Abstract 15686: Electron Tomography and Live Tissue Confocal Imaging Reveal Contrasting Ultrastructural Abnormalities in Calcium Release Units of CPVT Mouse Models Due to Calsequestrin and Ryanodine Receptor Mutations

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a disease characterized by life-threatening ventricular arrhythmias (VA). Major CPVT mutations involve two proteins of the calcium release unit (CRU): the junctional sarcoplasmic (jSR) membrane ryanodine receptor (RyR 2 ) and jSR luminal Ca 2+ binding protein calsequestrin (CASQ 2 ). Hypothesis: CRU ultrastructure variations are mutations specific. Methods: Ultrastructural analysis of CRU including RyR 2 , jSR, T- tubules (TT) their interface and mitochondrial dynamics in Casq 2-/- and RyR2 A4860G+/- mouse CPVT models by 3D electron tomography and confocal imaging of immune-labeled tissue. Results: CASQ2 mutation: Casq 2-/- null mutation did not affect docking of jSR to TT or to surface membrane but the jSR appeared empty without Casq2 densities and large size variability (Fig B) as compared to WT (Fig A). Size variations are likely due to parallel changes in presence of two proteins that mediate CASQ 2 -to-RyR 2 connection: Triadin and Junctin. No mitochondrial abnormalities were observed. RyR 2 mutation: Abnormal RyR 2 function did not affect RyR 2 , jSR counts or TT morphology or their interface. However, we observed an unusual mitochondrial response: the extrusion of extended nanotunneling (NT) at a frequency 3 fold greater than in WT (Fig C). While enhancing long range inter-mitochondrial signal transfer, immunostaining showed that NTs actually slow down the rate of inter-mitochondrial exchanges (by 50%). 3-D tomograms of rapidly frozen tissue demonstrated that NTs are part of an extensive mitochondrial network. Tomograms and immunostaining showed direct proximity between microtubules and mitochondria with no major rearrangement of microtubule architecture. Conclusions: While functional abnormal Ca 2+ release from CRUs may underlie arrhythmogenesis, abnormal mitochondrial disposition and inter-mitochondrial signal transfer may play an important, yet unrecognized role.