Abstracts

Rationale: Sudden Unexpected Death in Epilepsy (SUDEP) is one of the most common causes of death in individuals with epilepsy. While the exact cause of SUDEP is unknown, cardiac arrhythmias are implicated as a mechanism contributing to SUDEP in Dravet Syndrome (DS). This study investigated if cardiac mitochondrial energetics are altered in the Scn1a^+/- mouse model of DS. We hypothesized that mitochondrial respiration and H₂O₂ production are compromised in Scn1a^+/- mouse hearts, leading to cardiac arrhythmias. In addition to measuring mitochondrial respiratory chain activity, we also analyzed its composition. Enzymes of the respiratory chain reversibly assemble into large, supramolecular complexes, known as “supercomplexes.” Supercomplexes are believed to boost mitochondrial efficiency, by increasing mitochondrial respiration and decreasing H₂O₂ production.

Methods: We first isolated mitochondria from Scn1a^+/- and Scn1a^+/+ hearts. Using high-resolution respirometry (O2k-Oroboros), we concurrently measured mitochondrial oxygen and H₂O₂ flux under different electron transport pathway states in our mitochondrial preparations. Lastly, we assessed the presence of different supercomplexes subtypes in our mitochondrial preparations, by isolating different protein complexes using a BN-PAGE gel.

Results: Initially, upon investigation of cardiac mitochondrial respiration in Scn1a^+/- and Scn1a^+/+, no differences were detected. However, when we separated data by sex, we found deficits in respiration between our male Scn1a^+/- and Scn1a^+/+ cohorts. Mitochondrial respiration was decreased in male Scn1a^+/- hearts after the addition of substrates supporting Complex II-linked respiration (succinate + ADP; 1.35-fold). These findings were confirmed through the addition of a Complex I inhibitor, rotenone, which still led to significantly decreased respiration (1.26-fold) in Scn1a^+/-. However, counter to our results concerning respiration, mitochondrial H₂O₂ production was not significantly different between hearts from Scn1a^+/- and Scn1a^+/+, or when comparing individual sexes. To assess potential sources of respiration deficits in male Scn1a^+/- hearts, we assessed supercomplex formation in our isolated mitochondria preparations. In Scn1a^+/- hearts, the expression of several supercomplex subtypes, especially those containing Complex I and III, was reduced compared to Scn1a^+/+.

Conclusions: Our results suggest that males of the Scn1a^+/- mouse model of DS possess distinct mitochondrial deficits, with significant impairments to Complex II-linked respiration that was not visible in female Scn1a^+/- mice. These sex differences may be due to more disruptable supercomplexes, ultimately leading to reduced mitochondrial efficiency. Ultimately, these alterations to mitochondrial function may underlie an increased risk of cardiac arrhythmias and SUDEP. Future experiments will further elucidate the relationship between sex and mitochondrial function in the Scn1a^+/- model.

Funding:

This work was supported by NIH grant R21NS116647 to C.R.F.