Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization

Abstract

Objectives: This study sought to study the role of junctophilin-2 (JPH2) in atrial fibrillation (AF).

Background: JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca(2+) handling and modulation of ryanodine receptor Ca(2+) channels (RyR2). Whereas defective RyR2-mediated Ca(2+) release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias.

Methods: Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudoknock-in (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation.

Results: PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice expressing a hypertrophic cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca(2+) release events. These changes were attributed to reduced binding of E169K-JPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca(2+) spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients and an increased frequency of spontaneous Ca(2+) release events.

Conclusions: Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2/RyR2 ratios can promote SR Ca(2+) leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.

Keywords: AF; Ca(2+); DAD; EAD; HCM; JMC; JPH2; NCX; NTg; Na(+)/Ca(2+) exchanger; PKI; RyR2; SR; Tg; WT; atrial fibrillation; calcium; delayed afterdepolarizations; early afterdepolarizations; hypertrophic cardiomyopathy; junctional membrane complex; junctophilin; junctophilin-2; nontransgenic; pseudoknock-in; ryanodine receptor type 2; ryanodine receptor; sarcoplasmic reticulum; transgenic; wild type.

Figure 1. E169K-JPH2 is associated with HCM and paroxysmal atrial fibrillation (pAF)

Pedigree showing presence of HCM and supraventricular arrhythmias [pAF, sino-atrial (SA) block, supraventricular tachycardia (SVT)] in proband (arrow) and his father (grey square), both mutation positive (heterozygous G>A), and his sister who was genotype negative (white circle) as indicated by the asterisk.

Figure 2. Higher susceptibility to pacing-induced AF in E169K-PKI but not A399S mice

A, Representative ECG tracings after rapid atrial burst pacing in E169K-PKI mouse revealing AF, compared with sinus rhythm in WT-PKI mouse and A399S-PKI mouse. B, Bar graph showing average duration of AF episodes following rapid atrial pacing bursts. Number of incidences inside bars. C, Bar graph showing increased incidence of pacing-induced AF (reproducible, and lasting > 1 s) in E169K-PKI mice compared with WT-PKI and A399S-PKI mice. Number of mice per group inside bars. *P<0.05.

Figure 3. Enhanced SR Ca²⁺ leak in E169K-PKI but not A399S-PKI mouse myocytes

A, Representative confocal line-scan images and profiles showing spontaneous cell-wide Ca²⁺ waves in atrial myocytes from E169K-PKI mice following 1 Hz field stimulation. B, Representative confocal line-scan images showing increased incidence of Ca²⁺ sparks in atrial myocytes from E169K-PKI mice following 1-Hz field stimulation. C–D, Bar graphs showing quantification of Ca²⁺ wave (SCR) and Ca²⁺ spark frequencies (CaSpF). *P<0.05.

Figure 4. JPH2 mutant E169K but not A399S reduces JPH2-RyR2 binding and JPH2 levels inversely correlate with AF inducibility

A, Western blots showing reduced JPH2 co-immunoprecipitation following RyR2 pull-down from cardiac lysates of E169K-PKI mouse heart compared with WT-PKI, A399S-PKI and NTg mice. B, Bar graph showing quantification of JPH2 co-immunoprecipitated with RyR2 pull-down. C, Western blots showing total JPH2 levels compared with GAPDH in atria from MCM-shJPH2-WT-Tg mice that overexpressed higher cardiac JPH2 levels (WT-Tg), control non-Tg MCM mice with normal JPH2 levels (NTg), or MCM-shJPH2 knockdown mice with reduced cardiac JPH2 levels (JPH2-KD). Bar graphs showing JPH2 levels normalized to GAPDH in these 3 groups, on a log scale. D, Stacked bar graph showing AF incidence after atrial burst pacing in the 3 groups of mice. Number of mice per group inside bars. *P<0.05, **P<0.01, ***P<0.001.

Figure 5. JPH2-derived small peptide stabilizes RyR2-mediated SR Ca²⁺ leak in atrial myocytes from JPH2 knockdown mice

A, Representative confocal line scan images showing spontaneous Ca²⁺ sparks recorded from permeabilized atrial myocytes treated with vehicle only (upper left), JPH2 peptide at 1 μM (upper right), JPH2 peptide at 10 μM (bottom left), and scrambled control peptide (10 μM, bottom right). B, Bar graph showing Ca²⁺ spark frequency (CaSpF), which was significantly decreased by JPH2-derived peptide but not by scrambled control peptides. **P<0.01, ***P<0.001. Numbers indicate number of cells (number of mice). C, Representative planar lipid bilayer RyR2 single channel tracings from Mer-Cre-Mer (Control) and JPH2 knockdown (JPH2-KD) mice at baseline (top) and with the addition of JPH2 peptide (bottom). D, Bar graph showing significant increase in RyR2 open probability (Po) in JPH2-KD vs control and significant reduction in RyR2 Po with the addition of JPH2 peptide in JPH2-KD mice. P <0.05

Figure 6. Reduced JPH2:RyR2 ratios in patients with paroxysmal AF

Representative Western blots (A) and bar graphs (B–C) showing RyR2 and JPH2 protein levels normalized to calsequestrin (CSQ) in atria from patients in sinus rhythm (SR) and paroxysmal AF (pAF). (D) Ratio of JPH2-to-RyR2 levels in human atria. Representative Western blots (E) and bar graphs (F–G) showing SERCA2a and PLB levels normalized to CSQ.

Figure 7. Enhanced pro-arrhythmic Ca²⁺ release events in patients with paroxysmal AF

A, Representative Ca²⁺ transient and membrane current (I_m) tracings from isolated atrial myocytes from SR and pAF patients following 1 min of pacing at 0.5-Hz. B, Bar graph showing incidence of spontaneous Ca²⁺ release events (SCR). C, Frequency (left), latency (middle), amplitude (right) of SCR. D, Amplitude of SCR-mediated NCX current. *P<0.05, **P<0.01. Numbers represent number of cells/number of patients.