Modulation of Abnormal Sodium Channel Currents in Heart and Brain: Hope for SUDEP Prevention and Seizure Reduction

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1 Current Literature In Basic Science Modulation of Abnormal Sodium Channel Currents in Heart and Brain: Hope for SUDEP Prevention and Seizure Reduction Scn2a Deletion Improves Survival and Brain Heart Dynamics in the Kcna1-Null Mouse Model of Sudden Unexpected Death in Epilepsy (SUDEP) Mishra V, Karumuri BK, Gautier NM, Liu R, Hutson TN, Vanhoof-Villalbal SL, Vlachos I, Iasemidis L, Glasscock E. Hum Mol Genet 2017;26: People with epilepsy have greatly increased probability of premature mortality due to sudden unexpected death in epilepsy (SUDEP). Identifying which patients are most at risk of SUDEP is hindered by a complex genetic etiology, incomplete understanding of the underlying pathophysiology and lack of prognostic biomarkers. Here we evaluated heterozygous Scn2a gene deletion (Scn2a +/ ) as a protective genetic modifier in the Kcna1 knockout mouse (Kcna1 / ) model of SUDEP, while searching for biomarkers of SUDEP risk embedded in electroencephalography (EEG) and electrocardiography (ECG) recordings. The human epilepsy gene Kcna1 encodes voltage-gated Kv1.1 potassium channels that act to dampen neuronal excitability whereas Scn2a encodes voltage-gated Nav1.2 sodium channels important for action potential initiation and conduction. SUDEP-prone Kcna1 / mice with partial genetic ablation of Nav1.2 channels (i.e., Scn2a +/ ; Kcna1 / ) exhibited a two-fold increase in survival. Classical analysis of EEG and ECG recordings separately showed significantly decreased seizure durations in Scn2a +/ ; Kcna1 / mice compared with Kcna1 / mice, without substantial modification of cardiac abnormalities. Novel analysis of the EEG and ECG together revealed a significant reduction in EEG ECG association in Kcna1 / mice compared with wild types, which was partially restored in Scn2a +/ ; Kcna1 / mice. The degree of EEG ECG association was also proportional to the survival rate of mice across genotypes. These results show that Scn2a gene deletion acts as protective genetic modifier of SUDEP and suggest measures of brain heart association as potential indices of SUDEP susceptibility. Cardiac Arrhythmia in a Mouse Model of Sodium Channel SCN8A Epileptic Encephalopathy Frasier CR, Wagnon JL, Bao YO, McVeigh LG, Lopez-Santiago LF, Meisler MH, Isom LL. Proc Natl Acad Sci U S A 2016 Oct 26. Patients with early infantile epileptic encephalopathy (EIEE) are at increased risk for sudden unexpected death in epilepsy (SUDEP). De novo mutations of the sodium channel gene SCN8A, encoding the sodium channel Nav1.6, result in EIEE13 (OMIM ), which has a 10% risk of SUDEP. Here, we investigated the cardiac phenotype of a mouse model expressing the gain of function EIEE13 patient mutation p.asn1768asp in Scn8a (Nav1.6-N1768D). We tested Scn8a N1768D/+ mice for alterations in cardiac excitability. We observed prolongation of the early stages of action potential (AP) repolarization in mutant myocytes vs. controls. Scn8a N1768D/+ myocytes were hyperexcitable, with a lowered threshold for AP firing, increased incidence of delayed afterdepolarizations, increased calcium transient duration, increased incidence of diastolic calcium release, and ectopic contractility. Calcium transient duration and diastolic calcium release in the mutant myocytes were tetrodotoxin-sensitive. A selective inhibitor of reverse mode Na/Ca exchange blocked the increased incidence of diastolic calcium release in mutant cells. Scn8a N1768D/+ mice exhibited bradycardia compared with controls. This difference in heart rate dissipated after administration of norepinephrine, and there were no differences in heart rate in denervated ex vivo hearts, implicating parasympathetic hyperexcitability in the Scn8a N1768D/+ animals. When challenged with norepinephrine and caffeine to simulate a catecholaminergic surge, Scn8a N1768D/+ mice showed ventricular arrhythmias. Two of three mutant mice under continuous ECG telemetry recording experienced death, with severe bradycardia preceding asystole. Thus, in addition to central neuron hyperexcitability, Scn8a N1768D/+ mice have cardiac myoycte and parasympathetic neuron hyperexcitability. Simultaneous dysfunction in these systems may contribute to SUDEP associated with mutations of Scn8a. Epilepsy Currents, Vol. 17, No. 5 (September/October) 2017 pp American Epilepsy Society 306

2 Commentary Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality with approximately 2,000 deaths per year in the United States and 15% of all epilepsyassociated mortality (1). Data from patients included in the mortality in epilepsy monitoring unit study (MORTEMUS) suggest that seizures disrupt normal respiratory and cardiac physiology leading to death. General risk factors for SUDEP include: an early age of epilepsy onset, a long duration of epilepsy, drug-refractory epilepsy, nocturnal seizures, and generalized tonic clonic seizures (2). However, the development of patient-specific prognostic biomarkers for SUDEP is needed to help identify patients at risk. This will enable personalized seizure management and SUDEP prevention. At least nine neurocardiac genes that may contribute to SUDEP susceptibility have been identified in animal and human studies, suggesting specific genes may have clinical utility as genomic biomarkers of SUDEP risk. Specifically, mutations in voltage-gated sodium channel (VGSC) genes are now known to be involved in the pathogenesis of epilepsy, especially epileptic encephalopathies, and lethal arrhythmias potentially associated with SUDEP based on detailed molecular genetic and physiological studies in heart and brain (3). Although the majority of mutations have been found in the SCN1A gene, variants in several other sodium channel genes have a significant role in epilepsy and may contribute to cardiac dysfunction predisposing patients to SUDEP. SCN1A mutations encoding the sodium channel Nav1.1 have been identified in generalized epilepsy with febrile seizures plus (GEFS+) and in severe myoclonic epilepsy of infancy (SMEI), also known as Dravet syndrome. Other sodium channel mutations associated with seizure disorders include SCN2A, SCN3A, SCN9A, and SCN8A. Recurrent mutations at SCN8A residues Arg1617 and Arg1872 (among other SCN8A gain of function mutations) result in Nav1.6 channel hyperactivity due to impaired transition from open state to inactivated state, that is, the predominant pathogenic mechanism in infantile epileptic encephalopathy (EIEE13; 4). Missense mutations of SCN2A have been identified in patients with phenotypes ranging from benign familial neonatal-infantile seizures (BFNIS), autism, GEFS+, and SMEI. SCN2A mutations demonstrate different clinical severities based on the associated biophysical function. For example, mice heterozygous for a Scn2a knockout allele were not reported to exhibit seizures (5), while Scn2a Q54 mice have seizures as a consequence of an abnormal neuronal persistent sodium current conducted by a mutant Nav1.2 transgene and decreased survival (6). Interactions between genetic variants have been described in animal models (e.g., double heterozygous mice inheriting mild alleles of Scn2a and Kcnq2) that demonstrate severe myoclonic seizures (7). Observed modifier effects in animal studies provide models for potential gene interactions underlying common human polygenic epilepsies and directions for future treatment development. Kcna1 encodes voltage-gated Kv1.1 potassium channels important in reducing neuronal excitability whereas the Scn2a encoded voltage-gated Nav1.2 sodium channels are important for initiation and conduction of action potentials (APs). Mishra and colleagues tested whether decreasing neuronal excitability via a partial Scn2a loss-of-function (Scn2a +/ ) would offset the increased excitability caused by Kcna1 loss-of-function (Kcna1 / ) to lower seizure burden and reduce predisposition to cardiac arrhythmia and death. Scn2a +/ ; Kcna1 / mice exhibited a significant two-fold increase in survival (i.e., 79% lived to 10 weeks) in contrast to premature death observed in Kcna1 / mice and perinatal lethality in Scn2a / mice. Scn2a +/ mice without deficient potassium channels had normal survival rates. Western blots confirmed approximately 50% decrease in Nav1.2 subunit protein expression in hippocampal, cortical, and brainstem homogenates from Scn2a +/ mice relative to wildtype (WT)-control mice. These results suggest a half dose of Nav1.2 is sufficient to promote survival in Kcna1 / SUDEP mice. Nav1.2 reduction decreases seizure durations in Kcna1 / mice but not seizure frequency or flurothyl-induced seizure threshold. Seizure burden, a direct reflection of seizure duration (i.e., total time seizing per hour), was significantly lower in Scn2a +/ and Kcna1 / mice relative to WTs. Overall, these results suggest that heterozygous Scn2a deletion decreases the incidence of premature death in Kcna1 / mice, in part due to suppression of seizure activity once it is generated without preventing seizure initiation. To address postictal generalized EEG suppression associated with cardiorespiratory dysfunction and SUDEP, power spectral densities were compared for short (<31 s), intermediate (31 60 s), and long-duration (>60 s) seizures during preictal and postictal epochs. Regardless of genotype, spectral ranges were distributed similarly across seizures of short and intermediate length. During long-duration seizures from Kcna1 / mice, postictal power in the delta frequency band increased, and both alpha and beta bandwidths decreased postictally relative to the preictal baseline. This is consistent with the finding that postictal EEG slowing is a characteristic of severe, tonic-clonic seizures (8), a seizure-type that places the patient at higher risk for SUDEP. Simultaneous EEG-ECG recordings were used to evaluate cardiac phenotypes and potential modifier effects of SCN2A deletion on Kcna1 / mice. Nav1.2 reduction does not ameliorate cardiac phentoypes in Kcna1 / mice. Kcna1 / mice demonstrate an increased rate of skipped heart beats (i.e., atrioventricular [AV] conduction blocks) as do Scn2 +/ mice, and Scn2 +/ ; Kcna1 / mice relative to WT mice. Heart rate, SDNN (a measure of autonomic variability), and RMSSD (a measure of sympathetic tone) do not differ across genotypes, suggesting that the protective effects of Nav1.2 reduction are not dependent on parasympathetic control of the heart. Correlations between cardiac measures (e.g., R-R intervals) and EEG signal complexity were assessed to better understand changes in brain heart dynamics. In general, WT mice exhibited a negative correlation between the duration of cardiac R-R intervals and EEG signal complexity (i.e., entropy in alpha and beta bands). No association was apparent between the EEG and ECG signals in Kcna1 / mice, but SCNA deletion partially restored the brain heart association. The authors suggest survival rate is directly proportional to the level of brain heart association based on a linear regression analysis and that this measure may be a useful physiological biomarker of SUDEP risk. Mishra et al. dissected potential phenotypic causation within a complex genetic landscape to identify a positive genetic modifier, providing valuable insight into disease pathophysiology and related SUDEP risk. SCN2A may represent 307

3 a potential gene target for therapeutic intervention, but the authors acknowledge that they did not address excitability at the cellular level in this study and potential changes in biophysical properties that may occur in both heart and brain when selectively targeting voltage-sensitive sodium channels. The Scn8a N1768D/+ gain of function mutation for Nav1.6, a major sodium channel in the CNS and PNS known to regulate AP initiation and propagation in both excitatory and inhibitory neurons, was developed as a model of EIEE13, which carries a 10% risk of SUDEP (9). This particular mutation was initially identified in a proband with seizure onset at 6 months of age and who eventually succumbed to SUDEP at 15 years old, and in vitro experiments demonstrated an increase in neuronal excitability with this mutation due to increased persistent sodium current (10). This was followed up by in vivo work utilizing this same mouse model demonstrating seizures and SUDEP in the Scn8a N1768D/+ mouse (11). In their current work, Frasier et al. examined the cardiac phenotype of myocytes derived from these mice, as well as the underlying cardiac pathologies. Scn8a, Scn1a, and Scn3a mrna, which encode the three tetrodotoxin-sensitive cardiac VGSC, are expressed at very low levels, accounting for about 1% of the VGSC in the heart. Levels of these transcripts, as well as that of Scn5a, which encodes the tetrodotoxin-resistant Nav1.5, the major cardiac VGSC, were not altered in Scn8a N1768D/+ mice versus WT nor was sodium current altered in ventricular myocytes. Examination of ventricular myocyte morphology did not show a change in cell length, width, or cell capacitance with the N1768D mutation compared to control mice. Examination of cardiac AP morphology revealed prolonged early phases of AP repolarization, with no change in total duration of the AP, suggesting an effect on intracellular calcium signaling in ventricular myocytes from Scn8a N1768D/+ mice, as well as a lower threshold for AP firing. Additionally, the presence of arrhythmogenic substrates was indicated by an elevated incidence of delayed afterdepolarizations (DAD). Further investigation of intracellular calcium handling in Scn8a N1768D/+ myocytes revealed an enhanced calcium transient duration and a higher incidence of diastolic calcium release (DCR) events, both of which were blocked by inhibition of TTX-S VGSCs, implicating current through Nav1.6. The authors further showed inhibition of reverse mode Na/Ca exchange prevented the occurrence of DCR events without affecting the calcium transient duration. Measures of myocyte contractility were unchanged in cells from Scn8a N1768D/+ mice; however, the authors did find an increased occurrence of ectopic beats. As Nav1.6 is expressed at the transverse tubules (t-tubules) of the heart, the authors conclude that the Scn8a N1768D/+ mice demonstrate hyperexcitable myocytes as a result of enhanced persistent sodium current at the t-tubules that may enhance calcium influx through reverse mode Na/Ca exchange which then results in increased incidence of DAD and DCR, and the occurrence of ectopic beats. This is reminiscent of recent work by this group in dissociated neurons from this same mouse model showing increased neuronal excitability associated with elevations in sodium current, and aberrant firing patterns in hippocampal brain slices (12), indicating mutations in VGSC have similar effects in brain and heart that contribute to epilepsy and arrhythmias which may then culminate in SUDEP. In vivo ECG measurements of anesthetized mice demonstrated bradycardia in Scn8a N1768D/+ mice, with no difference in the heart rate from ex vivo denervated hearts, suggesting enhanced parasympathetic activity in Scn8a N1768D/+ mice. The authors further probed cardiac arrhythmias and alterations in autonomic tone in Scn8a N1768D/+ mice by administering norepinephrine and caffeine to mimic a catecholaminergic surge and found the occurrence of premature ventricular contractions and brief periods of tachycardia. Telemeters were implanted in three Scn8a N1768D/+ and two WT mice to measure ECG activity, and the terminal events of two Scn8a N1768D/+ mice were recorded. Severe bradycardia prior to asystole was found in both Scn8a N1768D/+ mice, with a worsening atrioventricular block 1 hour before death in one mouse. In their investigation into the cardiac phenotype of the Scn8a N1768D/+ mouse, Frasier et al. utilized both cellular mechanistic experiments in myocytes as well as in vivo whole animal physiology measurements to demonstrate increases in parasympathetic activity and changes in cardiac excitability resulting in bradycardia and ventricular arrhythmias and eventual death in this mouse model of EIEE13. This suggests SCN8A is another neurocardiac gene implicated in SUDEP, and patients with mutations in this VGSC should be monitored for increased risk for cardiac arrhythmias. The studies by Mishra and Frasier both implicate alterations in VGSC expression or currents in the underlying neurocardiac mechanisms of SUDEP. Collectively, the hyperexcitability of the PNS and the t-tubules of the heart with increased activity of Nav1.6 demonstrated by Frasier et al. as well as the findings of Mishra et al. that reduced Nav1.2 expression results in suppression of seizure activity, increased survival, and normalization of brain heart interactions support the possibility that methods to decrease sodium currents in cardiac tissue and excitatory neurons may reduce both seizure susceptibility and the risk for SUDEP. These findings are consistent with studies in acquired and genetic models of epilepsy and SUDEP. Recently, in the rat kainate model of acquired epilepsy, increases in sodium current through neuronal sodium channels were found in cardiac tissue, and this was associated with enhanced expression of neuronal Nav1.1 in myocytes (13). Bradycardia and other cardiac abnormalities known to occur with SUDEP were found in two separate studies utilizing different mouse models of Dravet syndrome with haploinsufficiency of Scn1a; however, one group implicated loss of Scn1a in GABAergic forebrain interneurons but not cardiac tissue in the cardiac arrhythmias (14), whereas Auerbach et al. found enhanced excitability of myocytes as well as increased Nav1.5 current in this model (15). Compensatory increases of other VGSC subtypes in cardiac tissue have also been shown in studies utilizing global Scn1b KO mice, another SUDEP model. Abnormal ECG recordings were associated with greater peak and persistent sodium currents as well as elevated levels of Nav1.5 mrna and protein in ventricular myocytes (16), and more recently the same group demonstrated increased TTX-S sodium current and levels of neuronal Scn3a mrna, suggesting enhanced expression of Nav1.3 (17). Interestingly, two recent papers have highlighted the antiepileptic effects of two drugs known to inhibit cardiac 308

4 sodium channels (18, 19). Ranolazine, a specific inhibitor of persistent sodium currents and an FDA-approved drug for angina pectoris, reduced seizure frequency by approximately 50% in the gain of function Scn2a Q54 mouse, however GS967, a novel compound with greater inhibition of persistent sodium currents, decreased seizure frequency by more than 90% and increased survival, in addition to preventing neuron loss in the dentate hilus and suppressing mossy fiber sprouting in the dentate gyrus (18). A follow-up study by the same group showed GS967 also reduced spontaneous seizures and increased survival in Scn1a +/- mice, a Dravet syndrome model, effects that were shown to involve changes in sodium current density, decreased in pyramidal neurons and enhanced in bipolar neurons. These effects on neuronal excitability were associated with decreased expression of hippocampal Nav1.6, with no change in Nav1.1 and Nav.1.2. Treatment with lamotrigine, a non subtype-specific inhibitor of VGSC, generally had the opposite effect on these measures, including a worsening of seizures in the Scn1a +/- mice, which is consistent with a clinical observation in patients with Dravet syndrome (20). It is known that conventional sodium channel blockers, which are thought to be nonspecific for VGSC subtypes, exacerbate seizures in certain types of epilepsy, such as Dravet syndrome (20, 21). The results found in the studies by Mishra et al. and Frasier et al. using genetic manipulation of VGSC subtypes, as well as those shown with GS967 via inhibition of persistent sodium currents, highlight the therapeutic potential of inhibitors of specific VGSC subtypes or certain types of sodium currents to suppress seizures and reduce SUDEP risk. However, the clinical efficacy of more specific VGSC inhibitors will depend on the continuing evolution of precision medicine and expansion of these methodologies to common clinical practice. Further, the studies by Mishra et al. and Frasier et al. involve monogenic models of SUDEP, although the genetic factors are likely to be much more complex. The presence of an additional genetic variant or an environmental factor such as certain AEDs, noncompliance with AEDs, stress, sleep disturbance, or frequent severe seizures may act to predispose patients to arrhythmias and SUDEP (22). Increased utilization of whole genome screening will enable the identification of all genetic variants versus screening for mutations in a few candidate genes known to cause epilepsy. Another limitation of the two studies highlighted here is the lack of respiratory monitoring in the mice undergoing SUDEP, and no evaluation of central respiratory circuits that likely rely on these same sodium and potassium channels. Nonetheless, the focus on cardiac mechanisms is a reasonable place to start given the arrhythmogenic potential of mutations in these ion channels. Future work may also involve the generation of neurons from fibroblasts of patients with epilepsy, which will allow clinicians to record from neuronal cells and test candidate drugs within the context of the patient's specific channelopathies and other genetic variants (23). Implementation of precision medicine practices of these types for patient care will provide greater recognition of those at increased risk for SUDEP, as well as additional information as to the optimal medications to suppress seizures while decreasing susceptibility to SUDEP. By Lindsey B. Gano, PhD and Heidi L. 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