14-3-3ε/YWHAE regulates the transcriptional expression of cardiac sodium channel NaV1.5.

BACKGROUND: Cardiac voltage-gated sodium channel alpha subunit 5 (NaV1.5) encoded by SCN5A is associated with arrhythmia disorders. However, the molecular mechanism underlying NaV1.5 expression remains to be fully elucidated. Previous studies have reported that the 14-3-3 family acts as an adaptor involved in regulating kinetic characteristics of cardiac ion channels. OBJECTIVE: The purpose of this study was to establish 14-3-3ε/YWHAE, a member of the 14-3-3 family, as a crucial regulator of NaV1.5 and to explore the potential role of 14-3-3ε in the heart. METHODS: Western blotting, patch clamping, real-time reverse transcription-polymerase chain reaction, RNA immunoprecipitation, electrocardiogram recording, echocardiography, and histologic analysis were performed. RESULTS: YWHAE overexpression significantly reduced the expression level of SCN5A mRNA and sodium current density, whereas YWHAE knockdown significantly increased SCN5A mRNA expression and sodium current density in HEK293/NaV1.5 and H9c2 cells. Similar results were observed in mice injected with adeno-associated virus serotype 9-mediated YWHAE knockdown. The effect of 14-3-3ε on NaV1.5 expression was abrogated by knockdown of TBX5, a transcription factor of NaV1.5. An interaction between 14-3-3ε protein and TBX5 mRNA was identified, and YWHAE overexpression significantly decreased TBX5 mRNA stability without affecting SCN5A mRNA stability. In addition, mice subjected to adeno-associated virus serotype 9-mediated YWHAE knockdown exhibited shorter R-R intervals and higher prevalence of premature ventricular contractions. CONCLUSION: Our data unveil a novel regulatory mechanism of NaV1.5 by 14-3-3ε and highlight the significance of 14-3-3ε in transcriptional regulation of NaV1.5 expression and cardiac arrhythmias.

Self-Reporting Microsensors Inspired by Noctiluca Scintillans for Small-Defect Positioning and Electrical-Stress Visualization in Polymers.

Small defects induce concentrated electrical stress in dielectric polymers, leading to premature failure of materials. Existing sensing methods fail to effectively visualize these defects owing to the invisible-energy state of the electric field. Thus, it is necessary to establish a nondestructive method for the real-time detection of small defects in dielectric polymers. In this study, a self-reporting microsensor (SRM) inspired by Noctiluca scintillans is designed to endow materials with the ability of self-detection for defects and electrical stress. The SRM leverages the energy of a nearby electric field to emit measurable fluorescence, enabling defect localization and diagnosis as well as electrical-stress visualization. A controllable dielectric microsphere is constructed to achieve an adjustable electroluminescence threshold for the SRM, thereby increasing its detection accuracy while decreasing the electroluminescence threshold. The potential degradation in the polymer performance owing to SRM implantation is addressed by assembling long molecular chains on the SRM surface to spontaneously generate an interpenetrating network. Results of finite element analyses and experiments demonstrate that the SRM can effectively realize nondestructive visualization and positioning of small defects and concentrated electrical stress in polymers, positioning it as a promising sensing method for monitoring the electric field and charge distribution in materials.

Identification of a novel missense SCN5A mutation in a Chinese Han family with Brugada syndrome.

Brugada syndrome is an inherited cardiac arrythmia causes sudden death usually associated with loss-of-function mutations of SCN5A, a gene encodes α subunit of cardiac sodium channel Nav1.5 which plays key role in cardiac function. SCN5A mutation screen is often applied to diagnosis of Brugada syndrome, while its genetic etiology remains not fully understood. In present study, we performed sequence analysis of SCN5A gene in a Chinese Han family with Brugada syndrome, and found a novel heterozygous mutation (c.4969 C > T, p.Leu1657Phe). Functional electrophysiological study showed that the mutation reduced ∼60% sodium current density and largely reduced Nav1.5 activation (positively shifted activation curve by 13.93 mV), which are the key features for the pathogenesis of Brugada syndrome. However, the mutation enhanced Nav1.5 function as it slightly decreased inactivation (positively shifted inactivation curve by 7.4 mV) and accelerated recovery (decreased fast recovery by 1.39 ms). In addition, the mutation acts in a dominant negatively manner as it reduced ∼49% sodium current densities in heterozygous state. In conclusion, the study describes a novel SCN5A mutation of p.Leu1657Phe associated with Brugada syndrome, the mutation reduced current density in a dominant negative manner and altered gating kinetics, which will benefit early clinical diagnosis of Brugada syndrome.

Identification and functional analysis of two new de novo KCNMA1 variants associated with Liang-Wang syndrome.

AIM: Loss-of-function KCNMA1 variants cause Liang-Wang syndrome (MIM #618729), a newly identified multiple malformation syndrome with a broad spectrum of developmental and neurological phenotypes. However, the full spectrum of clinical features and underlying pathogenic mechanisms need full elucidation. METHODS: Exome sequencing was used to identify pathogenic variants. Patch-clamp recordings were performed to access the effects of KCNMA1 variants on BK channels. Total and membrane protein expression levels of BK channels were characterized using Western blotting. RESULTS: We report identification and functional characterization of two new de novo loss-of-function KCNMA1 variants p.(A172T) and p.(A314T) with characteristics of Liang-Wang syndrome. Variant p.(A172T) is associated with developmental delay, cognitive impairment and ataxia. Mechanistically, p.(A172T) abolishes BK potassium current, inhibits Mg2+ -dependent gating, but shifts conductance-voltage (G-V) curves to more positive potentials when complexed with WT channels. Variant p.(A314T) is associated with developmental delay, intellectual disability, cognitive impairment, mild ataxia and generalized epilepsy; suppresses BK current amplitude; and shifts G-V curves to more positive potentials when expressed with WT channels. In addition, two new patients with previously reported gain-of-function variants p.(N536H) and p.(N995S) are found to show epilepsy and paroxysmal dyskinesia as reported previously, but also exhibit additional symptoms of cognitive impairment and dysmorphic features. Furthermore, variants p.(A314T) and p.(N536H) reduced total and membrane levels of BK proteins. CONCLUSION: Our findings identified two new loss-of-function mutations of KCNMA1 associated with Liang-Wang syndrome, expanded the spectrum of clinical features associated with gain-of-function KCNMA1 variants and emphasized the overlapping features shared by gain-of-function and loss-of-function mutations.

Mechanistic insights into the interaction of cardiac sodium channel Nav1.5 with MOG1 and a new molecular mechanism for Brugada syndrome.

BACKGROUND: Mutations in cardiac sodium channel Nav1.5 cause Brugada syndrome (BrS). MOG1 is a chaperone that binds to Nav1.5, facilitates Nav1.5 trafficking to the cell surface, and enhances the amplitude of sodium current INa. OBJECTIVE: The purpose of this study was to identify structural elements involved in MOG1-Nav1.5 interaction and their relevance to the pathogenesis of BrS. METHODS: Systematic analyses of large deletions, microdeletions, and point mutations, and glutathione S-transferases pull-down, co-immunoprecipitation, cell surface protein quantification, and patch-clamping of INa were performed. RESULTS: Large deletion analysis defined the MOG1-Nav1.5 interaction domain to amino acids S476-H585 of Nav1.5 Loop I connecting transmembrane domains I and II. Microdeletion and point mutation analyses further defined the domain to F530T531F532R533R534R535. Mutations F530A, F532A, R533A, and R534A, but not T531A and R535A, significantly reduced MOG1-Nav1.5 interaction and eliminated MOG1-enhanced INa. Mutagenesis analysis identified D24, E36, D44, E53, and E101A of MOG1 as critical residues for interaction with Nav1.5 Loop I. We then characterized 3 mutations at the MOG1-Nav1.5 interaction domain: p.F530V, p.F532C, and p.R535Q reported from patients with long QT syndrome and BrS. We found that p.F532C reduced MOG1-Nav1.5 interaction and eliminated MOG1 function on INa; p.R535Q is also a loss-of-function mutation that reduces INa amplitude in a MOG1-independent manner, whereas p.F530V is benign as it does not have an apparent effect on MOG1 and INa. CONCLUSION: Our findings define the MOG1-Nav1.5 interaction domain to a 5-amino-acid motif of F530T531F532R533R534 in Loop I. Mutation p.F532C associated with BrS abolishes Nav1.5 interaction with MOG1 and reduces MOG1-enhanced INa density, thereby uncovering a novel molecular mechanism for the pathogenesis of BrS.

Ubiquitination-activating enzymes UBE1 and UBA6 regulate ubiquitination and expression of cardiac sodium channel Nav1.5.

Cardiac sodium channel Nav1.5 is associated with cardiac arrhythmias and heart failure. Protein ubiquitination is catalyzed by an E1-E2-E3 cascade of enzymes. However, the E1 enzyme catalyzing Nav1.5 ubiquitination is unknown. Here, we show that UBE1 and UBA6 are two E1 enzymes regulating Nav1.5 ubiquitination and expression. Western blot analysis and patch-clamping recordings showed that overexpression of UBE1 or UBA6 increased the ubiquitination of Nav1.5 and significantly reduced Nav1.5 expression and sodium current density, and knockdown of UBE1 or UBA6 expression significantly increased Nav1.5 expression and sodium current density in HEK293/Nav1.5 cells. Similar results were obtained in neonatal cardiomyocytes. Bioinformatic analysis predicted two ubiquitination sites at K590 and K591. Mutations of K590 and K591 to K590A and K591A abolished the effects of overexpression or knockdown of UBE1 or UBA6 on Nav1.5 expression and sodium current density. Western blot analysis showed that the effects of UBE1 or UBA6 overexpression on the ubiquitination and expression of Nav1.5 were abolished by knockdown of UBC9, a putative E2 enzyme reported for Nav1.5 ubiquitination by us. Interestingly, real-time RT-PCR analysis showed that the expression level of UBE1, but not UBA6, was significantly up-regulated in ventricular tissues from heart failure patients. These data establish UBE1 and UBA6 as the E1 enzymes involved in Nav1.5 ubiquitination, and suggest that UBE1 and UBA6 regulate ubiquitination of Nav1.5 through UBC9. Our study is the first to reveal the regulatory role of the UBE1 or UBA6 E1 enzyme in the ubiquitination of an ion channel and links UBE1 up-regulation to heart failure.

Identification of rare variants in cardiac sodium channel ß4-subunit gene SCN4B associated with ventricular tachycardia.

Ventricular tachycardia (VT) causes sudden cardiac death, however, the majority of risk genes for VT remain unknown. SCN4B encodes a ß-subunit, Navß4, for the voltage-gated cardiac sodium channel complex involved in generation and conduction of the cardiac action potential. We hypothesized that genomic variants in SCN4B increase the risk of VT. We used high-resolution melt analysis followed by Sanger sequencing to screen 199 VT patients to identify nonsynonymous variants in SCN4B. Two nonsynonymous heterozygous variants in SCN4B were identified in VT patients, including p.Gly8Ser in four VT patients and p.Ala145Ser in one VT patient. Case-control association studies were used to assess the association between variant p.Gly8Ser and VT in two independent populations for VT (299 VT cases vs. 981 controls in population 1 and 270 VT patients vs. 639 controls in population 2). Significant association was identified between p.Gly8Ser and VT in population 1 (P = 1.21 × 10-4, odds ratio or OR = 11.04), and the finding was confirmed in population 2 (P = 0.03, OR = 3.62). The association remained highly significant in the combined population (P = 3.09 × 10-5, OR = 6.17). Significant association was also identified between p.Gly8Ser and idiopathic VT (P = 1.89 × 10-5, OR = 7.27). Functional analysis with Western blotting showed that both p.Gly8Ser and p.Ala145Ser variants significantly reduced the expression level of Navß4. Based on 2015 ACMG Standards and Guidelines, p.Gly8Ser and p.Ala145Ser can be classified as the pathogenic and likely pathogenic variant, respectively. Our data suggest that SCN4B is a susceptibility gene for common VT and idiopathic VT and link rare SCN4B variants with large effects (OR = 6.17-7.27) to common VT.

Significant association of rare variant p.Gly8Ser in cardiac sodium channel ß4-subunit SCN4B with atrial fibrillation.

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Nav 1.5 (encoded by the SCN5A gene) and one or more auxiliary ß-subunits, including Nav ß1 to Nav ß4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10-4 , odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.

UBC9 regulates cardiac sodium channel Nav1.5 ubiquitination, degradation and sodium current density.

Voltage-gated sodium channel Nav1.5 is critical for generation and conduction of cardiac action potentials. Mutations and expression level changes of Nav1.5 are associated with cardiac arrhythmias and sudden death. The ubiquitin (Ub) conjugation machinery utilizes three enzyme activities, E1, E2, and E3, to regulate protein degradation. Previous studies from us and others showed that Nedd4-2 acts as an E3 ubiquitin-protein ligase involved in ubiquitination and degradation of Nav1.5, however, more key regulators remain to be identified. In this study, we show that UBC9, a SUMO-conjugating enzyme, regulates ubiquitination and degradation of Nav1.5. Overexpression of UBC9 significantly decreased Nav1.5 expression and reduced sodium current densities, whereas knockdown of UBC9 expression significantly enhanced Nav1.5 expression and increased sodium current densities, in both HEK293 cells and primary neonatal cardiomyocytes. Overexpression of UBC9 increased ubiquitination of Nav1.5, and proteasome inhibitor MG132 blocked the effect of UBC9 overexpression on Nav1.5 degradation. Co-immunoprecipitation showed that UBC9 interacts with Nedd4-2. UBC9 with mutation C93S, which suppresses SUMO-conjugating activity of UBC9, was as active as wild type UBC9 in regulating Nav1.5 levels, suggesting that UBC9 regulates Nav1.5 expression levels in a SUMOylation-independent manner. Our findings thus identify a key structural element of the ubiquitin-conjugation machinery for Nav1.5 and provide important insights into the regulatory mechanism for ubiquitination and turnover of Nav1.5.

Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Nav1.5.

BACKGROUND: The cardiac sodium channel Nav1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Nav1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Nav1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Nav1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood. METHODS AND RESULTS: Here we investigated the role of the SAR1 GTPases in trafficking of Nav1.5. Overexpression of dominant-negative mutant SAR1A (T39N or H79G) or SAR1B (T39N or H79G) significantly reduces the expression level of Nav1.5 on cell surface, and decreases the peak sodium current density (INa) in HEK/Nav1.5 cells and neonatal rat cardiomyocytes. Simultaneous knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces the INa density, whereas single knockdown of either SAR1A or SAR1B has minimal effect. Computer modeling showed that the three-dimensional structure of SAR1 is similar to RAN. RAN was reported to interact with MOG1, a small protein involved in regulation of the ER exit of Nav1.5. Co-immunoprecipitation showed that SAR1A or SAR1B interacted with MOG1. Interestingly, knockdown of SAR1A and SAR1B expression abolished the MOG1-mediated increases in both cell surface trafficking of Nav1.5 and the density of INa. CONCLUSIONS: These data suggest that SAR1A and SAR1B are the critical regulators of trafficking of Nav1.5. Moreover, SAR1A and SAR1B interact with MOG1, and are required for MOG1-mediated cell surface expression and function of Nav1.5.

Genetic Regulation of the Thymic Stromal Lymphopoietin (TSLP)/TSLP Receptor (TSLPR) Gene Expression and Influence of Epistatic Interactions Between IL-33 and the TSLP/TSLPR Axis on Risk of Coronary Artery Disease.

The thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) axis is involved in multiple inflammatory immune diseases, including coronary artery disease (CAD). To explore the causal relationship between this axis and CAD, we performed a three-stage case-control association analysis with 3,628 CAD cases and 3,776 controls using common variants in the genes TSLP, interleukin 7 receptor (IL7R), and TSLPR. Three common variants in the TSLP/TSLPR axis were significantly associated with CAD in a Chinese Han population [rs3806933T in TSLP, Padj = 4.35 × 10-5, odds ratio (OR) = 1.18; rs6897932T in IL7R, Padj = 1.13 × 10-7, OR = 1.31; g.19646A>GA in TSLPR, Padj = 2.04 × 10-6, OR = 1.20]. Reporter gene analysis demonstrated that rs3806933 and rs6897932 could influence TSLP and IL7R expression, respectively. Furthermore, the "T" allele of rs3806933 might increase plasma TSLP levels (R2 = 0.175, P < 0.01). In a stepwise procedure, the risk for CAD increased by nearly fivefold compared with the maximum effect of any single variant (Padj = 6.99 × 10-4, OR = 4.85). In addition, the epistatic interaction between TSLP and IL33 produced a nearly threefold increase in the risk of CAD in the combined model of rs3806933TT-rs7025417TT (Padj = 3.67 × 10-4, OR = 2.98). Our study illustrates that the TSLP/TSLPR axis might be involved in the pathogenesis of CAD through upregulation of mRNA or protein expression of the referenced genes and might have additive effects on the CAD risk when combined with IL-33 signaling.

αB-Crystallin Interacts with Nav1.5 and Regulates Ubiquitination and Internalization of Cell Surface Nav1.5.

Nav1.5, the pore-forming α subunit of the cardiac voltage-gated Na(+) channel complex, is required for the initiation and propagation of the cardiac action potential. Mutations in Nav1.5 cause cardiac arrhythmias and sudden death. The cardiac Na(+) channel functions as a protein complex; however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Nav1.5-interacting protein, αB-crystallin. GST pull-down, co-immunoprecipitation, and immunostaining analyses validated the interaction between Nav1.5 and αB-crystallin. Whole-cell patch clamping showed that overexpression of αB-crystallin significantly increased peak sodium current (INa) density, and the underlying molecular mechanism is the increased cell surface expression level of Nav1.5 via reduced internalization of cell surface Nav1.5 and ubiquitination of Nav1.5. Knock-out of αB-crystallin expression significantly decreased the cell surface expression level of Nav1.5. Co-immunoprecipitation analysis showed that αB-crystallin interacted with Nedd4-2; however, a catalytically inactive Nedd4-2-C801S mutant impaired the interaction and abolished the up-regulation of INa by αB-crystallin. Nav1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Nav1.5 ubiquitination and increases of INa density. Two disease-causing mutations in αB-crystallin, R109H and R151X (nonsense mutation), eliminated the effect of αB-crystallin on INa This study identifies αB-crystallin as a new binding partner for Nav1.5. αB-Crystallin interacts with Nav1.5 and increases INa by modulating the expression level and internalization of cell surface Nav1.5 and ubiquitination of Nav1.5, which requires the protein-protein interactions between αB-crystallin and Nav1.5 and between αB-crystallin and functionally active Nedd4-2.

Candidate pathway-based genome-wide association studies identify novel associations of genomic variants in the complement system associated with coronary artery disease.

BACKGROUND: Genomic variants identified by genome-wide association studies (GWAS) explain <20% of heritability of coronary artery disease (CAD), thus many risk variants remain missing for CAD. Identification of new variants may unravel new biological pathways and genetic mechanisms for CAD. To identify new variants associated with CAD, we developed a candidate pathway-based GWAS by integrating expression quantitative loci analysis and mining of GWAS data with variants in a candidate pathway. METHODS AND RESULTS: Mining of GWAS data was performed to analyze variants in 32 complement system genes for positive association with CAD. Functional variants in genes showing positive association were then identified by searching existing expression quantitative loci databases and validated by real-time reverse transcription polymerase chain reaction. A follow-up case-control design was then used to determine whether the functional variants are associated with CAD in 2 independent GeneID Chinese populations. Candidate pathway-based GWAS identified positive association between variants in C3AR1 and C6 and CAD. Two functional variants, rs7842 in C3AR1 and rs4400166 in C6, were found to be associated with expression levels of C3AR1 and C6, respectively. Significant association was identified between rs7842 and CAD (P=3.99×10(-6); odds ratio, 1.47) and between rs4400166 and CAD (P=9.30×10(-3); odds ratio, 1.24) in the validation cohort. The significant findings were confirmed in the replication cohort (P=1.53×10(-5); odds ratio, 1.37 for rs7842; P=8.41×10(-3); odds ratio, 1.21 for rs4400166). CONCLUSIONS: Integration of GWAS with biological pathways and expression quantitative loci is effective in identifying new risk variants for CAD. Functional variants increasing C3AR1 and C6 expression were shown to confer significant risk of CAD for the first time.