In a mouse model of Dravet Syndrome, mitochondrial dysfunction may contribute to SUDEP.

Dravet syndrome (DS) is a severe, pediatric-onset epilepsy disorder linked to loss-of-function mutations in the sodium channel gene SCN1B. DS patients have a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). Cardiac arrhythmias have been implicated as a potential cause underlying SUDEP. An exact pathway for how mutations in SCN1B leads to arrhythmia in DS is unclear. One cellular component linked to regulation of cardiac homeostasis are mitochondria, known as “the powerhouse of the cell” due to their ability to produce cellular energy (ATP) via the electron transport chain (ETC). The ETC is a major producer of reactive oxygen species (ROS). Typically, ROS are buffered by cellular antioxidants, to prevent oxidative stress, an imbalance of ROS that can lead to cell damage. Our previous work indicates that cardiac arrhythmias may result from mitochondrial instability and imbalances between ROS production and buffering. We analyzed whether Scn1b-/-mice are susceptible to arrhythmias due to altered mitochondrial ATP generation, ROS production, and compromised cellular antioxidant defenses. We isolated cardiac mitochondria from postnatal day (P) 15-20 KO and Scn1b+/+ (WT) mice. To assess mitochondrial ATP and ROS production, high-resolution respirometry (O2k, Oroboros) was used to measure mitochondrial O2 and H2O2 flux. We used a substrate-uncoupler inhibitor (SUIT) protocol to elucidate flux under different ETC pathways, including Complex I- and II-linked respiration. As a next step, we evaluated expression of superoxide dismutase (Sod) proteins associated with mitochondrial antioxidant defenses, including Cu/Zn-Sod (Sod1) and Mn-Sod (Sod2) in hearts from KO and WT mice pre- (P10) and post- (P17) seizure development. After addition of substrates supporting Complex-II linked respiration (succinate, ADP) there were no differences in O2 flux between mitochondria isolated from KO and WT hearts. Upon further addition of pyruvate to mitochondria to stimulate Complex I, O2 flux was significantly reduced (p \u3c 0.0001) in mitochondria from KO mice, when compared to WT. Moreover, upon titration of rotenone (a Complex I inhibitor) its negative effect on O2 flux was not as substantial in KO mitochondria as in WT, suggesting that mitochondria from KO have deficits in Complex-I linked respiration. Furthermore, we detected significant differences in ROS production by mitochondria isolated from KO animals. Under conditions of reverse electron flow (succinate as substrate), a state where ROS production is highest, H2O2 flux was elevated significantly (p = 0.048) in mitochondria isolated from KO mice, compared to those isolated from WT. During our analysis of Sod expression, we found that Sod1 (p = 0.01) and Sod2 (p = 0.01) expression is significantly decreased at P17 in KO hearts compared to WT. Overall, our results suggest imbalances between mitochondrial activity and antioxidant defenses, which may underlie increased arrhythmia susceptibility in KO mice