Voltage-gated Sodium Channel Alpha Subunit Research Articles

Gene therapy as an emerging treatment for Scn2a mutation-induced autism spectrum disorders

Autism spectrum disorder (ASD) is a complex neurological and developmental disorder that affects how a person acts, communicates, learns, and interacts with others. It affects the structure and function of the brain and nervous system. How ASD is caused remains uncertain and there is no effective treatment for this disorder. Searching for new technologies for treatment of this disorder becomes a priority. Genetic alterations have been implicated in the generation of this disorder. One of the most promising genes for potential treatment of ASD is sodium voltage-gated channel alpha subunit 2 gene (SCN2A). SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to ASD. Here, we discuss the implications of loss of function (LOF) mutations in SCN2A and the potential efficacy of gene therapy by highlighting the usage of CRISPR restoration of various Scn2a-insufficiencies. Various therapeutics exist that can be extrapolated to address the needs of Scn2a LOF induced ASD. The current treatment that exists for ASD can be seen as outdated in comparison to the advent of new technologies that build upon CRISPR. Due to complications in treatment of ASD, genetic therapies may induce alterations such as insertion–deletion mutations, which may lead to further complications along with a negative public outlook on CRISPR technologies. Gene therapy can be applied to ASD but much work is yet to be done in order to address both biochemical and ethical considerations.

Classical trigeminal neuralgia is associated with gephyrin and sodium voltage-gated channel alpha subunit 8

Trigeminal neuralgia is highly debilitating, and its etiology is still undefined. The goal of this work was to define associations between well-characterized trigeminal neuralgia cases and common genetic variants in the population. two hundred and fifty-seven individuals diagnosed with classical trigeminal neuralgia were compared to 865 individuals without classical trigeminal neuralgia and with an assessment for lower or higher pain threshold based on the amount of anesthetic required for routine dental treatment. Genotypes of 24 variants marking genes in the VGSC (voltage-gated sodium channels) or GABA (gamma-aminobutyric acid) pathways were obtained using TaqMan chemistry end end-point analysis. Chi-square was used for all comparisons with an alpha of 0.002. An association between classical trigeminal neuralgia and individuals requiring less or more anesthetic for routine dental treatments showed associations with SCN8A rs1601012 and GPHN rs723432 (p = 0.0009 and p = 0.0002, respectively). In conclusion, classical trigeminal neuralgia is associated with SCN8A and GPHN and markers rs1601012 rs723432 may be useful to determine individual risks for the condition.

SCN1A: bioinformatically informed revised boundaries for promoter and enhancer regions.

Pathogenic variations in the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene are responsible for multiple epilepsy phenotypes, including Dravet syndrome, febrile seizures (FS) and genetic epilepsy with FS plus. Phenotypic heterogeneity is a hallmark of SCN1A-related epilepsies, the causes of which are yet to be clarified. Genetic variation in the non-coding regulatory regions of SCN1A could be one potential causal factor. However, a comprehensive understanding of the SCN1A regulatory landscape is currently lacking. Here, we summarized the current state of knowledge of SCN1A regulation, providing details on its promoter and enhancer regions. We then integrated currently available data on SCN1A promoters by extracting information related to the SCN1A locus from genome-wide repositories and clearly defined the promoter and enhancer regions of SCN1A. Further, we explored the cellular specificity of differential SCN1A promoter usage. We also reviewed and integrated the available human brain-derived enhancer databases and mouse-derived data to provide a comprehensive computationally developed summary of SCN1A brain-active enhancers. By querying genome-wide data repositories, extracting SCN1A-specific data and integrating the different types of independent evidence, we created a comprehensive catalogue that better defines the regulatory landscape of SCN1A, which could be used to explore the role of SCN1A regulatory regions in disease.

Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

SCN1A polymorphisms influence the antiepileptic drugs responsiveness in Jordanian epileptic patients.

The aim of this study was to evaluate whether the voltage-gated sodium channel alpha subunit 1 (SCN1A) gene polymorphisms influence the responsiveness of Jordanian epileptic patients to antiepileptic drugs (AEDs). A total of 72 AEDs-treated epileptics were polymerase chain reaction (PCR)-genotyped for six single nucleotide polymorphisms (SNPs), including SCN1A rs2298771, rs3812718, rs3812719, rs2217199, rs2195144 and rs1972445. Genotype and allele distributions in drug-responsive and drug-resistant patients were compared. The six SNPs haplotypes were examined, and the linkage disequilibrium (LD) was assessed. The genotypes of drug-resistant and drug-responsive groups were in Hardy-Weinberg equilibrium. Three genetic polymorphisms of the SCN1A gene seemed to influence the resistance to AEDs, on the level of alleles and genotypes. Data revealed that rs2298771 G allele, rs3812719 C allele, and rs2195144 T allele increased the risk of developing AEDs-resistance (OR=2.9; 95%CI= 1.4-5.9, p=0.003; OR=2.4; 95%CI=1.2-4.7, p=0.01; OR=2.3; 95%CI=1.2-4.7, p=0.01), respectively. Haplo type analysis of SCN1A polymorphisms revealed high-degree LD associated with resistance to AEDs. A synergetic effect appears with highly significant association in GCCATG haplotype of rs2298771, rs3812718, rs3812719, rs2217199, rs2195144, and rs1972445 respectively (OR=2.8; 95%CI=1.5-6.2, p=0.002). Data suggests that SCN1A polymorphisms could influence the resistance to AEDs in Jordanian epileptics at three SNPs (rs2298771; rs3812719; rs2195144). Additionally, haplotype analysis indicated a substantial degree of LD between the six SCN1A polymorphisms. Further investigation with larger sample size is needed to confirm the results of the current study.

Effects of chronic intermittent hypoxia on atrial electrical remodeling in rats

The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Nav1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Cav1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/Kv4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD90 and APD50 were analyzed and compared between the two groups. In voltage clamp mode, INa, ICa-L, Ito and their kinetic parameters were recorded and compared between the two groups. Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Nav1.5, Cav1.2 and Kv4.3 were decreased (P＜0.05). APD90 and APD50 in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of INa, ICa-L(P＜0.01) and Ito(P＜0.01). CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Complex interactions between p.His558Arg and linked variants in the sodium voltage-gated channel alpha subunit 5 (Na V 1.5).

Common genetic polymorphisms may modify the phenotypic outcome when co-occurring with a disease-causing variant, and therefore understanding their modulating role in health and disease is of great importance. The polymorphic p.His558Arg variant of the sodium voltage-gated channel alpha subunit 5 (NaV1.5) encoded by the SCN5A gene is a case in point, as several studies have shown it can modify the clinical phenotype in a number of cardiac diseases. To evaluate the genetic backgrounds associated with this modulating effect, we reanalysed previous electrophysiological findings regarding the p.His558Arg variant and further assessed its patterns of genetic diversity in human populations. The NaV1.5 p.His558Arg variant was found to be in linkage disequilibrium with six other polymorphic variants that previously were also associated with cardiac traits in GWAS analyses. On account of this, incongruent reports that Arg558 allele can compensate, aggravate or have no effect on NaV1.5, likely might have arose due to a role of p.His558Arg depending on the additional linked variants. Altogether, these results indicate a major influence of the epistatic interactions between SCN5A variants, revealing also that phenotypic severity may depend on the polymorphic background associated to each individual genome.

Primary erythromelalgia mainly manifested by hypertensive crisis: A case report and literature review.

BackgroundPrimary erythrocytic (PEM) is a rare autosomal dominant single gene disease. Most of the changes of gene loci can be found by whole exon gene sequencing, and the clinical symptoms and patient survival can be improved by specific site-to-site drug treatment. The other manifestations of this patient population are not remarkable. After the application of common drugs, the toxicity and side effects can be limiting. In addition to other common clinical manifestations, we found that the only unique manifestation of this patient was hypertensive crisis. Following multidisciplinary diagnosis and treatment (MDT), we decided to first control hypertension to alleviate the acute and critical patients. However, after controlling the hypertensive crisis, we unexpectedly found that the clinical symptoms of the patients had been significantly improved. Therefore, we concluded that the use of antihypertensive drugs can treat erythematous limb pain with the clinical manifestation of hypertensive crisis. Here, we describe a typical PEM disease, primary clinical features, diagnosis and treatment.MethodsMedical records of an 8-year-old boy with PEM were analyzed retrospectively, which included clinical characteristics, follow-up information, and SCN9A (Sodium Voltage-Gated Channel Alpha Subunit 9) gene analysis.ResultsThe 8-year-old boy had complained of abnormal paresthesia in his feet and ankles with burning sensation and pain for 2 years. The skin of both lower legs was red and underwent ichthyosis and lichenification. Genetic analysis confirmed the existence of a SCN9A gene mutation. The symptoms were gradually improved by treating with intravenous drip and oral administration of nitroglycerin to slow his heart rhythm.ConclusionPrimary erythrocytic is characterized by skin ulceration, redness, elevated temperature, and severe burning pain primarily in both lower extremities. PEM can be diagnosed by genetic analysis. As this case demonstrates, treating with nitroglycerin as the drug of choice to control the hypertensive crisis significantly improved the symptoms of PEM and hypertension in this patient.

Migraine with Brainstem Aura Associated with SCN2A Mutation: A Case Report

Abstract Background Migraine with brainstem aura (MBA) is a rare subtype of migraine with aura that associates headache with paroxysmal episodes of ataxia, dysarthria, and diplopia among other symptoms attributed to brainstem involvement. The manifestation of this disease in the absence of typical aura symptoms (i.e., fully reversible visual, sensory and/or speech/language symptoms) is even more infrequent. Case Report In this article, we reported a case of a 16-year-old male patient with MBA associated with a sodium voltage-gated channel alpha subunit 2 (SCN2A) de novo mutation. The patient did not have typical aura symptoms. Ataxia was the most prominent clinical feature, leading to initial diagnosis of episodic ataxia. Patient responded partially to treatment with onabotulinum toxin A. Conclusion MBA needs to be taken into consideration in the differential diagnosis of recurrent ataxia, even in the absence of typical aura symptoms. Finding a de novo SCN2A mutation in our patient helps in further understanding of MBA genetics.

Whole-Exome Sequencing Identifies Novel SCN1A and CACNB4 Genes Mutations in the Cohort of Saudi Patients With Epilepsy.

Epilepsy is a neurological disorder described as recurrent seizures mild to severe convulsions along with conscious loss. There are many different genetic anomalies or non-genetic conditions that affect the brain and cause epilepsy. The exact cause of epilepsy is unknown so far. In this study, whole-exome sequencing showed a family having novel missense variant c.1603C>T, p. Arg535Cys in exon 10 of Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) gene. Moreover, targeted Sanger sequencing analysis showed c.1212A>G p.Val404Ile in SCN1A gene in 10 unrelated patients and a mutation in Calcium Voltage-Gated Channel Auxiliary Subunit Beta 4 gene where one base pair insertion of “G” c.78_79insG, p.Asp27Glyfs*26 in the exon 3 in three different patients were observed from the cohort of 25 epileptic sporadic cases. The insertion changes the amino acid sequence leading to a frameshift mutation. Here, we have described, for the first time, three novel mutations that may be associated with epilepsy in the Saudi population. The study not only help us to identify the exact cause of genetic variations causing epilepsy whereas but it would also eventually enable us to establish a database to provide a foundation for understanding the critical genomic regions to control epilepsy in Saudi patients.

Electrophysiological characterization of schizophrenia-associated variants in NaV1.2 sodium channel

IntroductionA major pathophysiological hypothesis of schizophrenia states an increased activity of glutamatergic neurons leading to an imbalance of neural excitation and inhibition (E/I-imbalance). One potential molecular mechanism of E/I-imbalance is a dysfunction of voltage-gated sodium channels, which are crucial for the generation of action potentials, the fundamental event of neuronal excitation. Indeed, patients with schizophrenia exhibit an increased burden of rare exonic variants of sodium channel genes, but the literature describing their electrophysiological effect is scarce.ObjectivesThe aim of this project is to assess the functional impact of three mutations of the Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A) gene / NaV1.2 channel which were identified in four patients with schizophrenia, using a heterologous expression system.MethodsThree variants of the human SCN2A gene (R850P, V1282F and S1656P) were created using site-directed mutagenesis. HEK293T cells transfected with either the mutant or wild type constructs are being investigated by voltage-clamp technique, applying activation, steady-state fast inactivation, use dependency and ramp protocols.ResultsAll three mutated constructs were successfully created. Preliminary recordings from the V1282F mutant indicate a shift of both the activation and steady-state fast inactivation to the hyperpolarized direction.ConclusionsIn a subgroup of patients, E/I imbalance may be a consequence of Nav1.2 mutations leading to increased excitability of glutamatergic neurons. By integrating insights from different mutations we aim to identify traits of a potentially shared disease pathway which may provide a basis for the development of novel therapeutics.DisclosureNo significant relationships.

SCN1A-Related Epilepsy: Novel Mutations and Rare Phenotypes.

ObjectivesTo expand the genotypes and phenotypes of sodium voltage-gated channel alpha subunit 1 (SCN1A)-related epilepsy.MethodsWe retrospectively collected the clinical and genetic information of 22 epilepsy patients (10 males, 12 females; mean: 9.2 ± 3.9 years; 3.9–20.3 years) carrying 22 variants of SCN1A. SCN1A mutations were identified by next-generation sequencing.ResultsTwenty-two variants were identified, among which 12 have not yet been reported. The median age at seizure onset was 6 months. Sixteen patients were diagnosed with Dravet syndrome (DS), two with genetic epilepsy with febrile seizures plus [one evolved into benign epilepsy with centrotemporal spikes (BECTS)], one with focal epilepsy, one with atypical childhood epilepsy with centrotemporal spikes (ABECTS) and two with unclassified epilepsy. Fourteen patients showed a global developmental delay/intellectual disability (GDD/ID). Slow background activities were observed in one patient and epileptiform discharges were observed in 11 patients during the interictal phase.SignificanceThis study enriches the genotypes and phenotypes of SCN1A-related epilepsy. The clinical characteristics of patients with 12 previously unreported variants were described.

BS-514-01 SCN10ASHORT AS A NOVEL GENE THERAPY TARGET TO TREAT CONDUCTION DEFECTS

Voltage-gated sodium channels play a critical role in the action potential (AP) upstroke velocity and impulse propagation in the heart. Sodium channel gene therapy is challenged by the relatively large transgene size of Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A), the main alpha-subunit of the cardiac sodium channel isoform. Our previous work demonstrated that SCN10Ashort, a small fragment comprising of the C-terminus of SCN10A, increased sodium current when co-expressed with SCN5A. In the present study we therefore explored SCN10Ashort as a novel gene therapy target.

Discovering the Triad between Nav1.5, Breast Cancer, and the Immune System: A Fundamental Review and Future Perspectives.

Nav1.5 is one of the nine voltage-gated sodium channel-alpha subunit (VGSC-α) family members. The Nav1.5 channel typically carries an inward sodium ion current that depolarises the membrane potential during the upstroke of the cardiac action potential. The neonatal isoform of Nav1.5, nNav1.5, is produced via VGSC-α alternative splicing. nNav1.5 is known to potentiate breast cancer metastasis. Despite their well-known biological functions, the immunological perspectives of these channels are poorly explored. The current review has attempted to summarise the triad between Nav1.5 (nNav1.5), breast cancer, and the immune system. To date, there is no such review available that encompasses these three components as most reviews focus on the molecular and pharmacological prospects of Nav1.5. This review is divided into three major subsections: (1) the review highlights the roles of Nav1.5 and nNav1.5 in potentiating the progression of breast cancer, (2) focuses on the general connection between breast cancer and the immune system, and finally (3) the review emphasises the involvements of Nav1.5 and nNav1.5 in the functionality of the immune system and the immunogenicity. Compared to the other subsections, section three is pretty unexploited; it would be interesting to study this subsection as it completes the triad.

Analysis of cancer-promoting genes related to chemotherapy resistance in esophageal squamous cell carcinoma

BackgroundAccording to histopathology, esophageal cancer can be divided into squamous cell carcinoma (SCC) and esophageal adenocarcinoma (adeno arcinoma). In China, 90% of esophageal cancer patients are squamous cell carcinoma. Cisplatin and fluaziridine are the main chemotherapy before and after surgery. Long-term drug treatment is often accompanied by the emergence of drug resistance of tumor cells. There are many mechanisms for the emergence of drug resistance of tumor cells, including the increase of drug efflux, the decrease of drug intake, the inhibition of cell apoptosis, and so on. This study aimed to investigate the key cancer-promoting genes related to chemotherapy resistance in esophageal squamous cell carcinoma (ESCC).MethodsTwo datasets from the Gene Expression Omnibus (GEO) database (GSE86099 and GSE50224) were retrieved. We performed microRNA (miRNA) and messenger RNA (mRNA) expression analysis to identify differentially expressed genes (DEGs). The intersection of the downregulated miRNA targets and the upregulated mRNAs were used for Gene Ontology (GO) enrichment analysis, and survival risk was assessed using data from The Cancer Genome Atlas (TCGA).ResultsThere were 35 common genes, of which, based on GO enrichment, most were related to the cardiac muscle cell action. Four genes showed significant association with the estimated half-maximal inhibitory concentration (IC50) of paclitaxel: bone morphogenetic protein 1 (BMP1), dumbbell former 4 protein (DBF4), angiogenin (ANG), and MAP7 domain containing 2 (MAP7D2). Four risk factors (MP1, HIP1, ANG, and MAP7D2) were selected to generate a signature using least absolute shrinkage and selection operator (LASSO) regression. Protein-protein interaction (PPI) analysis showed guanine nucleotide-binding protein subunit beta 4 (GNB4), calcium voltage-gated channel auxiliary subunit beta 2 (CACNB2), and sodium voltage-gated channel alpha subunit 1 (SCN1A) were located at key positions of the network. Among potential risk genes, only the high expression of dedicator of cytokinesis 8 (DOCK8) was associated with poorer survival.ConclusionsThe 35 oncogenes may be involved in mechanisms of chemotherapy resistance in ESCC, as well as the corresponding enrichment and regulatory network. The signature containing 4 key risk genes merits further investigation and may provide a deeper understanding of the molecular mechanisms in ESCC treatment failure.

Neuropathologic damage induced by radiofrequency ablation at different temperatures &amp;ndash; An Experimental Study

&lt;p&gt;&lt;strong&gt;Background and Objective:&lt;/strong&gt; Radiofrequency ablation is a safe and less invasive technique that uses an electric current to damage nerve tissue to stop it from sending pain signals. This study was aimed to determine the molecular mechanism of neuropathologic damage induced by radiofrequency ablation at different temperatures.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;A total of 36 Sprague Dawley (SD) rats were used as model with neuropathological injury. These rats were divided into 6 groups based on temperature stimulation at 42℃, 47℃, 52℃, 57℃, 62℃ and 67℃. Conduction time, distance and velocity were recorded after thermal injury. Hematoxylin and eosin (H&amp;amp;E) staining was used to observe the histopathological changes of sciatic nerve. Neural ion channel proteins such as Sodium voltage-gated channel alpha subunit 9 (SCN9A), Sodium channel B3 subunit (SCN3B) and Neurofascin (NFASC) expression &amp;nbsp;in sciatic nerve tissue was &amp;nbsp;detected by Western Blot.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; Nerve conduction velocity gradually decreased with the increase in temperature and neuronal damage was seen at 67℃. H&amp;amp; E staining showed increased degeneration of neurons with an increase in temperature from 47℃ to 67℃. SCN9A and SCN3B expression at 57℃, 62℃, and 67℃ was much higher however, NFASC expression was lower at the same temperatures.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; Neuropathological damage caused by RFA at different temperatures shows&amp;nbsp; &amp;nbsp;positive correlation with nerve conduction velocity. Heat transfer injury affects the expression of SCN9A, SCN3B and NFASC in sciatic nerve tissue.&lt;/p&gt;

Novel SCN5A and GPD1L Variants Identified in Two Unrelated Han-Chinese Patients With Clinically Suspected Brugada Syndrome.

Brugada syndrome (BrS) is a complexly genetically patterned, rare, malignant, life-threatening arrhythmia disorder. It is autosomal dominant in most cases and characterized by identifiable electrocardiographic patterns, recurrent syncope, nocturnal agonal respiration, and other symptoms, including sudden cardiac death. Over the last 2 decades, a great number of variants have been identified in more than 36 pathogenic or susceptibility genes associated with BrS. The present study used the combined method of whole exome sequencing and Sanger sequencing to identify pathogenic variants in two unrelated Han-Chinese patients with clinically suspected BrS. Minigene splicing assay was used to evaluate the effects of the splicing variant. A novel heterozygous splicing variant c.2437-2A>C in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) and a novel heterozygous missense variant c.161A>T [p.(Asp54Val)] in the glycerol-3-phosphate dehydrogenase 1 like gene (GPD1L) were identified in these two patients with BrS-1 and possible BrS-2, respectively. Minigene splicing assay indicated the deletion of 15 and 141 nucleotides in exon 16, resulting in critical amino acid deletions. These findings expand the variant spectrum of SCN5A and GPD1L, which can be beneficial to genetic counseling and prenatal diagnosis.

Mangiferin: Analgesic properties in neuropathic pain, molecular docking and meta-analysis

Aim: To investigate the analgesic effects of mangiferin (MAF) in experimental neuropathic pain (NPP) models and underlying mechanism. Methods: Databases including Pubmed, Web of Science, Embase, Cochrane Library, China national knowledge infrastructure (CNKI), Wangfang, and Weipu were used to search for the related studies. The search terms such as MAF, NPP, meta-analysis, rat, and mouse were used in various scientific databases. Meta-analysis was used to assay the analgesic efficacy of MAF. Further, molecular docking analysis was used to measure bindings of MAF and NPP-related proteins. Results: 6 studies were eventually included for the meta-analysis according to the inclusion and exclusion criteria. The meta-analysis results demonstrated that MAF significantly inhibited acetic acid-induced writhing responses, formalin-induced pain behavior, thermal analgesia, and mechanical allodynia as compared with the model control. However, risk of bias especially blinding existed in the included studies. The publication bias was checked by Begg's and Egger's tests. The molecular docking analysis indicated that MAF bound to 62 pain-related proteins at different levels. The top 10 predicted target proteins of MAF were glutamate carboxypeptidase II (GCPII), dopamine transporter (DAT), N-methyl-d-aspartic acid receptor 2B (NMDAR2B), transient receptor potential member 8 (TRPM8), monoamine oxidase type B (MAO-B), sodium voltage-gated channel alpha subunit 9A (SCN9A), fatty acid amide hydrolase (FAAH), gamma-secretase (GS), angiopoietin-1 (ANGPT1), and ANGPT2 based on the scores assessed by hydrogen bonding and hydrophobic interactions. Conclusions: In summary, MAF exerts the analgesic efficacy in the murine experimental pain models, which is associated with this compound binding to the NPP-related target proteins.

Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome.

Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.

Three-Week-Old Rabbit Ventricular Cardiomyocytes as a Novel System to Study Cardiac Excitation and EC Coupling.

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.