Decreased Reactive Oxygen Species Buffering Capacity May Underlie Arrhythmia Susceptibility in the Scn1b-/- Mouse Model of Dravet Syndrome

Dravet syndrome (DS) is a severe pediatric-onset epilepsy disorder that mostly arises from loss-of-function mutations in voltage-gated sodium channel genes. Patients with DS have an elevated risk (~17%) of Sudden Unexpected Death in Epilepsy (SUDEP), a fatal complication of seizure disorders. While the exact physiological mechanisms underlying SUDEP remain unclear, cardiac arrhythmias have been heavily implicated. Previous work indicates that reactive oxygen species (ROS) accumulation during oxidative stress, a condition where ROS generation exceeds antioxidant capacity, can lead to mitochondrial instability and cardiac arrhythmias. The purpose of this investigation was to determine how ROS scavenging and antioxidant levels may be altered in the Scn1b-/- mouse model of DS. In the heart, the primary antioxidant pathway is the glutathione (GSH) system. This study tested the hypothesis that Scn1b-/- (KO) mice are more susceptible to arrhythmias due to a decreased ability to buffer ROS accumulations during prolonged oxidative stress through the GSH system. First, qPCR analysis was conducted on hearts from KO and Scn1b+/+ (WT) mice to determine differences in gene expression of key enzymes in the GSH system, glutathione peroxidase (Gpx) and glutathione reductase (Gsr). Gpx is responsible for the reduction of H2O2 to H2O. Gsr maintains GSH in the reduced state. In P17 (after seizure onset) KO mice, Gpx expression was decreased (0.27-fold; p = 0.03) but Gsr remained unchanged. The enzymatic activity of GPx and GR were also measured. Counter to the qPCR data, the results suggested that there are no differences in enzyme activity in KO mice. To determine if there were differences at the cellular level, isolated cardiac cells were subjected to a prolonged oxidative challenge. Fluorescence microscopy was used to assess changes in the signal of CM-DCF, a fluorescent ROS indicator, following the addition of 80 mM diamide to cells. Diamide causes oxidative stress by depleting intracellular stores of GSH. Cells isolated from KO mice subject to prolonged oxidative stress die at much faster rates than cells isolated from WT mice. In addition, death occurs at earlier timepoints (as early as 1 min). The mean time to death in KO cells was significantly shorter (p = 0.03) and occurred on average 4.25 minutes faster than in WT cells. In addition, CM-DCF signals intensified shortly before cell death, indicating ROS accumulation precedes cell death. Finally, to test for arrhythmia susceptibility at the whole organ level, isolated hearts were perfused with diamide and scored for arrhythmia susceptibility and severity. Over the first 15 and 30 minutes of perfusion KO hearts had significantly higher scores. In conclusion, the results of this study indicate that hearts from Scn1b-/- mice have a decreased capability to handle ROS accumulations during prolonged oxidative stress. This may be the result of deficits in the GSH system that compromise ROS scavenging in the heart

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

Teriflunomide Treatment Exacerbates Cardiac Ischemia Reperfusion Injury in Isolated Rat Hearts

PURPOSE: Previous work suggests that Dihydroorotate dehydrogenase (DHODH) inhibition via teriflunomide (TERI) may provide protection in multiple disease models. To date, little is known about the effect of TERI on the heart. This study was performed to assess the potential effects of TERI on cardiac ischemia reperfusion injury. METHODS: Male and female rat hearts were subjected to global ischemia (25 min) and reperfusion (120 min) on a Langendorff apparatus. Hearts were given either DMSO (VEH) or teriflunomide (TERI) for 5 min prior to induction of ischemia and during the reperfusion period. Left ventricular pressure, ECG, coronary flow, and infarct size were determined using established methods. Mitochondrial respiration was assessed via respirometry. RESULTS: Perfusion of hearts with TERI led to no acute effects in any values measured across 500 pM-50 nM doses. However, following ischemia-reperfusion injury, we found that 50 nM TERI-treated hearts had an increase in myocardial infarction (p \u3c 0.001). In 50 nM TERI-treated hearts, we also observed a marked increase in the severity of contracture (p \u3c 0.001) at an earlier time-point (p = 0.004), as well as reductions in coronary flow (p = 0.037), left ventricular pressure development (p = 0.025), and the rate-pressure product (p = 0.008). No differences in mitochondrial respiration were observed with 50 nM TERI treatment (p = 0.24-0.87). CONCLUSION: This study suggests that treatment with TERI leads to more negative outcomes following cardiac ischemia reperfusion, and administration of TERI to at-risk populations should receive special considerations