Beavers, David L.

Background: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young people. Recently, a patient with HCM with paroxysmal atrial fibrillation (AF) and sinoatrial block was found to have a missense mutation, E169K, in the junctophilin 2 (JPH2) protein. Loss of JPH2 has been shown to affect intracellular Ca2+ release in the heart. To date, no study has evaluated the effect of E169K mutation on Ca2+ handling and arrhythmogenesis. Hypothesis: The JPH2 E169K mutation promotes sarcoplasmic reticulum Ca2+ leak causing abnormal atrial conduction and AF. Methods and Results: We evaluated our hypothesis by generating 3 mice that overexpressed 1) JPH2 with E169K mutation (EK-Tg), 2) WT JPH2 (WT-Tg) and 3) JPH2 with inactive P584S mutation (PS-Tg). On Western Blot, JPH2 expression was increased by 2-3 fold in all the mouse groups compared to non-transgenic controls. Echocardiography at 6 months of age revealed normal fractional shortening in all mice. At baseline, EK-Tg mice had a shorter RR interval (104±1 ms, n=10) vs. WT-Tg (123±5 ms, n=8, p=0.001) and PS-Tg mice (121±5 ms, n=5, p<0.001). EK-Tg mice also had a shorter PR interval (36±1 ms, n=10) vs. WT-Tg (41±1 ms, n=8, p=0.01) and PS-Tg mice (39±1 ms, n=5, p=0.15). On intracardiac electrophysiology studies, EK-Tg mice had shorter AVERP (39±2 ms, n=10) vs. WT-Tg (51±2 ms, n=8, p=0.005) and PS-Tg mice (48±3 ms, n=5, p=0.05). On overdrive pacing of the atria in the presence of epinephrine and caffeine, EK-Tg mice had a 57% incidence of AF (n=10) vs. 21% in WT-Tg (n=8, p<0.05) and 33% in PS-Tg mice (n=5, p=0.2). Isolated cardiomyocytes from EK-Tg mice had a higher Ca2+ spark frequency (6.7±0.8 events/100 um/s, n=20) vs. cardiomyocytes from WT-Tg (1.8±0.5 events/100 um/s, n=10, p<0.001) and PS-Tg mice (3.3±1.0 events/100 um/s, n=16, p<0.05). Lastly, JPH2 levels were lower in RyR2 channels immunoprecipitated from EK-Tg mice vs. RyR2 from WT-Tg mice (65% vs. 100%, p<0.05). Conclusion: The JPH2 mutation, E169K, leads to reduced binding of JPH2 with RyR2, increased SR Ca2+ leak, abnormal atrial conduction and AF. The data suggest that JPH2 may serve as a critical stabilizing molecule for RyR2; further approaches to stabilize this interaction may lead to improved control of AF in susceptible populations.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development.