Structural and Functional Alteration of RyR Clusters After Remodeling in Persistent Atrial Fibrillation

Abstract

In chronic atrial fibrillation (AF), abnormalities in Ca2+ release from RyR have been implicated as major factors contributing to arrhythmia and contractile dysfunction, but the relation to RyR organization remains unknown. Using STED microscopy we examined RyR cluster morphology in isolated atrial myocytes from sheep with persistent AF (N=6, 16-23 weeks of AF) and age matched control (Ctrl) animals; in parallel experiments we measured Ca2+ sparks in permeabilized myocytes. STED measurements revealed RyR clusters typically contained 15 contiguous RyR and on average this did not differ between AF and Ctrl. However, the nearest neighbor distance between clusters was reduced in AF. Grouping of clusters within 150 nm as functional Ca2+ release units (CRU) indicated that in AF these units exhibit increased fragmentation, with more clusters per functional unit. Measurement of Ca2+ sparks in permeabilized myocytes revealed a >50% increase in spark frequency and a higher prevalence of macrosparks. Spark time-to-peak (TTP) and duration were also increased, but width was reduced. Measurement of the intrinsic cellular buffer capacity showed this was reduced in AF. Using computational modeling it was found that the increased TTP and duration can be ascribed to the increased number of clusters per functional grouping in AF. The observed increased CRU fragmentation and reduction in Ca2+ buffering, can increased occurrence of sparks and macrosparks in AF. In conclusion, ultrastructural reorganization of RyR clusters within the functional units contributes to overactive Ca2+ release with increased chance of propagating events in AF.