest during development, modulates the expression of LA ion channels maintaining the RMP and modulates the antiarrhythmic effects of Na-channel blockers.

TRANSLATIONAL OUTLOOK: Clinical studies are needed to assess whether reduced PITX2 expression identifies patients with AF who respond favorably to Na-channel blocking drugs.