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Scn1b plays essential roles in the heart, where it encodes β 1 -subunits that serve as modifiers of gene expression, cell surface channel activity, and cardiac conductivity. Reduced β 1 function is linked to electrical instability in various diseases with cardiac manifestations and increased susceptibility to arrhythmias. Recently, we demonstrated that loss of Scn1b in mice leads to compromised mitochondria energetics and reactive oxygen species (ROS) production. In this study, we examined the link between increased ROS and arrhythmia susceptibility in Scn1b -/- mice. In addition, ROS-scavenging capacity can be overwhelmed during prolonged oxidative stress, increasing arrhythmia susceptibility. Therefore, we isolated whole hearts and cardiomyocytes from Scn1b -/- and Scn1b +/+ mice and subjected them to an oxidative challenge with diamide, a glutathione oxidant. Next, we analyzed gene expression and activity of antioxidant enzymes in Scn1b -/- hearts. Cells isolated from Scn1b -/- hearts died faster and displayed higher rates of ROS accumulation preceding cell death compared with those from Scn1b +/+ . Furthermore, Scn1b -/- hearts showed higher arrhythmia scores and spent less time free of arrhythmia. Lastly, we found that protein expression and enzymatic activity of glutathione peroxidase is increased in Scn1b -/- hearts compared with wild type. Our results indicate that Scn1b -/- mice have decreased capability to manage ROS during prolonged oxidative stress. ROS accumulation is elevated and appears to overwhelm ROS scavenging through the glutathione system. This imbalance creates the potential for altered cell energetics that may underlie increased susceptibility to arrhythmias or other adverse cardiac outcomes. NEW & NOTEWORTHY Using an oxidative challenge, we demonstrated that isolated cells from Scn1b -/- mice are more susceptible to cell death and surges in reactive oxygen species accumulation. At the whole organ level, they were also more susceptible to the formation of cardiac arrhythmias. This may in part be due to changes to the glutathione antioxidant system.

Dravet Syndrome (DS) is a pediatric-onset epilepsy with an elevated risk of Sudden Unexpected Death in Epilepsy (SUDEP). Most individuals with DS possess mutations in the voltage-gated sodium channel gene Scn1a , expressed in both the brain and heart. Previously, mutations in Scn1a have been linked to arrhythmia. We used a Scn1a -/+ DS mouse model to investigate changes to cardiac mitochondrial function that may underlie arrhythmias and SUDEP. We detected significant alterations in mitochondrial bioenergetics that were sex-specific. Mitochondria from male Scn1a -/+ hearts had deficits in maximal ( p = 0.02) and Complex II-linked respiration ( p = 0.03). Male Scn1a -/+ mice were also more susceptible to cardiac arrhythmias under increased workload. When isolated cardiomyocytes were subjected to diamide, cardiomyocytes from male Scn1a -/+ hearts were less resistant to thiol oxidation. They had decreased survivability compared to Scn1a +/+ ( p = 0.02) despite no whole-heart differences. Lastly, there were no changes in mitochondrial ROS production between DS and wild-type mitochondria at basal conditions, but Scn1a -/+ mitochondria accumulated more ROS during hypoxia/reperfusion. This study determines novel sex-linked differences in mitochondrial and antioxidant function in Scn1a -linked DS. Importantly, we found that male Scn1a -/+ mice are more susceptible to cardiac arrhythmias than female Scn1a -/+ mice. When developing new therapeutics to address SUDEP risk in DS, sex should be considered., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chad R. Frasier reports financial support was provided by National Institute of Neurological Disorders and Stroke. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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 < 0.001). In 50 nM TERI-treated hearts, we also observed a marked increase in the severity of contracture (p < 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans., Methods: To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants., Results: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles., Conclusion: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.