Abstract
King DR, Demirtas M, Tarasov M, Struckman HL, Meng X, Nassal D, Moise N, Miller A, Min D, Soltisz AM, Anne MNK, Alves DPA, Wagnon JL, Weinberg SH, Hund TJ, Veeraraghavan R, Radwański PB. JACC Clin. Electrophysiol. 2024;10(5):829–842. doi:10.1016/j.jacep.2024.01.003. Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss-of-function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear. In this study, the authors sought to provide evidence implicating remodeling of Na+- and Ca2+-handling machinery, including NaV 1.6 and Na+/Ca2+ exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias. The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS. DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+ release channels, the ryanodine receptors (RyR2), and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death. These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca2+-mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.
Vanoye CG, Abramova TV, DeKeyser J-M, Ghabra NF, Oudin MJ, Burge CB, Helbig I, Thompson CH, George AL. JCI Insight 2024; 9(12). doi:10.1172/jci.insight.177530. Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
Commentary
On the road to personalized medicine, it is difficult to know if we are following the right path. This is particularly important when developing treatments to postpone or prevent sudden death in epilepsy (SUDEP). Clinically, the incidence of mortality due to SUDEP is increased in people with Dravet syndrome (DS) in two age ranges: children under 4 years of age, and between the ages of 10–20 years. 1 Because previous studies have mostly focused on SUDEP in pediatric DS populations, it is unknown whether the etiology of SUDEP differs between the younger and older ages.
But, before we dive further into the importance of age, we first need to introduce two salty characters, voltage-gated sodium channels (Nav)1.1 and Nav1.6. Abnormalities in Nav channels, including those that cause a loss of Nav1.1 channel function or a gain of Nav1.6 channel function, associate with a spectrum of genetic epilepsies and encephalopathies. 2 The Nav1.1 channel alpha subunit is encoded by the SCN1A gene (A for “alpha subunit” which makes the sodium-selective pore) and is predominately expressed in inhibitory interneurons. 3 Loss-of-function mutations in SCN1A associate with 80%–90% of DS, and the higher incidence of SUDEP in DS is widely attributed to SCN1A haploinsufficiency. 4 The Nav1.6 channel, on the other hand, is encoded by the SCN8A gene (A also for alpha subunit) and regulates neuronal excitation. 3 Gain-of-function SCN8A variants are implicated in early onset developmental and epileptic encephalopathy (SCN8A-DEE), which is characterized by epilepsy phenotype and higher SUDEP probability. 5
Interestingly, DS preclinical models with SCN1A haploinsufficiency (Scn1a+/−) and a loss of Nav1.1 channel function are found to also have a possible gain of Nav1.6 channel function. Indeed, suppressing the gain of Nav1.6 function mitigates seizures and, more importantly, reduces mortality in preclinical models of DS and SCN8A-DEE, which suggests that Nav1.6 contributes to mortality in DS and may represent a novel target.6-8 However, in the salty battle between Nav1.1 and Nav1.6, both of which are expressed in the brain and heart, the exact mechanisms by which a gain of Nav1.6 channel function contributes to SUDEP pathophysiology in DS is unclear. Furthermore, circling back to age, Nav1.6 has two age-dependent splice isoforms, which are rarely talked about a younger/neonatal isoform that dominates during the first year of life and then a second adult isoform that takes over during the rest of life. 9 Two recent studies help have shed light on whether the differential age-related predisposition to SUDEP in DS involves the Nav1.6 isoforms.
The first study by King and colleagues used the Scn1a+/− DS model (loss of Nav1.1 function) and generated two new Scn1a+/− DS models: 1 Scn1a+/− DS model with cardiac-specific deletion of Nav1.6, and 1 Scn1a+/− DS model with global Nav1.6 haploinsufficiency. The Scn1a+/− DS mice showed a “younger” and “older” peak in mortality (similar to humans). Interestingly, the cardiac-specific Nav1.6 deletion protected only against mortality in older cohorts. This confirms that an indirect effect of the Scn1a+/− haploinsufficiency is cardiac Nav1.6 channel remodeling toward a gain-of-function. This also suggests that cardiac Nav1.6 contributes to mortality in older DS cohorts, but not younger cohorts.
Whereas previous studies in the DS field indicate the primary etiology of sudden death is brain-driven cardiac dysfunction, King's study supports a shift in the current thinking toward the possibility that indirect, secondary remodeling of cardiac function (ie, increased Nav1.6 function) can be maladaptive and directly contribute to sudden death in the older DS population. Scn1a+/− DS mice with either global reduction or cardiac-specific deletion of Nav1.6 were protected against arrhythmias. Previous whole cell patch clamp recordings from DS cardiomyocytes had reported an increase in arrhythmogenic late sodium currents of unidentified origin. 4 Zooming in with the elegant scanning-ion-conductance-microscopy-guided-smart-patch-clamp-electrophysiology technique, King and group used glass nanopipettes to scan cell surface protrusions and indentations, generate a topographic map, and record single channel currents across the cardiomyocyte surface. They found that the arrhythmogenic late sodium currents in DS cardiomyocytes arose from Nav1.6-rich T-tubule subcellular regions, which mediate cardiac myofibril contraction via sarcoplasmic reticulum Ca2+ release. 10 Using sophisticated proximity ligation assays and stochastic optical reconstruction microscopy, they found augmented Nav1.6 clusters near calcium handling players within the T-tubules, the sodium-calcium exchanger, and Ryanodine receptor 2. Confocal microscopy also confirmed spontaneous arrhythmogenic calcium waves in DS hearts. An important takeaway from this salty battle is that Nav1.1 haploinsufficiency indirectly caused Nav.1.6 cardiac remodeling which then drove arrhythmias and directly contributed to DS mortality in older cohorts, irrespective of its role in brain and seizures.
A limitation of this study, as noted by the authors, is that common data elements such as seizures and postictal apnea were not monitored before sudden death and thus their contribution to mortality cannot be ruled out. Considering that not all DS mice experience early mortality, whether the Nav1.6 remodeling predicts 100% of SUDEP cases needs to be studied. In addition, “younger” and “older” are relative and unique to each species. Whether these findings are generalizable needs further evaluation. If future studies confirm specific mechanisms that uniquely contribute to younger and older mortality in DS, then prospective treatment strategies may include temporally titrated pharmaco-preventative strategies.
The second study by Vanoye and colleagues zoomed in on the young versus old battle, further illustrating the importance of timing. During the first year of life, the neonatal Nav1.6 isoform is expressed (Nav1.6N). The adult isoform (Nav1.6A) then comes online and eventually dominates for the rest of life. Each has distinct electrochemical properties. A limitation of previous studies is that many used the Nav1.6N isoform to examine the functional consequences of potentially pathogenic SCN8A variants, thus, interpretations may have translational relevance limited to early disease phenotypes. In addition, studies that did express Nav1.6A and Nav1.6N in cell lines engineered the Nav1.6 isoforms to be insensitive to tetrodotoxin (TTX) to distinguish them from endogenous sodium currents of the cell lines. This alteration can affect the channel electrochemical properties of the SCNA8 variants studied. This is a confounding variable for many SCN8A studies and limits their physiological relevance. Vanoye and colleagues addressed all these shortcomings and generated a novel low endogenous Na+ current cell line. Human Nav1.6N and Nav1.6A isoforms were then expressed in this cell line and automated patch clamp assays revealed significant differences in activation and inactivation thresholds between Nav1.6N and Nav1.6A isoforms. At a typical age of onset of SCN8A-associated encephalopathies, there is a mix of Nav1.6N and Nav1.6A isoforms. The group studied 15 disease-associated variants and found biophysical properties of Nav1.6N and Nav1.6A isoforms were uniquely affected, ranging in gain- and loss-of-functions across the various electrophysiological parameters. The importance of identifying differential effects of pathologic variants on Nav1.6N and Nav1.6A isoforms is that it enables future studies to use similar high throughput platforms as screening tools for understanding Nav1.6 variants and developing variant precision therapeutics for DS and SCN8A-DEE patients. A limitation of this study is that while variants that cause biophysical changes in the channel often correlate to neuronal dysfunction, neuronal function was not assessed. Because the variants lead to different changes in the neonatal and adult isoforms, future pharmacological studies are needed to determine whether targeting each isoform can be therapeutic for SUDEP prevention in DS and SCN8a-DEE.
Overall, in vivo data combined with sophisticated super-resolution microscopy, electrophysiology, and computational simulation indicate that the Nav1.6 remodeling within functional domains in DS hearts is secondary to the loss of Nav1.1 channels in DS. This introduces an important premise that, in addition to the Nav1.1 mutation having direct effects, the phenotype of the disease may also include indirect secondary effects such as remodeling of other Nav channels. A popular notion in the field is that Nav1.1 and Nav1.6 channels have a feng shui balance in maintaining network inhibition and activation, respectively, and there may be benefits in targeting either channel in DS and SCN8A-DEE. King and colleagues extend this notion to cardiac-associated SUDEP susceptibility in DS. Similarly, Vanoye and the team remind us that the road to translational relevance is difficult to traverse and we cannot reach the right destination by walking on the wrong path. It would be interesting to determine whether similar Nav1.6 channel remodeling also contributes to respiratory-associated SUDEP susceptibility. Future studies are needed to determine whether the neonatal and adult isoforms exhibit unique pharmacological profiles that can be exploited for SUDEP prevention in DS and SCN8A-DEE.
