Autoimmune Atrial Fibrillation

Abstract

Background: Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF.

Methods: Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization.

Results: A common autoantibody response against K_ir3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. K_ir3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K⁺ current, I_KACh. Functional studies on human induced pluripotent stem cell-derived atrial cardiomyocytes showed that anti-K_ir3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of I_KACh, both key mediators of AF. To establish a causal relationship, we developed a mouse model of K_ir3.4 autoimmunity. Electrophysiological study in K_ir3.4-immunized mice showed that K_ir3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF.

Conclusions: To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of K_ir3.4 autoantibody-mediated AF.

Keywords: G protein-coupled inwardly-rectifying potassium channel 4; atrial fibrillation; autoantibody; autoimmunity; inward rectifier potassium channel.

Figure 1.

Overview of analysis workflow. Diagram showing the analysis workflow of the present study: 37 patients with AF, 37 age- and sex-matched healthy controls, and 14 patients before the development of AF (pre-AF) were enrolled. Using peptide microarray, plasma samples were screened for the presence of autoantibodies targeting a panel of cardiac ion channels. The heat map of peptides was analyzed for immunoreactivity across all plasma samples. The identified autoantibody was then purified using affinity chromatography and tested on human atrial cardiomyocytes. Mice immunized with the target peptide produced autoantibodies to replicate the human condition. No structural heart abnormalities were evident, and electrophysiological in vivo study confirmed an increased AF susceptibility in immunized mice. AF indicates atrial fibrillation; EP, electrophysiological; iPS, induced pluripotent stem cell; and SR, sinus rhythm.

Figure 2.

Cardiac action potentials and I_KACh current recorded in human induced pluripotent stem cell–derived atrial cardiomyocytes (hiPSC-aCMCs). A, Representative action potential traces recorded in hiPSC-aCMCs under control condition, in response to 10 µmol/L carbachol and ±0.5 µg/mL anti-K_ir3.4 IgG. B, Mean action potential duration±SEM determined at 90% repolarization (APD₉₀) obtained in control cells (390.2±50.5 ms; n=6), in response to 10 µmol/L carbachol (307.2±24.0 ms; n=8), in the presence of 0.5 µg/mL anti-K_ir3.4 IgG (261.5±17.5 ms; n=8) vs 0.5 µg/mL anti-K_ir3.4 IgG in addition to 10 µmol/L carbachol (203.2±8.3 ms; n=8). Statistical significance was determined using 1-way ANOVA with Tukey multiple comparisons test. C, Representative I_KACh currents normalized to cell capacitance recorded in control cells, in response to 10 µmol/L carbachol, in the presence of 0.5 µg/mL anti-K_ir3.4 IgG with and without the addition of 10 µmol/L carbachol. The voltage step stimulation protocol is outlined. D, Mean current density-voltage relationships (±SEM) of cells under control condition (n=4), in response to 10 µmol/L carbachol (n=7), in the presence of 0.5 µg/mL anti-K_ir3.4 IgG with (n=5) and without the addition of 10 µmol/L carbachol (n=6). To record I_KACh, 10 µmol/L nifedipine (I_Ca,L inhibition), 2 mmol/L 4-aminopyridine (I_Kur and I_to inhibition), and 10 µmol/L ML-133 (I_K1 inhibition) were added to the external solution. All data were measured at 37 °C. Statistical significance was determined using Kruskal-Wallis followed by Dunn multiple-comparisons test. APD90 indicates action potential duration at 90% repolarization; I_Ca,L, L-type calcium current; I_K1, inward rectifier potassium current; I_KAch, acetylcholine-activated inwardly rectifying potassium current; I_Kur, ultrarapid delayed rectifier potassium channel; I_to, transient outward potassium current; K_ir3.4, inwardly rectifying potassium channel subunit 3.4.

Figure 3.

Electrophysiological phenotyping of mice with K_ir3.4 autoantibodies. A, Study design of the experimental autoimmune AF model with Balb/c mice. B, Representative surface ECG traces derived from limb leads I and II, recorded from a sham-immunized and K_ir3.4-immunized mouse. C, Bar graphs overlaid with dot plots present mean ECG interval values±SD recorded in sham- (n=14) and K_ir3.4-immunized mice (n=10). Statistical significance was determined using the Student t test (PR, QRS, QTc, and JTc) and Mann-Whitney U test (RR). D, Representative bipolar intracardiac electrogram recordings at the level of the right ventricle (RV) and right atrium (RA). STIM denotes right atrial stimulation. ECG in lead II configuration shows the corresponding surface ECG signals. E, Bar graphs overlaid with dot plots present mean intracardiac electrophysiological data±SD acquired in sham- (n=14) and K_ir3.4-immunized mice (n=10). Statistical significance was determined using the Student t test (SNRT, cSNRT, AERP, and AVERP) and Mann-Whitney U test (WCL). F, Bar graph shows the proportion of sham- (4 of 14) and K_ir3.4-immunized mice (8 of 10) with burst pacing–induced AF. Points indicate mean AF duration±SD (P=0.214). Statistical significance was determined by Mann-Whitney U test (AF duration), and Fisher exact test was used to assess the induced AF rate. AERP indicates atrial effective refractory period; AF, atrial fibrillation; AVERP, atrioventricular effective refractory period; cSNRT, corrected sinus node recovery time; EGM, electrogram; EPS, electrophysiological study; SNRT, sinus node recovery time; and WCL, Wenckebach cycle length.