Novel Genetic Variants Expand the Functional, Molecular, and Pathological Diversity of KCNA1 Channelopathy.

The KCNA1 gene encodes Kv1.1 voltage-gated potassium channel α subunits, which are crucial for maintaining healthy neuronal firing and preventing hyperexcitability. Mutations in the KCNA1 gene can cause several neurological diseases and symptoms, such as episodic ataxia type 1 (EA1) and epilepsy, which may occur alone or in combination, making it challenging to establish simple genotype-phenotype correlations. Previous analyses of human KCNA1 variants have shown that epilepsy-linked mutations tend to cluster in regions critical for the channel's pore, whereas EA1-associated mutations are evenly distributed across the length of the protein. In this review, we examine 17 recently discovered pathogenic or likely pathogenic KCNA1 variants to gain new insights into the molecular genetic basis of KCNA1 channelopathy. We provide the first systematic breakdown of disease rates for KCNA1 variants in different protein domains, uncovering potential location biases that influence genotype-phenotype correlations. Our examination of the new mutations strengthens the proposed link between the pore region and epilepsy and reveals new connections between epilepsy-related variants, genetic modifiers, and respiratory dysfunction. Additionally, the new variants include the first two gain-of-function mutations ever discovered for KCNA1, the first frameshift mutation, and the first mutations located in the cytoplasmic N-terminal domain, broadening the functional and molecular scope of KCNA1 channelopathy. Moreover, the recently identified variants highlight emerging links between KCNA1 and musculoskeletal abnormalities and nystagmus, conditions not typically associated with KCNA1. These findings improve our understanding of KCNA1 channelopathy and promise to enhance personalized diagnosis and treatment for individuals with KCNA1-linked disorders.

Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897­914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

Kv1.1 subunits localize to cardiorespiratory brain networks in mice where their absence induces astrogliosis and microgliosis.

Cardiorespiratory collapse following a seizure is a suspected cause of sudden unexpected death in epilepsy (SUDEP), the leading cause of epilepsy-related mortality. In the commonly used Kcna1 gene knockout (Kcna1-/-) mouse model of SUDEP, cardiorespiratory profiling reveals an array of aberrant breathing patterns that could contribute to risk of seizure-related mortality. However, the brain structures mediating these respiratory abnormalities remain unknown. We hypothesize that Kv1.1 deficiency in respiratory control centers of the brain contribute to respiratory dysfunction in Kcna1-/- mice leading to increased SUDEP risk. Thus, in this study, we first used immunohistochemistry to map expression of Kv1.1 protein in cardiorespiratory brain regions of wild-type Kcna1+/+ (WT) mice. Next, GFAP and Iba1 immunostaining was used to test for the presence of astrogliosis and microgliosis, respectively, in the cardiorespiratory centers of Kcna1-/- mice, which could be indicative of seizure-related brain injury that could impair breathing. In WT mice, we detected Kv1.1 protein in all cardiorespiratory centers examined, including the basolateral amygdala, dorsal respiratory group, dorsal motor nucleus of vagus, nucleus ambiguus, ventral respiratory column, and pontine respiratory group, as well as chemosensory centers including the retrotrapezoid and median raphae nuclei. Extensive gliosis was observed in the same areas in Kcna1-/- mice suggesting that seizure-associated brain injury could contribute to respiratory abnormalities.

Kv1.1 deficiency alters repetitive and social behaviors in mice and rescues autistic-like behaviors due to Scn2a haploinsufficiency.

BACKGROUND: Autism spectrum disorder (ASD) and epilepsy are highly comorbid, suggesting potential overlap in genetic etiology, pathophysiology, and neurodevelopmental abnormalities; however, the nature of this relationship remains unclear. This work investigated how two ion channel mutations, one associated with autism (Scn2a-null) and one with epilepsy (Kcna1-null), interact to modify genotype-phenotype relationships in the context of autism. Previous studies have shown that Scn2a+/- ameliorates epilepsy in Kcna1-/- mice, improving survival, seizure characteristics, and brain-heart dynamics. Here, we tested the converse, whether Kcna1 deletion modifies ASD-like repetitive and social behaviors in Scn2a+/- mice. METHODS: Mice were bred with various combinations of Kcna1 and Scn2a knockout alleles. Animals were assessed for repetitive behaviors using marble burying, grooming, and nestlet shredding tests and for social behaviors using sociability and social novelty preference tests. RESULTS: Behavioral testing revealed drastic reductions in all repetitive behaviors in epileptic Kcna1-/- mice, but relatively normal social interactions. In contrast, mice with partial Kcna1 deletion (Kcna1+/- ) exhibited increased self-grooming and decreased sociability suggestive of ASD-like features similar to those observed in Scn2a+/- mice. In double-mutant Scn2a+/- ; Kcna1+/- mice, the two mutations interacted to partially normalize ASD-like behaviors associated with each mutation independently. CONCLUSIONS: Taken together, these findings suggest that Kv1.1 subunits are important in pathways and neural networks underlying ASD and that Kcna1 may be a therapeutic target for treatment of Scn2a-associated ASD.

Kv1.1 potassium channel subunit deficiency alters ventricular arrhythmia susceptibility, contractility, and repolarization.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.

Decreased bioavailability of hydrogen sulfide links vascular endothelium and atrial remodeling in atrial fibrillation.

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Clinical Spectrum of KCNA1 Mutations: New Insights into Episodic Ataxia and Epilepsy Comorbidity.

Mutations in the KCNA1 gene, which encodes voltage-gated Kv1.1 potassium channel α-subunits, cause a variety of human diseases, complicating simple genotype-phenotype correlations in patients. KCNA1 mutations are primarily associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1). However, some patients have EA1 in combination with epilepsy, whereas others have epilepsy alone. KCNA1 mutations can also cause hypomagnesemia and paroxysmal dyskinesia in rare cases. Why KCNA1 variants are associated with such phenotypic heterogeneity in patients is not yet understood. In this review, literature databases (PubMed) and public genetic archives (dbSNP and ClinVar) were mined for known pathogenic or likely pathogenic mutations in KCNA1 to examine whether patterns exist between mutation type and disease manifestation. Analyses of the 47 deleterious KCNA1 mutations that were identified revealed that epilepsy or seizure-related variants tend to cluster in the S1/S2 transmembrane domains and in the pore region of Kv1.1, whereas EA1-associated variants occur along the whole length of the protein. In addition, insights from animal models of KCNA1 channelopathy were considered, as well as the possible influence of genetic modifiers on disease expressivity and severity. Elucidation of the complex relationship between KCNA1 variants and disease will enable better diagnostic risk assessment and more personalized therapeutic strategies for KCNA1 channelopathy.

Neuron-specific Kv1.1 deficiency is sufficient to cause epilepsy, premature death, and cardiorespiratory dysregulation.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality, but the precise cellular substrates involved remain elusive. Epilepsy-associated ion channel genes with co-expression in brain and heart have been proposed as SUDEP candidate genes since they provide a singular unifying link between seizures and lethal cardiac arrhythmias. Here, we generated a conditional knockout (cKO) mouse with neuron-specific deletion of Kcna1, a SUDEP-associated gene with brain-heart co-expression, to test whether seizure-evoked cardiac arrhythmias and SUDEP require the absence of Kv1.1 in both brain and heart or whether ablation in neurons is sufficient. To obtain cKO mice, we developed a floxed Kcna1 mouse which we crossed to mice with the Synapsin1-Cre transgene, which selectively deletes Kcna1 in most neurons. Molecular analyses confirmed neuron-specific Kcna1 deletion in cKO mice and corresponding loss of Kv1.1 except in cerebellum where Synapsin1-Cre is not highly expressed. Survival studies and electroencephalography, electrocardiography, and plethysmography recordings showed that cKO mice exhibit premature death, epilepsy, and cardiorespiratory dysregulation but to a lesser degree than global knockouts. Heart rate variability (HRV) was increased in cKO mice with peaks during daytime suggesting disturbed diurnal HRV patterns as a SUDEP biomarker. Residual Kv1.1 expression in cKO cerebellum suggests it may play an unexpected role in regulating ictal cardiorespiratory dysfunction and SUDEP risk. This work demonstrates the principle that channelopathies with brain-heart expression patterns can increase death risk by brain-driven mechanisms alone without a functionally compromised heart, reinforcing seizure control as a primary clinical strategy for SUDEP prevention.

Directed Connectivity Analysis of the Neuro-Cardio- and Respiratory Systems Reveals Novel Biomarkers of Susceptibility to SUDEP.

Goal: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality and its pathophysiological mechanisms remain unknown. We set to record and analyze for the first time concurrent electroencephalographic (EEG), electrocardiographic (ECG), and unrestrained whole-body plethysmographic (Pleth) signals from control (WT - wild type) and SUDEP-prone mice (KO- knockout Kcna1 animal model). Employing multivariate autoregressive models (MVAR) we measured all tri-organ effective directional interactions by the generalized partial directed coherence (GPDC) in the frequency domain over time (hours). When compared to the control (WT) animals, the SUDEP-prone (KO) animals exhibited (p < 0.001) reduced afferent and efferent interactions between the heart and the brain over the full frequency spectrum (0-200Hz), enhanced efferent interactions from the brain to the lungs and from the heart to the lungs at high (>90 Hz) frequencies (especially during periods with seizure activity), and decreased feedback from the lungs to the brain at low (<40 Hz) frequencies. These results show that impairment in the afferent and efferent pathways in the holistic neuro-cardio-respiratory network could lead to SUDEP, and effective connectivity measures and their dynamics could serve as novel biomarkers of susceptibility to SUDEP and seizures respectively.

Deletion of the Cardiomyocyte Glucocorticoid Receptor Leads to Sexually Dimorphic Changes in Cardiac Gene Expression and Progression to Heart Failure.

Background The contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, we sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, we conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model). Methods and Results Deletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. Functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA-Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR-deficient cardiomyocytes presented a response comparable with controls. Conclusions These data suggest that GR regulates calcium responses in a sex-biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

Kv1.1 channel subunits in the control of neurocardiac function.

Voltage-gated Kv1.1 potassium channel α-subunits are broadly expressed in the nervous system where they act as critical regulators of neuronal excitability. Mutations in the KCNA1 gene, which encodes Kv1.1, are associated with the neurological diseases episodic ataxia and epilepsy. Studies in mouse models have shown that Kv1.1 is important for neural control of the heart and that Kcna1 deletion leads to cardiac dysfunction that appears to be brain-driven. Traditionally, KCNA1 was not believed to be expressed in the heart. However, recent studies have revealed that Kv1.1 subunits are not only present in cardiomyocytes, but that they also make an important heart-intrinsic functional contribution to outward K+ currents and action potential repolarization. This review recounts the winding history of discovery of KCNA1 gene expression and neurocardiac function from fruit flies to mammals and from brain to heart and looks at some of the salient questions that remain to be answered regarding emerging cardiac roles of Kv1.1.

Cardiorespiratory profiling reveals primary breathing dysfunction in Kcna1-null mice: Implications for sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality, but the relative importance of underlying cardiac and respiratory mechanisms remains unclear. To illuminate the interactions between seizures, respiration, cardiac function, and sleep that contribute to SUDEP risk, here we developed a mouse epilepsy monitoring unit (EMU) to simultaneously record video, electroencephalography (EEG), electromyography (EMG), plethysmography, and electrocardiography (ECG) in a commonly used genetic model of SUDEP, the Kcna1 knockout (Kcna1-/-) mouse. During interictal periods, Kcna1-/- mice exhibited an abnormal absence of post-sigh apneas and a 3-fold increase in respiratory variability. During spontaneous convulsive seizures, Kcna1-/- mice displayed an array of aberrant breathing patterns that always preceded cardiac abnormalities. These findings support respiratory dysfunction as a primary risk factor for susceptibility to deleterious cardiorespiratory sequelae in epilepsy and reveal a new role for Kcna1-encoded Kv1.1 channels in the regulation of basal respiratory physiology.

Genetic ablation or pharmacological inhibition of Kv1.1 potassium channel subunits impairs atrial repolarization in mice.

Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1-/-) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1-/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1-/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1-/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Pharmacogenetics of KCNQ channel activation in 2 potassium channelopathy mouse models of epilepsy.

OBJECTIVES: Antiseizure drugs are the leading therapeutic choice for treatment of epilepsy, but their efficacy is limited by pharmacoresistance and the occurrence of unwanted side effects. Here, we examined the therapeutic efficacy of KCNQ channel activation by retigabine in preventing seizures and neurocardiac dysfunction in 2 potassium channelopathy mouse models of epilepsy with differing severity that have been associated with increased risk of sudden unexpected death in epilepsy (SUDEP): the Kcna1-/- model of severe epilepsy and the Kcnq1A340E/A340E model of mild epilepsy. METHODS: A combination of behavioral, seizure threshold, electrophysiologic, and gene expression analyses was used to determine the effects of KCNQ activation in mice. RESULTS: Behaviorally, Kcna1-/- mice exhibited unexpected hyperexcitability instead of the expected sedative-like response. In flurothyl-induced seizure tests, KCNQ activation decreased seizure latency by ≥50% in Kcnq1 strain mice but had no effect in the Kcna1 strain, suggesting the influence of genetic background. However, in simultaneous electroencephalography and electrocardiography recordings, KCNQ activation significantly reduced spontaneous seizure frequency in Kcna1-/- mice by ~60%. In Kcnq1A340E/A340E mice, KCNQ activation produced adverse cardiac effects including profound bradycardia and abnormal increases in heart rate variability and atrioventricular conduction blocks. Analyses of Kcnq2 and Kcnq3 mRNA levels revealed significantly elevated Kcnq2 expression in Kcna1-/- brains, suggesting that drug target alterations may contribute to the altered drug responses. SIGNIFICANCE: This study shows that treatment strategies in channelopathy may have unexpected outcomes and that effective rebalancing of channel defects requires improved understanding of channel interactions at the circuit and tissue levels. The efficacy of KCNQ channel activation and manifestation of adverse effects were greatly affected by genetic background, potentially limiting KCNQ modulation as a way to prevent neurocardiac dysfunction in epilepsy and thereby SUDEP risk. Our data also uncover a potential role for KCNQ2-5 channels in autonomic control of chronotropy.

Scn2a deletion improves survival and brain-heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP).

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.

Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.

BACKGROUND: Fused in sarcoma (FUS) is an RNA-binding protein associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. ALS manifests in patients as a progressive paralysis which leads to respiratory dysfunction and failure, the primary cause of death in ALS. We expressed human FUS in rats to determine if FUS would induce ALS relevant respiratory changes to serve as an early stage disease indicator. The FUS expression was initiated in adult rats by way of an intravenously administered adeno-associated virus vector serotype 9 (AAV9) providing an adult onset model. RESULTS: The rats developed progressive motor impairments observed as early as 2-3 weeks post gene transfer. Respiratory abnormalities manifested 4-7 weeks post gene transfer including increased respiratory frequency and decreased tidal volume. Rats with breathing abnormalities also had arterial blood acidosis. Similar detailed plethysmographic changes were found in adult rats injected with AAV9 TDP-43. FUS gene transfer to adult rats yielded a consistent pre-clinical model with relevant motor paralysis in the early to middle stages and respiratory dysfunction at the end stage. Both FUS and TDP-43 yielded a similar consistent disease state. CONCLUSIONS: This modeling method yields disease relevant motor and respiratory changes in adult rats. The reproducibility of the data supports the use of this method to study other disease related genes and their combinations as well as a platform for disease modifying interventional strategies.

Gradient Index Microlens Implanted in Prefrontal Cortex of Mouse Does Not Affect Behavioral Test Performance over Time.

Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and five postsurgical sets of behavioral tests were performed over seven weeks. Behavioral tests included rotarod, foot fault, and Morris water maze. No significant differences were found between the three groups, suggesting that microlens implantation did not affect performance. The results for the current study clear the way for combining behavioral studies with gradient index lens imaging in the prefrontal cortex, and potentially other regions of the mouse brain, to study structural, functional, and behavioral relationships in the brain.

Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation.

Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson's trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.