Reduction in Junctophilin 2 Expression in Cardiac Nodal Tissue Results in Intracellular Calcium-Driven Increase in Nodal Cell Automaticity

Abstract

BACKGROUND: Spontaneously depolarizing nodal cells comprise the pacemaker of the heart. Intracellular calcium (Ca 2+ ) plays a critical role in mediating nodal cell automaticity and understanding this so-called Ca 2+ clock is critical to understanding nodal arrhythmias. We previously demonstrated a role for Jph2 (junctophilin 2) in regulating Ca 2+ -signaling through inhibition of RyR2 (ryanodine receptor 2) Ca 2+ leak in cardiac myocytes; however, its role in pacemaker function and nodal arrhythmias remains unknown. We sought to determine whether nodal Jph2 expression silencing causes increased sinoatrial and atrioventricular nodal cell automaticity due to aberrant RyR2 Ca 2+ leak. METHODS: A tamoxifen-inducible, nodal tissue-specific, knockdown mouse of Jph2 was achieved using a Cre-recombinase-triggered short RNA hairpin directed against Jph2 (Hcn4:shJph2). In vivo cardiac rhythm was monitored by surface ECG, implantable cardiac telemetry, and intracardiac electrophysiology studies. Intracellular Ca 2+ imaging was performed using confocal-based line scans of isolated nodal cells loaded with fluorescent Ca 2+ reporter Cal-520. Whole cell patch clamp was conducted on isolated nodal cells to determine action potential kinetics and sodium-calcium exchanger function. RESULTS: Hcn4:shJph2 mice demonstrated a 40% reduction in nodal Jph2 expression, resting sinus tachycardia, and impaired heart rate response to pharmacologic stress. In vivo intracardiac electrophysiology studies and ex vivo optical mapping demonstrated accelerated junctional rhythm originating from the atrioventricular node. Hcn4:shJph2 nodal cells demonstrated increased and irregular Ca 2+ transient generation with increased Ca 2+ spark frequency and Ca 2+ leak from the sarcoplasmic reticulum. This was associated with increased nodal cell AP firing rate, faster diastolic repolarization rate, and reduced sodium-calcium exchanger activity during repolarized states compared to control. Phenome-wide association studies of the JPH2 locus identified an association with sinoatrial nodal disease and atrioventricular nodal block. CONCLUSIONS: Nodal-specific Jph2 knockdown causes increased nodal automaticity through increased Ca 2+ leak from intracellular stores. Dysregulated intracellular Ca 2+ underlies nodal arrhythmogenesis in this mouse model.

WITHDRAWN: Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure

“…Similar observations were recently reported by Landstrom et al in tamoxifen-inducible, pacemaker tissue-specific (Hcn4-targeted), junctophilin 2 knockdown mice (Hcn4:shJph2). 33 Junctophilin 2 provides a structural bridge between the sarcolemma and SR membranes in atrial and ventricular working myocytes, facilitating the functional coupling between LTCCs and RyR2 at dyadic junctions. 34 However, it remains unknown whether junctophilin 2 serves a similar function in pacemaker cells.…”

Atrial cardiomyocytes contribute to the inflammatory status associated with atrial fibrillation in right heart disease

“… 43 Moreover, the knockdown of junctophilin-2 (JPH2), which is abundant in the T-tubules has been described as interacting with the Ca 2+ -handling machinery via the RYR2 by provoking Ca 2+ -leak from the sarcoplasmic reticulum and inducing increased automaticity and arrhythmogenesis. 44 Consistent with these observations, in our model of right-sided atrial remodelling, we discovered that JPH2 was significantly decreased in RA from RHD rats compared to sham ( Figure 10 ).…”

Junctional Ectopic Tachycardia Caused by Junctophilin-2 Expression Silencing Is Selectively Sensitive to Ryanodine Receptor Blockade

“… 11 , 12 , 13 We have previously demonstrated that Jph2 knockdown in murine nodal cells causes increased automaticity of nodal cells owing to Ca 2+ leak from the SR in Hcn4:shJph2 mice. 14 These findings support a potential pharmacologic target for correction of Ca 2+ dysregulation. 15 Recently, a novel compound, 2-(diethylamino) ethyl 4-(butylamino)-2-methoxybenzoate (EL20) has been shown to decrease Ca 2+ leak from the SR through targeting RyR2 in ventricular cardiac myocytes.…”

“…Compared with tamoxifen-treated control mice (Hcn4:WT mice), knockdown mice demonstrated an approximately 40% reduction of Jph2 transcript and protein expression. 14 These mice demonstrate no arrhythmias at rest nor inducible arrhythmias with intracardiac electrical stimulation, but rapid infusion of isoproterenol in ex vivo Langendorff perfused hearts consistently triggered a rapid AV-dissociated tachycardia; thus, we hypothesized that these mice could serve as an in vivo model of JET. 14 Initially, central venous isoproterenol and/or epinephrine injection via a customized lumened electrophysiologic catheter did not consistently trigger AV-dissociated tachycardia in vivo.…”

“… 14 These mice demonstrate no arrhythmias at rest nor inducible arrhythmias with intracardiac electrical stimulation, but rapid infusion of isoproterenol in ex vivo Langendorff perfused hearts consistently triggered a rapid AV-dissociated tachycardia; thus, we hypothesized that these mice could serve as an in vivo model of JET. 14 Initially, central venous isoproterenol and/or epinephrine injection via a customized lumened electrophysiologic catheter did not consistently trigger AV-dissociated tachycardia in vivo. Given the well established literature identifying temperature, mechanical “stretch” of the heart, and increased catecholamines as risk factors for the development of postoperative JET in children, 17 , 18 we sought to establish a novel methodology to simulate JET in mice ( Figures 1A and 1B ).…”