Search

Gap junctions in the rabbit sinoatrial node. Am J Physiol Heart Circ Physiol 280: H2103-H2115, 2001.--In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels. connexin; sinus node; electrophysiology; immunohistochemistry

Background Dravet syndrome is a severe genetic encephalopathy, caused by pathogenic variants in SCN1A. Low-grade parental mosaicism occurs in a substantial proportion of families (7%-13%) and has important implications for recurrence risks. However, parental mosaicism can remain undetected by methods regularly used in diagnostics. In this study, we use single-molecule molecular inversion probes (smMIP), a technique with high sensitivity for detecting lowgrade mosaic variants and high cost-effectiveness, to investigate the incidence of parental mosaicism of SCN1A variants in a cohort of 90 families and assess the feasibility of this technique. Methods Deep sequencing of SCN1A was performed using smMIPs. False positive rates for each of the proband's pathogenic variants were determined in 145 unrelated samples. If parents showed corresponding variant alleles at a significantly higher rate than the established noise ratio, mosaicism was confirmed by droplet digital PCR (ddPCR). Results Sequence coverage of at least 100× at the location of the corresponding pathogenic variant was reached for 80 parent couples. The variant ratio was significantly higher than the established noise ratio in eight parent couples, of which four (5%) were regarded as true mosaics, based on ddPCR results. The false positive rate of smMIP analysis without ddPCR was therefore 50%. Three of these variants had previously been considered de novo in the proband by Sanger sequencing. Conclusion smMIP technology combined withnext generation sequencing (NGS) performs better than Sanger sequencing in the detection of parental mosaicism. Because parental mosaicism has important implications for genetic counselling and recurrence risks, we stress the importance of implementing highsensitivity NGS-based assays in standard diagnostics. [ABSTRACT FROM AUTHOR]

ALDH7A1 and PNPO deficiencies are rare inborn errors of vitamin B6 metabolism causing perinatal seizure disorders. The phenotypic variability, however, is broad. To assess the frequency of these deficiencies in unexplained infantile epilepsy, we screened 113 patients for mutations in both genes. We identified 1 patient with an epilepsy phenotype resembling Dravet syndrome and likely pathogenic mutations in ALDH7A1. Presenting features were highly atypical of pyridoxine-dependent epilepsy, including febrile seizures, response to anticonvulsive drugs, and periods of seizure freedom without pyridoxine treatment. “Hidden” vitamin B6 deficiencies might be rare but treatable causes of unexplained epilepsy extending beyond the classical phenotypes. [ABSTRACT FROM PUBLISHER]

Objectives: To determine the prevalence of Dravet syndrome, an epileptic encephalopathy caused by SCN1A-mutations, often with seizure onset after vaccination, among infants reported with seizures following vaccination. To determine differences in characteristics of reported seizures after vaccination in children with and without SCN1A-related Dravet syndrome. Methods: Data were reviewed of 1,269 children with seizures following immunization in the first two years of life, reported to the safety surveillance system of the Dutch national immunization program between 1 January 1997 and 31 December 2006. Selective, prospective follow-up was performed of children with clinical characteristics compatible with a diagnosis of Dravet syndrome. Results: In 21.9% (n = 279) of children, a diagnosis of Dravet syndrome could not be excluded based on available clinical data (median age at follow-up 16 months). Additional follow-up data were obtained in 83.9% (n = 234) of these children (median age 8.5 years). 15 (1.2% of 1,269; 95%CI:0.6 to 1.8%) children were diagnosed with SCN1A-related Dravet syndrome. Of all reported seizures following vaccinations in the first year of life, 2.5% (95%CI:1.3 to 3.6%) were due to SCN1A-related Dravet syndrome, as were 5.9% of reported seizures (95%CI:3.1 to 8.7%) after 2nd or 3rd DTP-IPV-Hib vaccination. Seizures in children with SCN1A-related Dravet syndrome occurred more often with a body temperature below 38.5°C (57.9% vs. 32.6%, p = 0.020) and reoccurred more often after following vaccinations (26.7% vs. 4.0%, p = 0.003), than in children without a diagnosis of SCN1A-related Dravet Syndrome. Conclusions: Although Dravet syndrome is a rare genetic epilepsy syndrome, 2.5% of reported seizures following vaccinations in the first year of life in our cohort occurred in children with this disorder. Knowledge on the specific characteristics of vaccination-related seizures in this syndrome might promote early diagnosis and indirectly, public faith in vaccination safety. [ABSTRACT FROM AUTHOR]

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures-plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Nav1.1. The four mutations were co-expressed with β1 and β2 subunits in tsA201 cells, and characterized using the whole-cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild-type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage-dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Nav1.1 gating. Interestingly, while loss of Nav1.1 function is common in DS, the R865G mutation may cause DS by overall gain-of-function defects. [ABSTRACT FROM AUTHOR]

Background: Severe myoclonic epilepsy in infancy (SMEI) and borderline SMEI (SMEB) are caused by a mutation in SCN1A, which encodes a voltage-gated sodium channel α1-subunit protein. Although many mutations in SCN1A have been associated with clinical features of SMEI or SMEB from different ethnic groups, there have been no such reports from the South-East Asian populations so far. Methods: Patients 1 and 2 were Indonesian children diagnosed as having SMEI and SMEB based on their clinical features. SCN1A was screened for mutations using a combination of polymerase chain reaction and denaturing high-performance liquid chromatography. Nucleotide substitutions were confirmed on direct sequencing. Results: In patient 1, a G-to-A heterozygous transition was detected at nucleotide 4834 (c.4834G>A) in exon 25, leading to substitution of valine with isoleucine at amino acid position 1612 (p.V1612I) in the SCN1A protein. In patient 2 a T-to-G heterozygous transversion was identified at nucleotide 5266 (c.5266T>G) in exon 26, leading to substitution of cysteine with glycine at amino acid 1756 (p.C1756G) in the SCN1A protein. Both amino acid substitutions might disrupt these highly conserved regions in species from drosophila to human, leading to dysfunction of the protein. p.V1612I and p.C1756G were determined as disease-causing mutations due to their absence in the control population. Conclusion: The first cases of SMEI and SMEB are reported in South-East Asian populations. Two novel SCN1A mutations are also identified in these patients, p.V1612I and p.C1756G, which may lead to neuronal excitability or convulsions. [ABSTRACT FROM AUTHOR]

Background: Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. Methods: We established the expression of seven main cardiac ion channel α-subunits and four β-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. Results: Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. Conclusion: Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins. [ABSTRACT FROM AUTHOR]

Introduction: Since altered expression of gap junction proteins (connexins) in diseased myocardial tissue may lead to abnormal electrical coupling between cardiomyocytes and hence contribute to arrhythmogenesis, the expression of connexin(Cx)40 and Cx43 was studied in atrial appendage from goats in sinus rhythm (SR) and persistent atrial fibrillation (AF). Methods and Results: Biopsies were taken from the left and right atrial appendages from goats in SR or after pacing-induced persistent AF. Analyses of Cx40 and Cx43 mRNA and protein levels, using quantitative (competitive) polymerase chain reaction and western blotting, respectively, revealed no significant changes in the overall expression of Cx40 and Cx43 as a result of persistent AF. At the cellular level, immunohistochemistry and confocal laser scanning microscopy showed a homogeneous distribution of either connexin in atrial sections taken during SR. After induction of AF, the distribution of Cx43 gap junctions was unchanged whereas the Cx40 pattern showed marked inhomogeneities with small areas (0.15 to 0.6 mm in diameter, 25% of section surface area) of low-density Cx40 located between larger areas of normal (unchanged) Cx40 density. Activation mapping (244 electrodes, spatial resolution 2.25 mm) of the right atrial wall did not reveal changes in atrial conduction velocity. Conclusion: Pacing-induced persistent AF in the goat gave rise to changes in the spatial organization of Cx40 gap junctions. Although the overall conduction velocity appeared not to have changed, microheterogeneities in conduction due to the local redistribution of Cx40 gap junctions might have contributed to the initiation and maintenance of AF. [ABSTRACT FROM AUTHOR]

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Nav1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Nav1.1 mutants--R859H (GEFS+) and R865G (DS)--at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Nav1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures. [ABSTRACT FROM AUTHOR]

Background: SCN1A is one of the most important epilepsy-related genes, with pathogenic variants leading to a range of phenotypes with varying disease severity. Different modifying factors have been hypothesized to influence SCN1A-related phenotypes. We investigate the presence of rare and more common variants in epilepsy-related genes as potential modifiers of SCN1A-related disease severity., Methods: 87 patients with SCN1A-related epilepsy were investigated. Whole-exome sequencing was performed by the Beijing Genomics Institute (BGI). Functional variants in 422 genes associated with epilepsy and/or neuronal excitability were investigated. Differences in proportions of variants between the epilepsy genes and four control gene sets were calculated, and compared to the proportions of variants in the same genes in the ExAC database., Results: Statistically significant excesses of variants in epilepsy genes were observed in the complete cohort and in the combined group of mildly and severely affected patients, particularly for variants with minor allele frequencies of <0.05. Patients with extreme phenotypes showed much greater excesses of epilepsy gene variants than patients with intermediate phenotypes., Conclusion: Our results indicate that relatively common variants in epilepsy genes, which would not necessarily be classified as pathogenic, may play a large role in modulating SCN1A phenotypes. They may modify the phenotypes of both severely and mildly affected patients. Our results may be a first step toward meaningful testing of modifier gene variants in regular diagnostics for individual patients, to provide a better estimation of disease severity for newly diagnosed patients., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Pathogenic variants in SCN1A cause variable epilepsy disorders with different disease severities. We here investigate whether common variation in the promoter region of the unaffected SCN1A allele could reduce normal expression, leading to a decreased residual function of Nav1.1, and therefore to more severe clinical outcomes in patients affected by pathogenic SCN1A variants., Methods: Five different SCN1A promoter-haplotypes were functionally assessed in SH-SY5Y cells using Firefly and Renilla luciferase assays. The SCN1A promoter region was analyzed in a cohort of 143 participants with SCN1A pathogenic variants. Differences in clinical features and outcomes between participants with and without common variants in the SCN1A promoter-region of their unaffected allele were investigated., Results: All non-wildtype haplotypes showed a significant reduction in luciferase expression, compared to the wildtype promoter-region (65%-80%, p = 0.039-0.0023). No statistically significant differences in clinical outcomes were observed between patients with and without common promoter variants. However, patients with a wildtype promoter-haplotype on their unaffected SCN1A allele showed a nonsignificant trend for milder phenotypes., Conclusion: The nonsignificant observed trends in our study warrant replication studies in larger cohorts to explore the potential modifying role of these common SCN1A promoter-haplotypes., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Variants in the SCN2A gene cause a broad spectrum of epilepsy syndromes of variable severity including benign neonatal-infantile epilepsy (BFNIE), developmental and epileptic encephalopathies (DEE), and other neuropsychiatric disorders. Here, we studied three newly identified variants, which caused distinct phenotypes observed in nine affected individuals of three families, including BFNIE, and DEE with intractable neonatal seizures. Whole cell patch-clamp recordings of transfected tsA201 cells disclosed an increased current density and an increased subthreshold sodium inward current upon an action potential stimulus (p.(Lys908Glu)), a hyperpolarizing shift of the activation curve (p.(Val208Glu) and p.(Thr773Ile)), and an increased persistent current (p.(Thr773Ile)). To evaluate genotype-phenotype correlations, we next developed scoring systems for both the extent of the electrophysiological dysfunction and the severity of the clinical phenotype and applied those to 21 previously and newly functionally characterized SCN2A variants. All inherited variants were associated with a mild clinical phenotype and a lower electrophysiological score compared to those occurring de novo and causing severe phenotypes. Our results thus reveal a nice correlation between the extent of channel dysfunction and the clinical severity., (© 2018 Wiley Periodicals, Inc.)

Background: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation., Methods: To increase the number of validated EE genes, we sequenced 26 known and 351 candidate genes for EE in 360 patients. Variants in 25 genes known to be involved in EE or related phenotypes were followed up in 41 patients. We prioritized the candidate genes, and followed up 31 variants in this prioritized subset of candidate genes., Results: Twenty-nine genotypes in known genes for EE (19) or related diseases (10), dominant as well as recessive or X-linked, were classified as likely pathogenic variants. Among those, likely pathogenic de novo variants were found in EE genes that act dominantly, including the recently identified genes EEF1A2, KCNB1 and the X-linked gene IQSEC2. A de novo frameshift variant in candidate gene HNRNPU was the only de novo variant found among the followed-up candidate genes, and the patient's phenotype was similar to a few recent publications., Conclusion: Mutations in genes described in OMIM as, for example, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life.

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant alpha-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.

Background: Recordings of the electrical activity of mouse bundle branches (BBs) suggest reduced conduction velocity (CV) in the midseptal compared with the proximal part of the BB. The present study was performed to elucidate the mechanism responsible for this slowing of conduction., Methods and Results: Hearts of 16 mice were isolated and Langendorff perfused. After the right and left ventricular free walls were removed, the extracellular activity of the BB was mapped with a 247-point electrode. Premature stimulation was used to estimate CV restitution in the BBs. Expression/distribution of connexin40 (Cx40), Cx43, and Cx45 was determined. Morphology of the conduction system was assessed by whole-mount acetylcholine esterase staining and in Cx40(+/KI-GFP) hearts. Effective CV in the midseptal part of the left and right BBs was reduced by 50% compared with the proximal BB. CV restitution in the proximal and midseptal parts of the BBs was similar. Myocytes labeled positive for Cx40 and Cx45 in the entire BB. Cx43 colocalized with Cx40 and Cx45 only in the very distal BB. Subcellular distribution of gap junctions differed between proximal and distal BBs. Geometry of the midseptal and distal BBs revealed on both sides a profuse network of interlacing fibers, whereas the proximal BB consisted of a single (right BB) or multiple (left BB) parallel fibers., Conclusions: Comparison of connexin expression/distribution, geometry of the BBs, and CV characteristics suggests that increased path length for activation resulting from BB geometry is responsible for the apparently reduced CV in the midseptal BB of the mouse heart.

Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level., Methods: P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp., Results: Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels., Conclusion: P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.