Oxidative Stress causes Mitochondrial and Electrophysiologic Dysfunction to Promote Atrial Fibrillation in Pitx2^+/− Mice

Abstract

Background:

The strongest genetic risk factors for AF are DNA variants on chromosome 4q25 near the transcription factor gene PITX2. Mice deficient in Pitx2 (Pitx2^+/−) have increased AF susceptibility, although the molecular mechanism(s) remains controversial. Pitx2 encodes a transcription factor that activates an antioxidant response to promote cardiac repair. Increased reactive oxygen species (ROS) causing oxidation of polyunsaturated fatty acids generates reactive lipid dicarbonyl moieties that adduct to proteins and other macromolecules to promote cellular injury. We tested the hypothesis that oxidative stress, and specifically isolevuglandins (IsoLGs), the most reactive lipid dicarbonyls identified, are increased in the setting of Pitx2 deficiency to promote proarrhythmic remodeling and atrial fibrillation (AF).

Methods:

Pitx2^+/− and Pitx2^+/+ wild type (WT) littermate control mice were treated orally with vehicle, the lipid dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA), or an inactive control compound at weaning, until study at age 16–18 weeks.

Results:

Pitx2^+/− mice demonstrated increased P wave duration indicative of slowed atrial conduction, as well as increased inducible AF burden and sustained AF, compared to WT, and these abnormalities were prevented by 2-HOBA. Both ROS and IsoLG protein adducts were elevated in Pitx2^+/− atria with reduced expression of ROS-protective genes. High resolution respirometry demonstrated impaired mitochondrial function in Pitx2^+/− atria, with disruption of mitochondrial integrity and cell-cell junctions with connexin lateralization, as well as decreased mitochondrial biogenesis gene expression. Proarrhythmic ionic current remodeling in Pitx2^+/− atrial myocytes included elevated resting membrane potential, abbreviated action potential duration, and reduced maximum phase 0 upstroke velocity compared to WT. Most of these abnormalities were ameliorated or prevented by 2-HOBA.

Conclusions:

These results demonstrate a critical role for lipid dicarbonyl mediators of oxidative stress in the proarrhythmic remodeling and AF susceptibility that occurs with Pitx2 deficiency, implying the possibility of genotype-specific therapy to prevent AF.

Graphical Abstract:

Introduction

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in the Western world, afflicting one in four persons over the age of 40 years and resulting in increased mortality and significant morbidity.¹ Treatment of AF is complicated by a lack of effective, long-term therapy, largely due to an incomplete understanding of the critical components that drive AF pathophysiology. While multiple acquired conditions such as hypertension and obesity have long been recognized as risk factors for AF, there is increasing recognition of a genetic component of AF risk as well.² This risk is characterized by rare mutations that cause familial AF, as well as common genetic variants identified by genome-wide association studies (GWAS) that associate with AF in the general population. Many common variants are located in non-coding regions of the genome near transcription factors, implying a role for transcriptional regulation as a mechanism of AF susceptibility.³

Based on multiple GWAS, the strongest genetic hits consistently cluster on chromosome 4q25, near the gene for the paired-like homeodomain transcription factor 2, or PITX2.^4–7 This protein controls left-right asymmetry in the embryonic heart, while in the adult heart it is predominantly expressed in the left atrium, as the PITX2c isoform.^{8, 9} Based on data from human atrial samples, PITX2 expression is variable in the setting of AF, with a decrease or increase reported,^{10, 11} although decreased expression of PITX2 in left atrial cardiomyocyte nuclei was associated with AF recurrence post ablation.¹² In animal models, Pitx2 deficiency is consistently associated with increased AF susceptibility.^{9, 10, 13} The phenotype of reduced Pitx2 expression causing vulnerability to AF has been controversial, with variable reports of electrophysiologic and ultrastructural abnormalities, as well as transcriptional changes in genes involved in calcium handling, cell-cell junctions, ion channels, and bioenergetic processes that could exert detrimental effects.¹⁴ While cardiac expression of Pitx2 normally falls postnatally, existing evidence indicates that expression is increased with cardiac injury (e.g., myocardial infarction).¹⁵ In this setting, Pitx2 activates a set of stress response genes including reactive oxygen species (ROS) scavengers and electron transport chain components, to effect an antioxidant response that promotes cardiac repair.¹⁵ Indeed, with total loss of Pitx2 in mice, myocardial ROS production following injury is enhanced compared to WT animals, as these genes fail to be activated. There is also evidence that implicates Pitx2 in redox-mediated regulation in skeletal muscle during development.^{16, 17} Pitx2 has been shown to interact with Nrf2 to modulate oxidative stress in cardiomyocytes, while it directly regulates the expression of the superoxide dismutase genes Sod1 and Sod2, as well as genes encoding components of complex I and complex IV. Based on this evidence, we tested the hypothesis that oxidative stress constitutes a unifying mechanism whereby Pitx2 deficiency promotes AF susceptibility. In support of this hypothesis, chromosome 4q25 variants predict increased AF risk following cardiac surgery and in the early period following catheter ablation, conditions linked to inflammation and oxidative stress.^{18, 19}

A preferred cellular target for ROS is polyunsaturated fatty acids in the cell membrane and circulation. This lipid peroxidation process results in the generation of multiple compounds containing two C=O (carbonyl) groups, the most reactive of which are isolevuglandins (IsoLGs). IsoLGs and other dicarbonyl moieties such as malondialdehyde bind and adduct proteins, DNA, and other lipids to interfere with their function.²⁰ Highly-specific, small molecule scavengers of lipid dicarbonyls such as 2-hydroxybenzylamine (2-HOBA) have been developed (Supplemental Figure 1), and accumulating evidence in preclinical studies indicates that these reactive lipid oxidation products are responsible for a major component of oxidative injury in multiple experimental models.²⁰ Therefore, we further hypothesized that lipid dicarbonyls are critical mediators in this process.

Methods

An expanded version of the Methods is contained in the online Supplemental Material. The authors declare that all supporting data are available within the main article or the Supplemental Material.

Animals

For the experiments described below, mice heterozygous for a Pitx2-null allele that removes all isoform function^{9, 21} (Pitx2^+/−) were studied at 16 to 18 weeks of age, with age-matched WT littermates (Pitx2^+/+) serving as controls (Supplemental Figure 2). Male mice were studied, as preliminary experiments indicated that for Pitx2^+/− animals, only males demonstrated increased AF susceptibility with atrial pacing compared to Pitx2^+/+ mice.²² At weaning, mice were treated with either 2-HOBA (3 g/L) or 4-HOBA (1 g/L) in the drinking water, with water as vehicle control. Surface ECGs were recorded and AF inducibility was assessed during transesophageal pacing with operator blinding,²² while atrial cardiomyocytes were isolated using standard Langendorff methods.²³

Cardiac morphology and histology

Echocardiographic studies were performed using mice anesthetized with isoflurane, with data acquisition and analysis conducted in a manner blinded to genotype and treatment. Mice were sacrificed by isoflurane overdose and cervical dislocation, hearts were formalin fixed and paraffin embedded, and sections were stained with H&E, Masson’s trichrome, or Congo red.

Detection of superoxide (O₂^•−) and IsoLG adducts

Dihydroethidium (DHE) staining was employed to assess intracellular O₂^•− production in frozen myocardial sections.²⁴ Immunohistochemistry was performed using formalin-fixed paraffin-embedded myocardial tissues and a custom-made anti-IsoLG-lysyl adduct single-chain antibody (D11 ScFv^{25, 26}) with 3,3’-diaminobenzidine staining to detect IsoLG protein adducts.

mRNA quantification

Real-time quantitative RT-PCR was performed using left atrial (LA) samples from Pitx2^+/− and Pitx2^+/+ mice as described previously,²³ with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used for normalization in all experiments. Oligonucleotide sequences are provided in the Supplemental Material (Supplemental Table 1). Data analysis was performed using the ΔΔCT method.

Western blotting and immunofluorescence

Mitochondrial oxidative phosphorylation (OXPHOS) proteins in LA tissue lysates were analyzed by Western blotting. Fresh frozen myocardial LA tissue sections were used for Tom20 (the translocase of the outer mitochondrial membrane 20) immunofluorescence staining. FITC-conjugated wheat germ agglutinin was used to identify cardiomyocyte cell membranes. Immunofluorescence for connexin (Cx)40 and Cx43 was also performed with frozen atrial sections. Specific antibodies are listed in the Supplemental Material.

Transmission electron microscopy (TEM)

Following fixation with glutaraldehyde and paraformaldehyde, LA sections were imaged using TEM, with ultrastructural changes evaluated by a researcher blinded to sample identity.

High-resolution respirometry

Following euthanasia, the left and right atria were isolated and permeabilized, followed by measurement of oxygen consumption at baseline and after sequential addition of different mitochondrial complex substrates using a polarographic oxygen sensor (Oxygraph-2k, Oroboros Instruments).^{27, 28}

Electrophysiology

Action potentials and ionic currents were recorded from single atrial cells isolated from Pitx2^+/− and Pitx2^+/+ mice using current clamp and voltage clamp techniques, respectively, as described in detail in the Supplemental Material.

Statistics

Results are presented as mean ± SEM. Normality was assessed using primarily the Shapiro-Wilk (or Anderson-Darling) test. Statistical significance was determined by one-way or two-way ANOVA with Tukey’s post hoc test, or Kruskal-Wallis using Dunn’s post hoc test, using Graph Pad Prism software (Version 9) as appropriate. Differences with a P value < 0.05 were considered significant.

Ethics statement

All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services, and they were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Results

Pitx2^+/− mice display abnormal atrial conduction and AF susceptibility that is prevented by 2-HOBA

At baseline, Pitx2^+/− mice had a significantly longer P wave duration compared to Pitx2^+/+ littermates (Figures 1A–B), while other ECG parameters including PR, QRS, and QT intervals were comparable to WT (Figures 1C–E; Supplemental Table 2). To investigate the role of lipid dicarbonyls in AF susceptibility, Pitx2^+/− mice were treated at weaning with either 2-HOBA or an inactive structural analog 4-hydroxybenzylamine (4-HOBA) for 3 months, followed by transesophageal pacing (Figure 1F). Consistent with previous reports, Pitx2^+/− mice displayed a greater pacing-induced AF burden as well as the prevalence of sustained or any AF compared to Pitx2^+/+ mice (Figures 1F–I). Chronic treatment with 2-HOBA prevented AF vulnerability, while 4-HOBA had essentially no effect. There was a nonsignificant trend for reduced P wave duration in Pitx2^+/− mice receiving 2-HOBA, whereas 4-HOBA had no effect (Figure 1B). These findings indicate that reactive lipid dicarbonyl products of oxidative stress are important mediators of atrial conduction abnormalities and AF susceptibility in Pitx2^+/+ mice.

Figure 1. Pitx2^+/− mice demonstrate increased P wave duration and AF susceptibility that is reduced by chronic 2-HOBA treatment.

(A) Representative surface ECG waveforms in wild type (WT: Pitx2^+/+, vehicle [Veh] treated) and Pitx2^+/− mice in the absence (Veh) or presence of 2-hydroxybenzylamine (2-HOBA) or 4-hydroxybenzylamine (4-HOBA). Summary data are illustrated for P wave duration (B) and the duration of the PR (C), QRS (D), and QT (E) intervals. Data are presented as mean ± S.E.M., One-way ANOVA with Tukey’s post hoc test (B) or Kruskal-Wallis with Dunn’s post hoc test (C, D, E), n=54 for Pitx2^+/+; n=35, 24, and 17 for Pitx2^+/− mice treated with vehicle, 2-HOBA, or 4-HOBA, respectively; **P<0.01; ns=nonsignificant. (F) Representative ECG tracings during transesophageal pacing. Atrial pacing is present at the beginning of each strip, and the red arrows indicate the subsequent first spontaneous sinus P wave after pacing is terminated. (G) Inducible atrial fibrillation (AF) was quantitated as total or cumulative AF burden. Data are presented as mean ± S.E.M., Kuskal-Wallis with Dunn’s post hoc test; n=34 for Pitx2^+/+; n=24, 19, and 16 for Pitx2^+/− mice treated with vehicle, 2-HOBA, or 4-HOBA, respectively. % Mice with sustained AF (H) and % mice with any AF (I) are also shown. P values are calculated using Fisher’s exact test; **P<0.01; ***P<0.001; ****P<0.0001; n values are shown on the graph (numerator: number of mice with sustained or any AF; denominator: total mice studied).

Pitx2^+/− mice have normal cardiac function and morphology

A propensity to develop AF is often linked to compromised cardiac structure, especially abnormal left ventricular function and LA dilatation. In light of the observed atrial conduction abnormalities, we investigated the structural phenotype of Pitx2^+/− mice in detail. As previously reported,¹³ transthoracic echocardiography demonstrated normal left ventricular function and LA geometry in the Pitx2^+/− mice compared to WT controls (Figures 2A–E, Supplemental Table 3). In addition, the histologic architecture in the atria was comparable in the hearts of WT and Pitx2^+/− mice treated with either vehicle or 2-HOBA, with no evidence of obvious histologic abnormalities, fibrosis, or amyloid that would alter conduction (Figures 2F).

Figure 2. Cardiac morphology and ventricular function are similar in Pitx2^+/+ and Pitx2^+/− mice.

(A) Representative B-mode and M-mode echocardiographic images of Pitx2^+/+ mice, and Pitx2^+/− mice in the absence or presence of treatment with 2-hydroxybenzylamine (2-HOBA) or 4-hydroxybenzylamine (4-HOBA). Summary data are shown for heart rate (B), left ventricular ejection fraction (C), left ventricular fractional shortening (D), and anteroposterior LA diameter in diastole (E). Results are presented as mean ± S.E.M., One-way ANOVA with Tukey’s post hoc test; n values on bars. (F) Representative hematoxylin and eosin (H&E), Masson Trichrome (MT), and Congo red stained micrographs of left and right atrial sections are displayed from Pitx2^+/+ mice, and Pitx2^+/− mice in the absence and presence of 2-HOBA treatment (n≤5 mice for H&E and MT; n≤3 for Congo red). Scale bars =100 μm.

Pitx2^+/− mice demonstrate increased atrial O₂^•−, IsoLG adducts, and altered ROS-related gene expression that are ameliorated by 2-HOBA

Pitx2 encodes an antioxidant response following cardiac injury,¹⁵ and data from single-nuclei RNA-seq experiments have suggested that Pitx2-deficient hearts are oxidatively stressed.²⁹ To test this hypothesis, LA from Pitx2^+/− hearts were subjected to staining with DHE to detect O₂^•− and immunohistochemistry using an anti-IsoLG-lysyl adduct antibody (D11 ScFv) to assay for IsoLG adducts.²⁶ Compared to WT littermates, there was a marked increase in O₂^•− production in the Pitx^+/− LA, and this was ameliorated by treatment with 2-HOBA (Figure 3A). Similar results were also observed for IsoLG adducts, with enhanced production in the Pitx2^+/− mouse atria and decreased adduct formation following 2-HOBA treatment (Figure 3B). Importantly, these findings occurred in the absence of atrial inflammation, nor an increase in any atrial leukocyte subset (Supplemental Figure 3). In addition, Pitx2 deficiency resulted in significantly decreased expression of major antioxidant genes including Sod1 and Sod2 (Gpx1 and Nrf2 expression also trended toward a nonsignificant decrease) with normalized expression in the setting of chronic 2-HOBA treatment (Figure 3C). These findings demonstrate that oxidative stress is substantially increased in the Pitx2^+/− mouse atria, implying additional bioenergetic disturbances.

Figure 3. Oxidative stress and isolevuglandin (IsoLG) adducts are elevated in atrial myocardium of Pitx2^+/− mice, with suppression by 2-hydroxybenzylamine (2-HOBA).

Representative confocal microscopy images are shown for LA tissue from Pitx2^+/+ mice, and Pitx2^+/− mice ± 2-HOBA treatment after exposure to (A) dihydroethidium (DHE) to detect O₂^•− (using antimycin A as a positive control; scale bars=50 μm) or (B) D11 ScFv to assay for IsoLG adducts (primary antibody incubation step omitted for negative control); n=3 mice for each; scale bars=50 μm. (C) Expression of cellular antioxidant genes (Sod1, Sod2, Gpx1, and Nrf2; normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) are displayed for the experimental groups. Data are presented as mean ± S.E.M.; One-way ANOVA with Tukey’s post hoc test (n shown on the graph). *P<0.05; **P<0.01; ns=nonsignificant.

Mitochondrial bioenergetics are impaired in Pitx2^+/− mouse atria but improve with scavenging lipid dicarbonyls

Mitochondrial dysfunction has been increasingly recognized as an important mechanism in AF pathogenesis.³⁰ To test the hypothesis that Pitx2 deficiency alters mitochondrial bioenergetics in mouse atria, we performed high-resolution respirometry with oxygen flux measurements in permeabilized atrial tissue at baseline and following the sequential addition of substrates for complex I, long and medium chain fatty acid oxidation, and complex II. Compared to Pitx2^+/+ mice, atrial samples from Pitx^+/− mice revealed significantly lower mitochondrial respiration during utilization of ADP-supported long chain fatty acid oxidation, medium chain fatty acid oxidation, and complex II-mediated respiration, all of which were corrected by chronic 2-HOBA treatment (Figures 4A and 4B). These data indicate major impairment of mitochondrial oxidative phosphorylation in the atria of Pitx^+/− mice that is significantly improved by scavenging lipid dicarbonyls.

Figure 4. Mitochondrial bioenergetics are impaired in the atria of Pitx2^+/− mice but restored by dicarbonyl scavenging.

(A) Representative Oroboros Oxygraph respiratory traces in pooled left and right atrial tissue from Pitx2^+/+ mice, and Pitx2^+/− mice in the absence or presence of 2-hydroxybenzylamine (2-HOBA) treatment. The solid blue and broken red lines represent oxygen flux per mass (pmol/(s*mg) for Pitx2^+/+ mice and Pitx2^+/−, respectively. The solid and broken grey lines represent oxygen concentration (nmol/ml) for Pitx2^+/+ mice and Pitx2^+/−, respectively. Substrate abbreviations: GM= glutamate + malate, PC= palmitoyl + carnitine, OC= octanoyl + carnitine, and Succ= succinate. (B) Summary data indicate a reduction in respiration due to adenosine diphosphate (ADP)-supported long chain fatty acid oxidation, medium chain fatty acid oxidation, and complex II-mediated respiration in Pitx2^+/− compared to Pitx2^+/+ mice, with significant improvement with 2-HOBA treatment. Data are presented as mean ± SEM; Two-way ANOVA with Tukey’s post hoc test; n=5–9 mice for each group. *P<0.05; ****P<0.0001.

Remodeling of cardiac ultrastructure and gap junction proteins are present in Pitx2^+/− mouse atrium that improve with 2-HOBA treatment.

Given the abnormal mitochondrial bioenergetics, TEM was performed to investigate atrial ultrastructure in Pitx2^+/− mice. Compared to WT control, montage images revealed that the overall density of mitochondria in the atria of Pitx2 deficient mice was reduced (Figure 5A, top panel). Indeed, average mitochondria cross-sectional area in Pitx2^+/− atria was decreased compared to WT (Figure 5B) in the absence or presence of 2-HOBA treatment. This resulted from a reduction in average mitochondrial area in the Pitx2^+/− mice that trended to improve with 2-HOBA treatment (Figure 5C), while the number of mitochondria was unchanged (Figure 5D). The finding of decreased mitochondrial area was corroborated by Tom20 immunostaining, which identifies the mitochondrial outer membrane (Supplemental Figure 4A). However, this occurred with no apparent loss of mitochondrial OXPHOS proteins in Pitx2 deficient atria (Supplemental Figures 4B and 4C). At higher magnifications, Pitx2^+/− atria revealed mitochondrial cristae loss and increased cristae width, which were abrogated by 2-HOBA treatment (Figure 5A, middle and lower panel, and Figure 5E). In addition, there was widening of the intercellular space that was not present in either WT mice or Pitx2^+/− mice receiving 2-HOBA (Supplemental Figure 4D). Confocal imaging showed abundant Cx40 signal at the intercalated disks of control LA, but this was disrupted in the Pitx2^+/− mice, with numerous intercalated disks devoid of the typical Cx staining pattern (Figure 5F). Instead, there were areas of Pitx2^+/− atria where Cx40 was more laterally distributed. In 2-HOBA treated mice, Cx40 signals were observed mostly at the intercalated disk although evidence of lateralization was still present in some areas. Similar findings were seen when staining was performed for Cx43 in LA sections (Figure 5G). In the setting of Pitx2 deficiency, gene expression analysis revealed that mitochondrial biogenesis gene Tfam was reduced (Supplemental Figure 4F), with similar but non-significant trends for Pparg1a and Nrf1 (Supplemental Figures 4E and 4G), and this decrease was prevented by 2-HOBA. These findings demonstrate a key role for oxidative stress-mediated lipid dicarbonyls in disruption of mitochondrial and gap junction ultrastructure in the setting of Pitx2 deficiency.

Figure 5. 2-HOBA improves ultrastructural abnormalities that are present in the Pitx2^+/− left atria.

(A) Upper panel: Tiled montage TEM images from the LA ultrastructure of Pitx2^+/+ and Pitx2^+/− mice with or without 2-hydroxybenzylamine (2-HOBA) treatment. Scale bars=5 μm. Middle panel: High magnification photomicrographs of LA myocardium. Mitochondria (M, mitochondria; black arrow, mitochondria with degenerated cristae structures; yellow arrow, atrial granules; white arrow, focal vacuolization and dissolution of mitochondrial cristae). Scale bars=500 nm. Lower panel: Focus on the indicated mitochondria region shown in the middle panel. Scale bars=200nm. TEM photomicrographs from 2–3 mice were analyzed with FIJI software for: (B) mitochondrial cross-sectional area (n=336 for Pitx2^+/+, n=310 for Pitx2^+/− and n= 355 for Pitx2^+/−+2-HOBA, respectively); data are mean ±SD; Kruskal-Wallis test followed by Dunn’s post hoc analysis was used to determine statistical significance; *P<0.05, **P<0.01; (C) mitochondrial area per field was calculated from 8–9 photomicrographs from 2–3 mice; One-way ANOVA followed by Dunnett’s post hoc test was used to determine statistical significance; *P<0.05, ns=non-significant; (D) mitochondrial number in per field; ns=non-significant; (E) mitochondrial cristae width (n=210 for Pitx2^+/+, n=223 for Pitx2^+/− and n= 145 for Pitx2^+/−+2-HOBA, respectively); data are mean ± SD; Kruskal-Wallis test followed by Dunn’s was used to determine statistical significance; ****P<0.0001. Confocal microscopy demonstrates localization of gap junction proteins connexin 40 (Cx40, F) and connexin 43 (Cx43, G) in Pitx2^+/+ and Pitx2^+/− mouse left atrium (α-SA, sarcomeric α-actinin). Hoechst was used to visualize the nuclei.

Proarrhythmic electrophysiologic remodeling in Pitx2^+/− atrial myocytes is mediated by lipid dicarbonyls

Previous studies have demonstrated evidence of electrophysiologic remodeling in the Pitx2 deficient mouse atrium, although results have been conflicting.^{10, 13, 31} Accordingly, action potential (AP) recordings were obtained from single atrial myocytes isolated from WT mice as well as Pitx2^+/− mice treated with vehicle or 2-HOBA. Compared to WT, atrial myocytes from Pitx2 deficient mice display more positive resting membrane potentials (RMP), abbreviation of AP duration (APD at 90% repolarization, or APD₉₀), and a reduction in the maximum rate of rise of voltage during phase 0 (V_max; Figures 6A–D). Treatment with 2-HOBA prevented these changes, with parameter values that were essentially unchanged from WT controls (Figures 6A–D). The nature of this remodeling is predicted to be proarrhythmic, as a reduction in APD to reduce wavelength and slowing of conduction would both promote reentry. In addition, depolarization of the RMP would not only further slow conduction but also increase automaticity to promote premature beats.

Figure 6. Action potentials and I_K1 from Pitx2^+/− mouse atria demonstrate proarrhythmic remodeling that is mitigated by 2-HOBA.

(A) Representative atrial action potentials are illustrated from Pitx2^+/+ wild type (WT) mice and Pitx2^+/− mice in the absence or presence of 2-hydroxybenzylamine (2-HOBA) treatment. The dashed red line represents the resting membrane potential (RMP) under control conditions. Summary data demonstrate a rise in RMP (B), reduced action potential duration at 90% repolarization (APD₉₀) (C), and depressed maximum phase 0 upstroke velocity (V_max) (D) which are improved by chronic 2-HOBA treatment (not significant for RMP). n=28, 26, and 41 cells; One-way ANOVA with Tukey’s post hoc test or Kruskal-Wallis test followed by Dunn’s post hoc analysis as appropriate. (E) Representative K⁺ currents (voltage clamp protocol in the inset), (F) current-voltage (IV) relationships, and (G) peak current at −130mV are shown for I_K1 for each group. n=14, 11, and 7 cells; One-way ANOVA with Tukey’s post hoc test. *P<0.05; **P<0.01; ***P>0.001; NS=nonsignificant.

Oxidative stress-mediated ionic current modulation underlies electrophysiologic remodeling in Pitx2^+/− mice

The ionic basis for the observed electrical remodeling was investigated using voltage clamp techniques in single atrial myocytes. To explore the mechanism for elevated RMP, inward rectifier K⁺ currents (I_K1) were recorded. In comparison with WT, current amplitude was significantly decreased in Pitx2^+/− mice as illustrated by representative currents, the current-voltage (IV) relationship, and summary data at −130 mV in Figures 6E–G, respectively, and this was effectively prevented by 2-HOBA treatment. While the aberrant distribution of Cx40 and Cx43 could account for the reduction in V_max, cardiac Na⁺ currents were also examined. Indeed, both peak and late I_Na were significantly depressed in Pitx2 deficient mice without an obvious change in voltage dependence of the current-voltage relationship (Figures 7A–E), changes that were also abrogated by 2-HOBA. Although the reduction in late I_Na would shorten APD, additional experiments were performed to investigate other candidate currents. With respect to repolarizing K⁺ currents, sustained currents (I_sus) were increased in Pitx2^+/− mice compared to controls (Figures 7F–G), as was also the case for transient outward currents (I_To) (Supplemental Figure 5A), with a reduction in both currents by 2-HOBA treatment. On the other hand, there was no difference in L-type Ca⁺⁺ currents with Pitx2 deficiency compared to Pitx2^+/+ control mice (Supplemental Figure 5B). Taken together, the reduction in late Na⁺ current along with the increase in repolarizing K⁺ currents would effectively shorten APD to account for this effect on the cardiac AP.

Figure 7. Reduction in cardiac Na⁺ currents and increased I_sus in Pitx2^+/− atrial cells are prevented by chronic treatment with 2-hydroxybenzylamine (2-HOBA).

(A) Representative families of Na⁺ currents are illustrated (voltage clamp protocol in the inset; WT=wild-type). Summary data are shown for IV curves (B) and peak I_Na at −30mV (C). n=9, 10, and 15. (D and E) Similar findings for representative and peak currents are demonstrated for late I_Na (n=9, 10, and 15 cells) and sustained outward K⁺ current I_sus recorded using subtraction with 4-aminopyridine (4-AP) (F and G; n=10, 9, and 6 cells); One-way ANOVA with Tukey’s post hoc test; *P<0.05; **P<0.01; ***P>0.001; NS=nonsignificant.

Discussion

In this study, we demonstrate for the first time that oxidative stress plays a unifying role in AF susceptibility and proarrhythmic remodeling that occurs in the setting of Pitx2 deficiency. In addition, ROS-generated lipid dicarbonyls function as novel mediators in the pathologic process and can potentially be targeted pharmacologically by small molecule scavengers such as 2-HOBA. In Pitx2^+/− mice, we identified evidence of slowed atrial conduction manifested by increased P wave duration along with arrhythmia vulnerability, both of which were prevented by the lipid dicarbonyl scavenger 2-HOBA. The molecular basis for these findings included abnormal mitochondrial structure and function, as well as gap junction and ion channel remodeling causing arrhythmogenic action potential changes, effects largely abrogated by scavenging lipid dicarbonyls.

Several lines of evidence have linked Pitx2 to redox balance, although a definitive role in atrial arrhythmia susceptibility and proarrhythmic remodeling has not been previously reported. With total loss of Pitx2 in conditional knock-out (KO) mice, ventricular myocardial ROS was increased and animals developed greater scar in response to ischemic injury.¹⁵ In addition, a cooperative interaction has been identified between Pitx2 and the transcription factor Nrf2 to regulate an antioxidant gene response, including components of the electron transport chain and genes that protect the cell from increased ROS, such as Sod1 and Sod2.¹⁵ Moreover, recent evidence indicates that Pitx2 signaling protects against cardiomyocyte ferroptosis, a form of regulated cell death related to excessive lipid peroxidation.³² While ferroptosis may play a role in the atrial abnormalities of Pitx2^+/− mice, experiments to test this hypothesis are beyond the scope of the current investigation. Findings indicating a lack of inflammation in the Pitx2 deficient atria further supports aberrant transcriptional regulation as the cause of increased ROS under these circumstances. Prior studies have identified lipid dicarbonyls, in particular highly-reactive IsoLGs, as primary mediators of oxidative stress-related injury,²⁰ and these were indeed identified in Pitx2^+/− mouse atria. Overall, the protective effect of 2-HOBA provides strong evidence of a pivotal role for ROS in the pathophysiology of Pitx2 deficiency-dependent AF. In addition, the antioxidant N-acetylcysteine was previously found to reduce arrhythmias in a Pitx2^−/− zebrafish model.³³ Inflammation and oxidative stress play an essential role in hypertension, and 2-HOBA has also been shown to prevent AF susceptibility in murine models of hypertension and inflammation.^{28, 34}

There is increasing evidence that mitochondrial dysfunction per se is proarrhythmic. Using high-resolution respirometry in acutely-isolated Pitx2^+/− mouse atria, we found that myocardial bioenergetics were depressed, with reduced oxygen consumption, especially during fatty acid oxidation and complex II-dependent respiration. In addition, deterioration of mitochondrial structural integrity was evident by TEM, which is not surprising given the role of Pitx2 to regulate transcription of multiple electron transport chain components.¹⁵ Overall, mitochondrial mass was diminished, with reduced expression of Tfam. These findings are consistent with previous reports of disturbed mitochondrial architecture in conditional KO and zebrafish models having total loss of Pitx2,^{29, 33} but they have not been previously described in the setting of heterozygous deficiency. Interestingly, in P19 cells lacking Pitx2, oxygen consumption measured using the Seahorse platform was reduced, while expression of electron transport chain components was disrupted.²⁹ Moreover, PITX2-deficient human induced pluripotent stem cell-derived atrial cardiomyocytes displayed evidence of mitochondrial dysfunction despite variable and aberrant electrical remodeling.³⁵ Findings such as these indicate that Pitx2 is required to maintain normal mitochondrial structure and function.

While Pitx2 deficiency is widely accepted to promote AF susceptibility, the other components of the electrophysiologic phenotype have been controversial. To our knowledge, this is the first report of P wave prolongation in Pitx2^+/− mice, while a previous study using the same mice reported normal atrial conduction velocity and activation pattern.¹³ The age of the mice in the latter investigation was not reported and thus, this discrepancy with our results may be due to different ages at the time of study. On the other hand, Pitx2^−/− zebrafish, and a subset of Pitx2^+/− fish, displayed significant P wave prolongation, albeit this occurred in the setting of a cardiomyopathic phenotype with cardiac fibrosis.³³ Interestingly, in a GWAS study of over 64,000 individuals, an exome variant in PITX2 was associated with maximum P wave duration, further supporting a role for this transcription factor in atrial conduction.³⁶ An early investigation of Pitx2^+/− mice reported mild QRS prolongation,⁹ but we and others¹³ did not reproduce this finding.

In single Pitx2^+/− atrial myocytes, our results revealed unique findings of proarrhythmic electrical remodeling, with an elevated RMP, abbreviated APD, and reduction in V_max. The molecular basis for these action potential changes included a reduction in I_K1, peak I_Na, and late I_Na, an increase in I_sus and I_To, and widening/disruption of intercalated discs with Cx lateralization. With a conditional KO model, a more positive RMP was identified with loss of Pitx2, as well as decreased expression of Na⁺ and K⁺ channels including Scn5a and Kcnj2.¹⁰ Using an isolated perfused heart model of Pitx2^+/− mice, RMP was more depolarized than in WT controls while APD was abbreviated at short pacing cycle lengths, with no difference in V_max, peak Na⁺ currents, or I_K1 (although background K⁺ currents were reduced).^{13, 31} The mice in at least one of these studies were younger at 12–16 weeks of age, potentially accounting for the differences with our findings. Consistent with our data, a previous study using a conditional KO model has also reported abnormal, widened intercalated discs,³⁷ and Pitx2 deficiency has been linked to altered expression of genes related to cell-cell junctions, ion channel physiology, and Ca⁺⁺ handling.^{9–11, 13, 37, 38}

Limitations

We did not investigate the effects of 2-HOBA on Ca⁺⁺ handling with Pitx2 deficiency. Recent evidence indicates that the presence of a 4q25 risk variant was associated with increased Ca⁺⁺ sparks and RyR2 activity in human atrial myocytes.³⁹ Moreover, similar findings were obtained in atrial cells from Pitx2^+/− mice, with suppression of isoproterenol-mediated AF vulnerability by the selective RyR2 channel inhibitor, ent-verticilide.⁴⁰ Given that ROS is well recognized to cause RyR2 hyperactivity, it is likely that this mechanism also participates in AF susceptibility under these conditions. In this investigation, experiments were not performed to determine whether increased atrial ROS in Pitx2^+/− mice originated from both cardiomyocyte and non-myocyte cells, given that this has previously shown to be the case for neonatal myocardium.¹⁵ Due to cost considerations, atrial mRNA experiments were performed using 4 animals per group.

Conclusions

These findings support a critical role for oxidative stress, and specifically reactive lipid dicarbonyl mediators, in the proarrhythmic remodeling and AF susceptibility that occurs with Pitx2 deficiency. Recently, 2-HOBA has undergone Phase I clinical trials and is safe and well tolerated in humans.^{41, 42} Based on the efficacy of 2-HOBA in animal models of hypertension and inflammation-mediated AF,^{28, 34} the therapeutic strategy of targeting lipid dicarbonyls for AF prevention warrants further clinical investigation in not only genetically-susceptible patients, but those with other AF risk factors as well.

What is Known?

• Although chromosome 4q25 variants near the PITX2 gene are the strongest genetic risk factor for atrial fibrillation, the mechanism(s) causing this proarrhythmia remains controversial.

• Pitx2 encodes a transcription factor that activates an antioxidant response, and Pitx2-deficient mice are susceptible to atrial fibrillation.

What the Study Adds

• Pitx2 deficiency increases atrial oxidative stress, which promotes mitochondrial dysfunction and electrical remodeling to increase AF susceptibility, with a key role for highly-reactive dicarbonyl mediators of lipid oxidation in the pathophysiologic process.

• Scavengers of lipid dicarbonyls represent a novel therapeutic approach to prevent AF and may enable genotype-specific therapy.

Nonstandard Abbreviations and Acronyms

AF

atrial fibrillation

IsoLG

isolevuglandin

WT

wild type

2-HOBA

2-hydroxybenzylamine

ROS

reactive oxygen species

GWAS

genome-wide association study

DHE

dihydroethidium

TEM

transmission electron microscopy

Cx

connexin

4-HOBA

4-hydroxybenzylamine

AP

action potential

RMP

resting membrane potential

APD₉₀

action potential duration at 90% repolarization

V_max

maximum phase 0 upstroke slope

KO

knock-out

AVERP

atrioventricular effective refractory period

WCL

Wenckebach cycle length