Biophysical mechanisms of myocardium sodium channelopathies.

Genetic variants of gene SCN5A encoding the alpha-subunit of cardiac voltage-gated sodium channel Nav1.5 are associated with various diseases, including long QT syndrome (LQT3), Brugada syndrome (BrS1), and progressive cardiac conduction disease (PCCD). In the last decades, the great progress in understanding molecular and biophysical mechanisms of these diseases has been achieved. The LQT3 syndrome is associated with gain-of-function of sodium channels Nav1.5 due to impaired inactivation, enhanced activation, accelerated recovery from inactivation or the late current appearance. In contrast, BrS1 and PCCD are associated with the Nav1.5 loss-of-function, which in electrophysiological experiments can be manifested as reduced current density, enhanced fast or slow inactivation, impaired activation, or decelerated recovery from inactivation. Genetic variants associated with congenital arrhythmias can also disturb interactions of the Nav1.5 channel with different proteins or drugs and cause unexpected reactions to drug administration. Furthermore, mutations can affect post-translational modifications of the channels and their sensitivity to pH and temperature. Here we briefly review the current knowledge on biophysical mechanisms of LQT3, BrS1 and PCCD. We focus on limitations of studies that use heterologous expression systems and induced pluripotent stem cells (iPSC) derived cardiac myocytes and summarize our understanding of genotype-phenotype relations of SCN5A mutations.

In silico analysis of TRPM4 variants of unknown clinical significance.

BACKGROUND: TRPM4 is a calcium-activated channel that selectively permeates monovalent cations. Genetic variants of the channel in cardiomyocytes are associated with various heart disorders, such as progressive familial heart block and Brugada syndrome. About97% of all known TRPM4 missense variants are classified as variants of unknown clinical significance (VUSs). The very large number of VUSs is a serious problem in diagnostics and treatment of inherited heart diseases. METHODS AND RESULTS: We collected 233 benign or pathogenic missense variants in the superfamily of TRP channels from databases ClinVar, Humsavar and Ensembl Variation to compare performance of 22 algorithms that predict damaging variants. We found that ClinPred is the best-performing tool for TRP channels. We also used the paralogue annotation method to identify disease variants across the TRP family. In the set of 565 VUSs of hTRPM4, ClinPred predicted pathogenicity of 299 variants. Among these, 12 variants are also categorized as LP/P variants in at least one paralogue of hTRPM4. We further used the cryo-EM structure of hTRPM4 to find scores of contact pairs between parental (wild type) residues of VUSs for which ClinPred predicts a high probability of pathogenicity of variants for both contact partners. We propose that 68 respective missense VUSs are also likely pathogenic variants. CONCLUSIONS: ClinPred outperformed other in-silico tools in predicting damaging variants of TRP channels. ClinPred, the paralogue annotation method, and analysis of residue contacts the hTRPM4 cryo-EM structure collectively suggest pathogenicity of 80 TRPM4 VUSs.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a "xCELLigence" real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Relationship between the Levels of lncRNA H19 in Plasma and Different Adipose Tissue Depots with Patients' Response to Bariatric Surgery.

Bariatric surgery represents a widespread approach to treating morbid obesity. The search for biomarkers to identify patients to whom this type of treatment will be most effective is needed. Our aim was to characterize the relationship of levels of lncRNA H19 in plasma and different adipose tissue depots with patients' response to bariatric surgery. The study includes control subjects, patients with obesity and patients with obesity accompanied by impaired carbohydrate metabolism (ICM). Quantitative analysis of lncRNA H19 levels has been performed using qPCR in plasma and subcutaneous (SAT) and visceral adipose tissue (VAT). Patients with obesity without ICM have higher levels of lncRNA H19 in VAT compared to SAT, and higher levels of lncRNA H19 in SAT compared to SAT of control individuals. One year after the intervention, levels of lncRNA H19 decreased in SAT of patients with obesity without ICM. The preoperative level of lncRNA H19 in VAT demonstrates a positive correlation with excess weight loss and a negative correlation with initial BMI. In conclusion, ICM affects expression of lncRNA H19 in SAT of patients with obesity. The preoperative level of lncRNA H19 in VAT can be used to predict excess weight loss in patients with obesity after bariatric surgery.

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome.

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Nav1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Nav1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at -20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNav1.5 homology models, which are based on the cryo-EM structure of rat Nav1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.

miR-21, miR-93, miR-191, miR-let-7b, and miR-499 Expression Level in Plasma and Cerebrospinal Fluid in Patients with Prolonged Disorders of Consciousness.

In recent decades, significant progress has been achieved in understanding the mechanisms of disturbance and restoration of consciousness in patients after severe brain damage resulting in prolonged disorders of consciousness (pDOC). MicroRNAs (miRs) may be potential candidates as possible biomarkers for the classification of disease subtypes, and prognosis in patients with pDOC. The aim of the study was to analyze miRs expression levels (hsa-miR-21-5p, hsa-miR-93-5p, hsa-miR-191-5p, mmu-miR-499-5p, hsa-let-7b-5p) by a real-time polymerase chain reaction in plasma and cerebrospinal fluid (CSF) from patients with pDOC and to identify a potential biomarker for dividing patients into groups according to disease severity. We analyzed the levels of investigated miRs in pDOC patients, divided by etiology, CRSI, and the total group compared with controls. Our results showed that dividing patients with pDOC into groups according to the etiology of the disease resulted in the most significant differences in the levels of miR-93, -21, and -191 in CSF and plasma samples between groups of patients. Among the analyzed miRs, we did not find a marker that would help to distinguish VS/UWS patient groups from MCS. Examining of miRs as possible prognostic markers in patients with pDOC, the starting point seems to be the cause that led to the development of the disease.

Intersegment Contacts of Potentially Damaging Variants of Cardiac Sodium Channel.

Over 1,500 missense variants of sodium channel hNav1.5, which are reported in the ClinVar database, are associated with cardiac diseases. For most of the variants, the clinical significance is uncertain (VUS), not provided (NP), or has conflicting interpretations of pathogenicity (CIP). Reclassifying these variants as pathogenic/likely pathogenic (P/LP) variants is important for diagnosing genotyped patients. In our earlier work, several bioinformatics tools and paralogue annotation method consensually predicted that 74 VUS/NP/CIP variants of 54 wild type residues (set w54) are potentially damaging variants (PDVs). Atomic mechanisms underlying dysfunction of the PDVs are unknown. Here we employed a recent cryo-EM structure of the hNav1.5 channel with likely inactivated pore domain (PD) and activated voltage-sensing domains (VSDs), and ad hoc models of the closed and open PD and resting VSDs to explore intersegment contacts of w54 residues. We found that 44 residues from set w54 contact 84 residues with 118 disease missense variants. These include 104 VUS/NP/CIP variants, most of which are associated with the loss-of-function Brugada syndrome (BrS1) or gain-of-function long QT syndrome (LQT3). Matrix representation of the PDVs and their contact variants facilitated recognition of coupled mutations associated with the same disease. In particular, BrS1-associated coupled mutations, which disturb the P-loops region with the selectivity filter slow inactivation gate, would cause the channel dysfunction. Other likely causes of the channel dysfunction include coupled BrS1-associated variants within VSDs that would destabilize their activated states and coupled LQT3-associated variants, which would stabilize the open PD or activated VSDs. Our study proposes mechanisms of channel dysfunction for scores of BrS1- and LQT3-associated variants, confirms status for 82% of PDVs, and suggests damaging status for their contact variants, which are currently categorized as VUS/NP/CIP variants.

Possible Interactions of Extracellular Loop IVP2-S6 With Voltage-Sensing Domain III in Cardiac Sodium Channel.

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson-Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.

L-Type Calcium Channel: Predicting Pathogenic/Likely Pathogenic Status for Variants of Uncertain Clinical Significance.

(1) Background: Defects in gene CACNA1C, which encodes the pore-forming subunit of the human Cav1.2 channel (hCav1.2), are associated with cardiac disorders such as atrial fibrillation, long QT syndrome, conduction disorders, cardiomyopathies, and congenital heart defects. Clinical manifestations are known only for 12% of CACNA1C missense variants, which are listed in public databases. Bioinformatics approaches can be used to predict the pathogenic/likely pathogenic status for variants of uncertain clinical significance. Choosing a bioinformatics tool and pathogenicity threshold that are optimal for specific protein families increases the reliability of such predictions. (2) Methods and Results: We used databases ClinVar, Humsavar, gnomAD, and Ensembl to compose a dataset of pathogenic/likely pathogenic and benign variants of hCav1.2 and its 20 paralogues: voltage-gated sodium and calcium channels. We further tested the performance of sixteen in silico tools in predicting pathogenic variants. ClinPred demonstrated the best performance, followed by REVEL and MCap. In the subset of 309 uncharacterized variants of hCav1.2, ClinPred predicted the pathogenicity for 188 variants. Among these, 36 variants were also categorized as pathogenic/likely pathogenic in at least one paralogue of hCav1.2. (3) Conclusions: The bioinformatics tool ClinPred and the paralogue annotation method consensually predicted the pathogenic/likely pathogenic status for 36 uncharacterized variants of hCav1.2. An analogous approach can be used to classify missense variants of other calcium channels and novel variants of hCav1.2.

Regulatory Action of Plasma from Patients with Obesity and Diabetes towards Muscle Cells Differentiation and Bioenergetics Revealed by the C2C12 Cell Model and MicroRNA Analysis.

Obesity and type 2 diabetes mellitus (T2DM) are often combined and pathologically affect many tissues due to changes in circulating bioactive molecules. In this work, we evaluated the effect of blood plasma from obese (OB) patients or from obese patients comorbid with diabetes (OBD) on skeletal muscle function and metabolic state. We employed the mouse myoblasts C2C12 differentiation model to test the regulatory effect of plasma exposure at several levels: (1) cell morphology; (2) functional activity of mitochondria; (3) expression levels of several mitochondria regulators, i.e., Atgl, Pgc1b, and miR-378a-3p. Existing databases were used to computationally predict and analyze mir-378a-3p potential targets. We show that short-term exposure to OB or OBD patients' plasma is sufficient to affect C2C12 properties. In fact, the expression of genes that regulate skeletal muscle differentiation and growth was downregulated in both OB- and OBD-treated cells, maximal mitochondrial respiration rate was downregulated in the OBD group, while in the OB group, a metabolic switch to glycolysis was detected. These alterations correlated with a decrease in ATGL and Pgc1b expression in the OB group and with an increase of miR-378a-3p levels in the OBD group.

Association of Common Genetic Risk Variants With Gestational Diabetes Mellitus and Their Role in GDM Prediction.

Objective: We aimed to explore the associations between common genetic risk variants with gestational diabetes mellitus (GDM) risk in Russian women and to assess their utility in the identification of GDM cases. Methods: We conducted a case-control study including 1,142 pregnant women (688 GDM cases and 454 controls) enrolled at Almazov National Medical Research Centre. The International Association of Diabetes and Pregnancy Study Groups criteria were used to diagnose GDM. A total of 11 single- nucleotide polymorphisms (SNPs), including those in HKDC1 (rs10762264), GCK (rs1799884), MTNR1B (rs10830963 and rs1387153), TCF7L2 (rs7903146 and rs12255372), KCNJ11 (rs5219), IGF2BP2 (rs4402960), IRS1 (rs1801278), FTO (rs9939609), and CDKAL1 (rs7754840) were genotyped using Taqman assays. A logistic regression model was used to calculate odds ratios (ORs) and their confidence intervals (CIs). A simple-count genetic risk score (GRS) was calculated using 6 SNPs. The area under the receiver operating characteristic curve (c-statistic) was calculated for the logistic regression model predicting the risk of GDM using clinical covariates, SNPs that had shown a significant association with GDM in our study, GRS, and their combinations. Results: Two variants in MTNR1B (rs1387153 and rs10830963) demonstrated a significant association with an increased risk of GDM. The association remained significant after adjustment for age, pre-gestational BMI, arterial hypertension, GDM in history, impaired glucose tolerance, polycystic ovary syndrome, family history of diabetes, and parity (P = 0.001 and P < 0.001, respectively). After being conditioned by each other, the effect of rs1387153 on GDM predisposition weakened while the effect of rs10830963 remained significant (P = 0.004). The risk of GDM was predicted by clinical variables (c-statistic 0.712, 95 % CI: 0.675 - 0.749), and the accuracy of prediction was modestly improved by adding GRS to the model (0.719, 95 % CI 0.682 - 0.755), and more by adding only rs10830963 (0.729, 95 % CI 0.693 - 0.764). Conclusion: Among 11 SNPs associated with T2D and/or GDM in other populations, we confirmed significant association with GDM for two variants in MTNR1B in Russian women. However, these variants showed limited value in the identification of GDM cases.

Different Expressions of Pericardial Fluid MicroRNAs in Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Ischemic Heart Disease Undergoing Ventricular Tachycardia Ablation.

Introduction: Pericardial fluid is enriched with biologically active molecules of cardiovascular origin including microRNAs. Investigation of the disease-specific extracellular microRNAs could shed light on the molecular processes underlying disease development. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease characterized by life-threatening arrhythmias and progressive heart failure development. The current data about the association between microRNAs and ARVC development are limited. Methods and Results: We performed small RNA sequence analysis of microRNAs of pericardial fluid samples obtained during transcutaneous epicardial access for ventricular tachycardia (VT) ablation of six patients with definite ARVC and three post-infarction VT patients. Disease-associated microRNAs of pericardial fluid were identified. Five microRNAs (hsa-miR-1-3p, hsa-miR-21-5p, hsa-miR-122-5p, hsa-miR-206, and hsa-miR-3679-5p) were found to be differentially expressed between patients with ARVC and patients with post-infarction VT. Enrichment analysis of differentially expressed microRNAs revealed their close linkage to cardiac diseases. Conclusion: Our data extend the knowledge of pericardial fluid microRNA composition and highlight five pericardial fluid microRNAs potentially linked to ARVC pathogenesis. Further studies are required to confirm the use of pericardial fluid RNA sequencing in differential diagnosis of ARVC.

LMNA Mutations G232E and R482L Cause Dysregulation of Skeletal Muscle Differentiation, Bioenergetics, and Metabolic Gene Expression Profile.

Laminopathies are a family of monogenic multi-system diseases resulting from mutations in the LMNA gene which include a wide range of neuromuscular disorders. Although lamins are expressed in most types of differentiated cells, LMNA mutations selectively affect only specific tissues by mechanisms that remain largely unknown. We have employed the combination of functional in vitro experiments and transcriptome analysis in order to determine how two LMNA mutations associated with different phenotypes affect skeletal muscle development and metabolism. We used a muscle differentiation model based on C2C12 mouse myoblasts genetically modified with lentivirus constructs bearing wild-type human LMNA (WT-LMNA) or R482L-LMNA/G232E-LMNA mutations, linked to familial partial lipodystrophy of the Dunnigan type and muscular dystrophy phenotype accordingly. We have shown that both G232E/R482L-LMNA mutations cause dysregulation in coordination of pathways that control cell cycle dynamics and muscle differentiation. We have also found that R482/G232E-LMNA mutations induce mitochondrial uncoupling and a decrease in glycolytic activity in differentiated myotubes. Both types of alterations may contribute to mutation-induced muscle tissue pathology.

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage.

Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are often debated. WES dominated large-scale resequencing projects because of lower cost and easier data storage and processing. Rapid development of 3rd generation sequencing methods and novel exome sequencing kits predicate the need for a robust statistical framework allowing informative and easy performance comparison of the emerging methods. In our study we developed a set of statistical tools to systematically assess coverage of coding regions provided by several modern WES platforms, as well as PCR-free WGS. We identified a substantial problem in most previously published comparisons which did not account for mappability limitations of short reads. Using regression analysis and simple machine learning, as well as several novel metrics of coverage evenness, we analyzed the contribution from the major determinants of CDS coverage. Contrary to a common view, most of the observed bias in modern WES stems from mappability limitations of short reads and exome probe design rather than sequence composition. We also identified the ~ 500 kb region of human exome that could not be effectively characterized using short read technology and should receive special attention during variant analysis. Using our novel metrics of sequencing coverage, we identified main determinants of WES and WGS performance. Overall, our study points out avenues for improvement of enrichment-based methods and development of novel approaches that would maximize variant discovery at optimal cost.

Clinical Response to Personalized Exercise Therapy in Heart Failure Patients with Reduced Ejection Fraction is Accompanied by Skeletal Muscle Histological Alterations.

Abstract: Heart failure (HF) is associated with skeletal muscle wasting and exercise intolerance. This study aimed to evaluate the exercise-induced clinical response and histological alterations. One hundred and forty-four HF patients were enrolled. The individual training program was determined as a workload at or close to the lactate threshold (LT1); clinical data were collected before and after 12 weeks/6 months of training. The muscle biopsies from eight patients were taken before and after 12 weeks of training: histology analysis was used to evaluate muscle morphology. Most of the patients demonstrated a positive response after 12 weeks of the physical rehabilitation program in one or several parameters tested, and 30% of those showed improvement in all four of the following parameters: oxygen uptake (VO2) peak, left ventricular ejection fraction (LVEF), exercise tolerance (ET), and quality of life (QOL); the walking speed at LT1 after six months of training showed a significant rise. Along with clinical response, the histological analysis detected a small but significant decrease in both fiber and endomysium thickness after the exercise training course indicating the stabilization of muscle mechanotransduction system. Together, our data show that the beneficial effect of personalized exercise therapy in HF patients depends, at least in part, on the improvement in skeletal muscle physiological and biochemical performance.

Characterization of a novel SCN5A genetic variant A1294G associated with mixed clinical phenotype.

Mutations in gene SCN5A, which encodes cardiac voltage-gated sodium channel Nav1.5, are associated with multiple clinical phenotypes. Here we describe a novel A1294G genetic variant detected in a male patient with combined clinical phenotype including atrioventricular II block, Brugada-like ECG, septal fibrosis, right ventricular dilatation and decreased left ventricular contractility. Residue A1294 is located in the IIIS3-S4 extracellular loop, in proximity to several residues whose mutations are associated with sodium channelopathies. The wild-type channel Nav1.5 and mutant Nav1.5-A1294G were expressed in the CHO-K1 and HEK293T cells and whole-cell sodium currents were recorded using the patch-clamp method. The A1294G channels demonstrated a negative shift of steady-state inactivation, accelerated fast and slow inactivation and decelerated recovery from intermediate inactivation. Our study reveals biophysical mechanism of the Nav1.5-A1294G dysfunction, which may underlie the combined phenotypic manifestation observed in the patient.

Atomic Mechanisms of Timothy Syndrome-Associated Mutations in Calcium Channel Cav1.2.

Timothy syndrome (TS) is a very rare multisystem disorder almost exclusively associated with mutations G402S and G406R in helix IS6 of Cav1.2. Recently, mutations R518C/H in helix IIS0 of the voltage sensing domain II (VSD-II) were described as a cause of cardiac-only TS. The three mutations are known to decelerate voltage-dependent inactivation (VDI). Here, we report a case of cardiac-only TS caused by mutation R518C. To explore possible impact of the three mutations on interdomain contacts, we modeled channel Cav1.2 using as templates Class Ia and Class II cryo-EM structures of presumably inactivated channel Cav1.1. In both models, R518 and several other residues in VSD-II donated H-bonds to the IS6-linked α1-interaction domain (AID). We further employed steered Monte Carlo energy minimizations to move helices S4-S5, S5, and S6 from the inactivated-state positions to those seen in the X-ray structures of the open and closed NavAb channel. In the open-state models, positions of AID and VSD-II were similar to those in Cav1.1. In the closed-state models, AID moved along the ß subunit (Cavß) toward the pore axis and shifted AID-bound VSD-II. In all the models R518 retained strong contacts with AID. Our calculations suggest that conformational changes in VSD-II upon its deactivation would shift AID along Cavß toward the pore axis. The AID-linked IS6 would bend at flexible G402 and G406, facilitating the activation gate closure. Mutations R518C/H weakened the IIS0-AID contacts and would retard the AID shift. Mutations G406R and G402S stabilized the open state and would resist the pore closure. Several Cav1.2 mutations associated with long QT syndromes are consistent with this proposition. Our results provide a mechanistic rationale for the VDI deceleration caused by TS-associated mutations and suggest targets for further studies of calcium channelopathies.

Association of tribbles homologue 1 gene expression in human umbilical vein endothelial cells with duration of intrauterine exposure to hyperglycaemia.

Maternal gestational diabetes mellitus (GDM) is considered to be an important factor that epigenetically predisposes offspring to metabolic and cardiovascular diseases. However, the mechanisms of how intrauterine hyperglycaemia affects offspring have not been thoroughly studied. The mammalian tribbles homologue 1 (TRIB1) gene is associated with plasma lipid concentrations and coronary artery disease (CAD). Our aim was to study the effect of GDM and its treatment terms on the level of TRIB1 gene expression in human umbilical vein endothelial cells (HUVECs) of newborns from women with and without GDM. The study included 50 women with GDM and 25 women without GDM (control group). Women with GDM were divided into three groups according to their gestational age when the treatment of GDM started: 24-28 weeks (GDM1, N = 16), 29-32 weeks (GDM2, N = 25) and >34 weeks (GDM3, N = 9). The levels of TRIB1 gene expression in GDM3, GDM2, GDM1 and control groups were 2.8 ± 1.1, 4.2 ± 2.4, 6.0 ± 3.4 and 8.1 ± 6.1, respectively (p = 0.001). After comparison in pairs the difference was significant for the following pairs: GDM2-control (p = 0.004), GDM3-control (p = 0.002), GDM1-GDM3 (p = 0.012). Notably, if treatment had been started before the 28th week of gestation, the difference in TRIB1 gene expression in HUVECs was not significant (p = 0.320 for comparison between GDM1 and control groups). Our findings support the hypothesis that TRIB1 gene expression in HUVECs depends on the duration of intrauterine exposure to hyperglycaemia.

Effect of gene-lifestyle interaction on gestational diabetes risk.

We hypothesized that the association of certain lifestyle parameters with gestational diabetes mellitus (GDM) risk would depend on susceptibility loci. In total, 278 Russian women with GDM and 179 controls completed questionnaires about lifestyle habits (food consumption, physical activity and smoking). GDM was diagnosed according to the criteria of the International Association of Diabetes and Pregnancy Study Groups. Maternal blood was sampled for genotyping single-nucleotide polymorphisms (SNPs) in MTNR1B (rs10830963 and rs1387153), GCK (rs1799884), KCNJ11 (rs5219), IGF2BP2 (rs4402960), TCF7L2 (rs7903146 and rs12255372), CDKAL1 (rs7754840), IRS1 (rs1801278) and FTO (rs9939609). Binary logistic regression revealed an interaction effect of sausage intake and the number of risk alleles of two SNPs (rs10830963 in MTNR1B and rs1799884 in GCK) on GDM risk (P < 0.001). Among women without risk alleles of these two SNPs, sausage consumption was positively associated with GDM risk (P trend = 0.045). This difference was not revealed in women carrying 1 or more risk alleles. The risk of GDM increased as the number of risk alles increased in participants with low and moderate sausage consumption (P trend <0.001 and 0.006, respectively), while the risk of GDM in women with high sausage consumption remained relatively high, independent of the number of risk alleles. These findings indicate that the association of sausage consumption with GDM risk can be determined based on the number of risk alleles of rs10830963 in MTNR1B and rs1799884 in GCK.

Chicken rRNA Gene Cluster Structure.

Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5'ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3'ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity.