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Mice are routinely used to investigate molecular mechanisms underlying the atrial fibrillation (AF) substrate. We sought to optimize transesophageal rapid atrial pacing (RAP) protocols for the detection of AF susceptibility in mouse models. Hypertensive and control C57Bl/6J mice were subjected to burst RAP at a fixed stimulus amplitude. The role of parasympathetic involvement in pacing-related atrioventricular (AV) block and AF was examined using an intraperitoneal injection of atropine. In a crossover study, burst and decremental RAP at twice diastolic threshold were compared for induction of AV block during pacing. The efficacy of burst and decremental RAP to elicit an AF phenotype was subsequently investigated in mice deficient in the lymphocyte adaptor protein (Lnk-/-) resulting in systemic inflammation, or the paired-like homeodomain 2 transcription factor (Pitx2+/-) as a positive control. When pacing at a fixed stimulus intensity, pacing-induced AV block with AF induction occurred frequently, so that there was no difference in AF burden between hypertensive and control mice. These effects were prevented by atropine administration, implicating parasympathetic activation due to ganglionic stimulation as the etiology. When mice with AV block during pacing were eliminated from analysis, male Lnk-/- mice displayed an AF phenotype only during burst RAP compared to controls whereas male Pitx2+/- mice showed AF susceptibility during burst and decremental RAP. Notably, Lnk-/- and Pitx2+/- females exhibited no AF phenotype. Our data support the conclusion that multiple parameters should be used to ascertain AF inducibility and facilitate reproducibility across models and studies.

Background: With aging, the human atrium invariably develops amyloid composed of ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide). Preamyloid oligomers are the primary cytotoxic species in amyloidosis, and they accumulate in the atrium during human hypertension and a murine hypertensive model of atrial fibrillation susceptibility. We tested the hypothesis that preamyloid oligomers derived from natriuretic peptides cause cytotoxic and electrophysiological effects in atrial cells that promote arrhythmia susceptibility and that oligomer formation is enhanced for a mutant form of ANP linked to familial atrial fibrillation. Methods: Oligomerization was assessed by Western blot analysis. Bioenergic profiling was performed using the Seahorse platform. Mitochondrial dynamics were investigated with immunostaining and gene expression quantitated using RT quantitative polymerase chain reaction. Action potentials and ionic currents were recorded using patch-clamp methods and intracellular calcium measured using Fura-2. Results: Oligomer formation was markedly accelerated for mutant ANP (mutANP) compared with WT (wild type) ANP. Oligomers derived from ANP, BNP, and mutANP suppressed mitochondrial function in atrial HL-1 cardiomyocytes, associated with increased superoxide generation and reduced biogenesis, while monomers had no effects. In hypertensive mice, atrial cardiomyocytes displayed reduced action potential duration and maximal dV/dT of phase 0, with an elevated resting membrane potential, compared with normotensive mice. Similar changes were observed when atrial cells were exposed to oligomers. mutANP monomers produced similar electrophysiological effects as mutANP oligomers, likely due to accelerated oligomer formation, while ANP and BNP monomers did not. Oligomers decreased Na + current, inward rectifier K + current, and L-type Ca ++ current, while increasing sustained and transient outward K + currents, to account for these effects. Conclusions: These findings provide compelling evidence that natriuretic peptide oligomers are novel mediators of atrial arrhythmia susceptibility. Moreover, the accelerated oligomerization by mutANP supports a role for these mediators in the pathophysiology of this mutation in atrial fibrillation.

Introduction: The strongest genetic risk factor for atrial fibrillation (AF) is variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene Pitx2 . Mice deficient in Pitx2 ( Pitx2 +/- ) have increased AF susceptibility, although the mechanism(s) remains controversial. Recent evidence indicates that with cardiac injury, Pitx2 encodes an antioxidant gene program that promotes repair. Isolevuglandins (IsoLGs) are highly reactive lipid peroxidation products that mediate a major component of oxidative stress-related injury. We used a small molecule scavenger of IsoLGs to test the hypothesis that oxidative stress is enhanced in the setting of Pitx2 deficiency to cause AF susceptibility. Methods: Pitx2 +/- and Pitx2 +/+ (wild type littermate control) mice were treated orally with either vehicle or the IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) starting at weaning. At age 16-18 weeks, animals underwent transesophageal atrial pacing, echocardiography, and tissue harvest or flow cytometry to measure atrial inflammation. Results: Pitx2 +/- mice demonstrated a significant increase in inducible AF burden compared to control mice (242.9±105.3 vs 23.6±11 sec; n=7, 14; P <0.05). There was also a trend for an increased incidence of sustained AF (71.4% vs 21.4; P=0.055). Flow cytometry revealed that there was no increase in the number of total leukocytes in the atria of Pitx2 +/- mice compared to control atria, nor were differences present in selected populations of immune cells (including CD3, CD19, NK1.1, F4/80, Ly6G, or CD11b/MHCII positive cells). For Pitx2 +/- mice treated with 2-HOBA, there was trend for a reduction in AF burden (39.6±14.4 sec; n=11; P =0.075) as well as sustained AF (27.2%), while the drug had no effect in control mice. Based on cardiac histology (n=5, 5) and echocardiography (n=11, 14), no major histologic, structural, or functional abnormalities were identified in Pitx2 +/- mice. Conclusions: The reduction in AF burden by the IsoLG scavenger 2-HOBA supports the hypothesis that enhanced oxidative stress is responsible for AF susceptibility in the setting of Pitx2 deficiency. These results suggest a potential role for genotype-specific AF therapy (in 4q25 variant carriers) using IsoLG scavengers.

Introduction: Inflammation and oxidative stress are linked to multiple risk factors for atrial fibrillation (AF), and to AF itself in the setting of sterile injury (e.g. after catheter ablation or cardiac surgery). However, anti-inflammatory therapies and conventional antioxidants cause adverse effects or are ineffective to prevent AF. Highly reactive mediators of lipid peroxidation such as isolevuglandins (IsoLGs) have been identified as a major component of oxidative stress-related injury. We hypothesized that during AF promoted by cardiac inflammation, a scavenger of IsoLG will decrease AF susceptibility. Methods: We studied mice with a systemic inflammatory phenotype due to deficiency in the lymphocyte adaptor protein ( Lnk -/- ), a negative regulator of cytokine signaling. At weaning, Lnk -/- mice and their wild-type (WT) littermates received either vehicle or a potent IsoLG scavenger, 2-hydroxybenzylamine (2-HOBA), by oral administration. At age 14 weeks, animals underwent transesophageal burst pacing, echocardiography, and tissue harvest or flow cytometry to measure atrial inflammation and IsoLG-adducts. Results: Cardiac histology and echocardiography revealed no major histologic or structural abnormalities in Lnk -/- mice. Nevertheless, Lnk -/- mice demonstrated a significant increase in AF burden compared to WT controls (124.8±43.3 vs 6.8±3 sec, respectively [mean±SEM, n=28, 12; P<0.05]), as well as increased sustained AF (> 30 sec; 48.1% vs 0%; P<0.01]). Leukocyte infiltration was present in the atria of Lnk -/- mice, with a significant increase in CD3, CD19, NK1.1, and CD11b/MHCII positive cells, compared to atria from WT control mice. Furthermore, there was a 2 to 4-fold increase in IsoLG-adducts for Lnk -/- atrial immune cells positive for CD3, CD19, NK1.1 and CD11b/MHCII, compared to cells from WT atria. Lnk -/- mice treated with 2-HOBA had significantly reduced AF burden (4.7±4.5 sec, n=7; P<0.05) with a trend towards reduction in sustained AF (0%, n=7; P=0.057). Conclusions: IsoLGs play a critical role in the pathogenesis of inflammation-mediated AF, and 2-HOBA, a scavenger of IsoLGs, represents a potentially novel therapeutic strategy for AF in this clinical setting.