Abstracts

Rationale: Spreading depolarization (SD) is a wave of cellular depolarization that transiently silences local neuronal function. We have recently shown that SD in the dorsal medulla occurs following recurrent seizures and leads to lethal cardiorespiratory arrest in SUDEP mouse models. Deletion of Kv1.1 and Scn1a were found to lower threshold for brainstem SD. SUDEP victims often carry multiple gene mutations and the discovery of genetic modifiers that modulate SD threshold should help to more precisely identify patients at risk. The ryanodine receptor 2 (RYR2) is an intracellular Ca2+ channel, and leaky RYR2 mutations have been reported in SUDEP victims. Leaky RYR2 mutations are known to increase risk of deleterious arrhythmia of heart and aberrant neuronal excitability in brain. We hypothesized that a leaky RYR2 mutation may contribute to sudden death not only by affecting cardiac function, but also by modifying autonomic neuronal excitability and lowering SD threshold in the autonomic brainstem.Methods: Mutant mice carrying a RYR2 mutation (R176Q, hereafter RQ) were examined. This mutation was identified in patients with arrhythmogenic right ventricular dysplasia and increases the channel open probability. Spontaneous cortical-cardiac activities were monitored by chronic EEG/ECG recording. SD threshold was evaluated both in vivo anesthetized mice (topical KCl application) and in acute cortical and brainstem slice models (KCl microinjection and oxygen glucose deprivation). The effect of the RYR2 inhibitor JTV-519 was tested in some experiments.Results: The RQ mutant mice showed frequent cortical EEG spikes and bradycardia/asystoles, suggesting unstable autonomic tone. No spontaneous seizure was detected in adult mice, and spontaneous death was rare. Consistent with increased cortical hyperactivity, the number of cortical SD episodes triggered by KCl application was significantly increased in the RQ mice. In vitro, OGD exposure triggered SD significantly faster in acute cortical slices prepared from the RQ mutant mice than in WT slices. Similarly, SD incidence in brainstem slices prepared from juvenile RQ mice (P16-21) also showed faster SD onset than their littermate controls. Acute exposure to the RYR2 inhibitor JTV-519 (5 µM) did not reverse the lower SD threshold. Thus the lowered SD threshold due to RQ mutation may arise from the developmental effects of chronic RYR2 dysfunction, rather than a direct contribution of intracellular Ca2+ release.Conclusions: This study revealed that a leaky RYR2 mutation is sufficient to cause cortical and cardiac arrhythmias. The leaky RYR2 mutation was not sufficient to create epilepsy in this model, but significantly lowed SD threshold in the brainstem cardiorespiratory centers. The lowered SD threshold in brainstem suggests that a leaky RYR2 mutation may increase risk of sudden death not only through cardiac dysfunction, but also by lowering brainstem SD threshold to initiate lethal cardiorespiratory deregulation in epilepsy.