Abstract 16594: Disruption of Caveolae-Confined Phosphorylation of Ryanodine Receptors Promotes Ca 2+ Handling Abnormalities and Arrhythmogenesis in Mouse Atrial Myocytes

Abstract

Rationale: Chronic atrial fibrillation has been linked to ectopic triggered activity driven by delayed afterdepolarizations in response to sarcoplasmic reticulum Ca 2+ leak from certain areas in atria. These activities were associated with hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain uncertain. Here, we hypothesized that mouse atria have areas with lower transversal-axial tubule system (TATS) organization, where phosphorylation of RyRs is mainly confines by caveolae nanodomains and might be firstly disturbed in disease conditions. Methods and results: In wild type (WT) mouse intact atria stained with RH-237, we found that myocytes located in the intercaval region (ICR, between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum) have a significantly less density of TATS than right atrial appendage (RAA) myocytes: 5.7±0.4% in ICR vs. 13.4±0.9% in RAA, P<0.01. Also an elevated frequency of spontaneous Ca 2+ sparks was observed in myocytes isolated from ICR vs. RAA (12.5±2.6 vs. 1.5±0.3 sparks/μm/s, P<0.01). ICR myocytes isolated from mice with a cardiac-specific knockout of the main structural protein of caveolae, caveolin-3, showed significant increase of Ca 2+ sparks frequency as compared to WT (27.3±4.1 sparks/μm/s, P<0.01). Immunofluorescence staining of RyRs, phosphorylated at protein kinase A/ Ca 2+ /calmodulin-dependent protein kinase-II phosphorylation sites: Ser 2808 -RyR and Ser 2814 -RyR, showed predominant subsarcolemmal localization, with a high co-localization with caveolin-3, in ICR and RAA cells. Myocytes isolated from caveolin-3 knockout mice showed extension of both Ser 2808- RyR and Ser 2808- RyR to whole-cell wide striated pattern in ICR, but not RAA. This redistribution was associated with development of delayed afterdepolarizations in ICR but not RAA cells. Conclusions: Our findings demonstrate that in ICR in mouse atria, caveolae form functional, spatially-confined cAMP nanodomains and control localized RyR phosphorylation. Disruption of caveolae structures, seen in various pathologies, may lead to hyperphosphorylation of RyRs promoting Ca 2+ handling abnormalities and arrhythmogenic triggered activity.