Genetic Burden of TNNI3K in Diagnostic Testing of Patients With Dilated Cardiomyopathy and Supraventricular Arrhythmias.

BACKGROUND: Genetic variants in TNNI3K (troponin-I interacting kinase) have previously been associated with dilated cardiomyopathy (DCM), cardiac conduction disease, and supraventricular tachycardias. However, the link between TNNI3K variants and these cardiac phenotypes shows a lack of consensus concerning phenotype and protein function. METHODS: We describe a systematic retrospective study of a cohort of patients undergoing genetic testing for cardiac arrhythmias and cardiomyopathy including TNNI3K. We further performed burden testing of TNNI3K in the UK Biobank. For 2 novel TNNI3K variants, we tested cosegregation. TNNI3K kinase function was estimated by TNNI3K autophosphorylation assays. RESULTS: We demonstrate enrichment of rare coding TNNI3K variants in DCM patients in the Amsterdam cohort. In the UK Biobank, we observed an association between TNNI3K missense (but not loss-of-function) variants and DCM and atrial fibrillation. Furthermore, we demonstrate genetic segregation for 2 rare variants, TNNI3K-p.Ile512Thr and TNNI3K-p.His592Tyr, with phenotypes consisting of DCM, cardiac conduction disease, and supraventricular tachycardia, together with increased autophosphorylation. In contrast, TNNI3K-p.Arg556_Asn590del, a likely benign variant, demonstrated depleted autophosphorylation. CONCLUSIONS: Our findings demonstrate an increased burden of rare coding TNNI3K variants in cardiac patients with DCM. Furthermore, we present 2 novel likely pathogenic TNNI3K variants with increased autophosphorylation, suggesting that enhanced autophosphorylation is likely to drive pathogenicity.

Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects.

A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.

Arrhythmic Phenotypes Are a Defining Feature of Dilated Cardiomyopathy-Associated SCN5A Variants: A Systematic Review.

BACKGROUND: Variants in the SCN5A gene, that encodes the cardiac sodium channel, Nav1.5, are associated with a highly arrhythmogenic form of dilated cardiomyopathy (DCM). Our aim was to review the phenotypes, natural history, functional effects, and treatment outcomes of DCM-associated rare SCN5A variants. METHODS: A systematic review of reported DCM-associated rare SCN5A variants was undertaken using PubMed and Embase. RESULTS: Eighteen SCN5A rare variants in 29 families with DCM (173 affected individuals) were identified. Eleven variants had undergone experimental evaluation, with 7 of these resulting in increased sustained current flow during the action potential (eg, increased window current) and at resting membrane potentials (eg, creation of a new gating pore current). These variants were located in transmembrane voltage-sensing domains and had a consistent phenotype characterized by frequent multifocal narrow and broad complex ventricular premature beats (VPB; 72% of affected relatives), ventricular arrhythmias (33%), atrial arrhythmias (32%), sudden cardiac death (13%), and DCM (56%). This VPB-predominant phenotype was not seen with 1 variant that increased late sodium current, or with variants that reduced peak current density or had mixed effects. In the latter groups, affected individuals mainly showed sinus node dysfunction, conduction defects, and atrial arrhythmias, with infrequent VPB and ventricular arrhythmias. DCM did not occur in the absence of arrhythmias for any variant. Twelve studies (23 total patients) reported treatment success in the VPB-predominant cardiomyopathy using sodium channel-blocking drug therapy. CONCLUSIONS: SCN5A variants can present with a diverse spectrum of primary arrhythmic features. A majority of DCM-associated variants cause a multifocal VPB-predominant cardiomyopathy that is reversible with sodium channel blocking drug therapy. Early recognition of the distinctive phenotype and prompt genetic testing to identify variant carriers are needed. Our findings have implications for interpretation and management of SCN5A variants found in DCM patients with and without arrhythmias.

Use of machine learning to classify high-risk variants of uncertain significance in lamin A/C cardiac disease.

BACKGROUND: Variation in lamin A/C results in a spectrum of clinical disease, including arrhythmias and cardiomyopathy. Benign variation is rare, and classification of LMNA missense variants via in silico prediction tools results in a high rate of variants of uncertain significance (VUSs). OBJECTIVE: The goal of this study was to use a machine learning (ML) approach for in silico prediction of LMNA pathogenic variation. METHODS: Genetic sequencing was performed on family members with conduction system disease, and patient cell lines were examined for LMNA expression. In silico predictions of conservation and pathogenicity of published LMNA variants were visualized with uniform manifold approximation and projection. K-means clustering was used to identify variant groups with similarly projected scores, allowing the generation of statistically supported risk categories. RESULTS: We discovered a novel LMNA variant (c.408C>A:p.Asp136Glu) segregating with conduction system disease in a multigeneration pedigree, which was reported as a VUS by a commercial testing company. Additional familial analysis and in vitro testing found it to be pathogenic, which prompted the development of an ML algorithm that used in silico predictions of pathogenicity for known LMNA missense variants. This identified 3 clusters of variation, each with a significantly different incidence of known pathogenic variants (38.8%, 15.0%, and 6.1%). Three hundred thirty-nine of 415 head/rod domain variants (81.7%), including p.Asp136Glu, were in clusters with highest proportions of pathogenic variants. CONCLUSION: An unsupervised ML method successfully identified clusters enriched for pathogenic LMNA variants including a novel variant associated with conduction system disease. Our ML method may assist in identifying high-risk VUS when familial testing is unavailable.

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.

INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.

Phosphorylation of Lamin A/C at serine 22 modulates Nav 1.5 function.

Variants in the LMNA gene, which encodes for Lamin A/C, are associated with cardiac conduction disease (CCD). We previously reported that Lamin A/C variants p.R545H and p.A287Lfs*193, which were identified in CCD patients, decreased peak INa in HEK-293 cells expressing Nav 1.5. Decreased peak INa in the cardiac conduction system could account for patients' atrioventricular block. We found that serine 22 (Ser 22) phosphorylation of Lamin A/C was decreased in the p.R545H variant and hypothesized that lamin phosphorylation modulated Nav 1.5 activity. To test this hypothesis, we assessed Nav 1.5 function in HEK-293 cells co-transfected with LMNA variants or treated with the small molecule LBL1 (lamin-binding ligand 1). LBL1 decreased Ser 22 phosphorylation by 65% but did not affect Nav 1.5 function. To test the complete loss of phosphorylation, we generated a version of LMNA with serine 22 converted to alanine 22 (S22A-LMNA); and a version of mutant R545H-LMNA that mimics phosphorylation via serine 22 to aspartic acid 22 substitution (S22D-R545H-LMNA). We found that S22A-LMNA inhibited Lamin-mediated activation of peak INa by 63% and shifted voltage-dependency of steady-state inactivation of Nav 1.5. Conversely, S22D-R545H-LMNA abolished the effects of mutant R545H-LMNA on voltage-dependency but not peak INa . We conclude that Lamin A/C Ser 22 phosphorylation can modulate Nav 1.5 function and contributes to the mechanism by which R545H-LMNA alters Nav 1.5 function. The differential impact of complete versus partial loss of Ser 22 phosphorylation suggests a threshold of phosphorylation that is required for full Nav 1.5 modulation. This is the first study to link Lamin A/C phosphorylation to Nav 1.5 function.

A Novel Missense Mutation in TNNI3K Causes Recessively Inherited Cardiac Conduction Disease in a Consanguineous Pakistani Family.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a life-threatening condition with high morbidity and mortality. It exhibits genetic and clinical heterogeneity with diverse pathomechanisms, but in most cases, it disrupts the synchronous activity of impulse-generating nodes and impulse-conduction underlying the normal heartbeat. In this study, we investigated a consanguineous Pakistani family comprised of four patients with CCD. We applied whole exome sequencing (WES) and co-segregation analysis, which identified a novel homozygous missense mutation (c.1531T>C;(p.Ser511Pro)) in the highly conserved kinase domain of the cardiac troponin I-interacting kinase (TNNI3K) encoding gene. The behaviors of mutant and native TNNI3K were compared by performing all-atom long-term molecular dynamics simulations, which revealed changes at the protein surface and in the hydrogen bond network. Furthermore, intra and intermolecular interaction analyses revealed that p.Ser511Pro causes structural variation in the ATP-binding pocket and the homodimer interface. These findings suggest p.Ser511Pro to be a pathogenic variant. Our study provides insights into how the variant perturbs the TNNI3K structure-function relationship, leading to a disease state. This is the first report of a recessive mutation in TNNI3K and the first mutation in this gene identified in the Pakistani population.

Variant Intronic Enhancer Controls SCN10A-short Expression and Heart Conduction.

BACKGROUND: Genetic variants in SCN10A, encoding the neuronal voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, encoding the major essential cardiac sodium channel NaV1.5. METHODS: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. RESULTS: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene (Scn10a-short). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a, the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short-encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts NaV1.5-mediated sodium current and heart rhythm. CONCLUSIONS: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

Molecular Pharmacology of K2P Potassium Channels.

Potassium channels of the tandem of two-pore-domain (K2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K2P channel blockers and activators.

Pathological findings of myocardium in a patient with cardiac conduction defect associated with an SCN5A mutation.

A 16-year-old Japanese man was admitted to our hospital because of syncope during exercise. His father and his younger brother had permanent pacemaker implantation because of sick sinus syndrome. Several examinations revealed first-degree atrioventricular block, complete right bundle branch block, sick sinus syndrome, and ventricular tachycardia with normal cardiac function. As no abnormalities were evident on coronary angiography, right ventricular endomyocardial biopsy was performed. It showed myocardial disarrangement and lipofuscin accumulation in hypertrophic myocytes. Moreover, electron microscopy showed a few degenerative myocytes, Z-band streaming, disarrangement, increased small capillaries with Weibel-Palade bodies in endothelial cells, and endothelial proliferations. Genetic analysis of the proband, his father, and his younger brother revealed a missense mutation, D1275N, in SCN5A, a gene which encodes sodium ion channel protein, are related to cardiomyopathy and arrhythmia. The proband was diagnosed with a cardiac conduction defect (CCD) and underwent permanent pacemaker implantation. These pathological findings suggest various myocardial changes presented in CCD patients with a missense mutation, D1275N, in SCN5A.

Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome.

Nav1.5 is the primary voltage-gated Na+ (Nav) channel in the heart. Mutations of Nav1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Nav1.5-E1784K variant at an overall resolution of 3.3 Å. The structure is nearly identical to that of the wild-type human Nav1.5 bound to quinidine. Structural mapping of 91- and 178-point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are clustered mainly to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our "door wedge" model for fast inactivation. The structural analysis presented here, with a focus on the impact of mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Nav1.5 channelopathies.

Connexin45 (GJC1) loss-of-function mutation contributes to familial atrial fibrillation and conduction disease.

BACKGROUND: Atrial fibrillation (AF) represents the most common clinical cardiac arrhythmia and substantially increases the risk of cerebral stroke, heart failure, and death. Although causative genes for AF have been identified, the genetic determinants for AF remain largely unclear. OBJECTIVE: This study aimed to investigate the molecular basis of AF in a Chinese kindred. METHODS: A 4-generation family with autosomal-dominant AF and other arrhythmias (atrioventricular block, sinus bradycardia, and premature ventricular contractions) was recruited. Genome-wide scan with microsatellite markers and linkage analysis as well as whole-exome sequencing analysis were performed. Electrophysiological characteristics and subcellular localization of the AF-linked mutant were analyzed using dual whole-cell patch clamps and confocal microscopy, respectively. RESULTS: A novel genetic locus for AF was mapped to chromosome 17q21.3, a 3.23-cM interval between markers D17S951 and D17S931, with a maximum 2-point logarithm of odds score of 4.2144 at marker D17S1868. Sequencing analysis revealed a heterozygous mutation in the mapping region, NM_005497.4:c.703A>T;p.(M235L), in the GJC1 gene encoding connexin45 (Cx45). The mutation cosegregated with AF in the family and was absent in 632 control individuals. The mutation decreased the coupling conductance in cell pairs (M235L/M235L, M235L/Cx45, M235L/Cx43, and M235L/Cx40), likely because of impaired subcellular localization. CONCLUSION: This study defines a novel genetic locus for AF on chromosome 17q21.3 and reveals a loss-of-function mutation in GJC1 (Cx45) contributing to AF and other cardiac arrhythmias.

Clinical Spectrum of SCN5A Channelopathy in Children with Primary Electrical Disease and Structurally Normal Hearts.

Sodium voltage-gated channel α subunit 5 (SCN5A)-mutations may cause an array of arrhythmogenic syndromes most frequently as an autosomal dominant trait, with incomplete penetrance, variable expressivity and male predominance. In the present study, we retrospectively describe a group of Mexican patients with SCN5A-disease causing variants in whom the onset of symptoms occurred in the pediatric age range. The study included 17 patients with clinical diagnosis of primary electrical disease, at least one SCN5A pathogenic or likely pathogenic mutation and age of onset <18 years, and all available first- and second-degree relatives. Fifteen patients (88.2%) were male, and sixteen independent variants were found (twelve missense, three truncating and one complex inframe deletion/insertion). The frequency of compound heterozygosity was remarkably high (3/17, 17.6%), with early childhood onset and severe disease. Overall, 70.6% of pediatric patients presented with overlap syndrome, 11.8% with isolated sick sinus syndrome, 11.8% with isolated Brugada syndrome (BrS) and 5.9% with isolated type 3 long QT syndrome (LQTS). A total of 24/45 SCN5A mutation carriers were affected (overall penetrance 53.3%), and penetrance was higher in males (63.3%, 19 affected/30 mutation carriers) than in females (33.3%, 5 affected/15 carriers). In conclusion, pediatric patients with SCNA-disease causing variants presented mainly as overlap syndrome, with predominant loss-of-function phenotypes of sick sinus syndrome (SSS), progressive cardiac conduction disease (PCCD) and ventricular arrhythmias.

Mechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function.

Cardiac action potentials are initiated by sodium ion (Na+) influx through voltage-gated Na+ channels. Na+ channel gain-of-function (GOF) can arise in inherited conditions due to mutations in the gene encoding the cardiac Na+ channel, such as Long QT syndrome type 3 (LQT3). LQT3 can be a "concealed" disease, as patients with LQT3-associated mutations can remain asymptomatic until later in life; however, arrhythmias can also arise early in life in LQT3 patients, demonstrating a complex age-associated manifestation. We and others recently demonstrated that cardiac Na+ channels preferentially localize at the intercalated disc (ID) in adult cardiac tissue, which facilitates ephaptic coupling and formation of intercellular Na+ nanodomains that regulate pro-arrhythmic early afterdepolarization (EAD) formation in tissue with Na+ channel GOF. Several properties related to ephaptic coupling vary with age, such as cell size and Na+ channel and gap junction (GJ) expression and distribution: neonatal cells have immature IDs, with Na+ channels and GJs primarily diffusively distributed, while adult myocytes have mature IDs with preferentially localized Na+ channels and GJs. Here, we perform an in silico study varying critical age-dependent parameters to investigate mechanisms underlying age-associated manifestation of Na+ channel GOF in a model of guinea pig cardiac tissue. Simulations predict that total Na+ current conductance is a critical factor in action potential duration (APD) prolongation. We find a complex cell size/ Na+ channel expression relationship: increases in cell size (without concurrent increases in Na+ channel expression) suppress EAD formation, while increases in Na+ channel expression (without concurrent increases in cell size) promotes EAD formation. Finally, simulations with neonatal and early age-associated parameters predict normal APD with minimal dependence on intercellular cleft width; however, variability in cellular properties can lead to EADs presenting in early developmental stages. In contrast, for adult-associated parameters, EAD formation is highly dependent on cleft width, consistent with a mechanism underlying the age-associated manifestation of the Na+ channel GOF.

Next-generation sequencing identifies a novel heterozygous I229T mutation on LMNA associated with familial cardiac conduction disease.

LMNA gene encodes Lamin A and C (Lamin A/C), which are intermediate filament protein implicating in DNA replication and transcription. Mutations in LMNA are validated to cause cardiac conduction disease (CCD) and cardiomyopathy.In a Chinese family, we identified 5 members harboring the identical heterozygous LMNA (c.686T>C, I229T) disease-causing mutation, which was not found in the 535 healthy controls. In silico analysis, we revealed structural alteration in Lamin A/C I229T mutant. Furthermore, molecular docking identified human polycomb repressive complex 2 and Lamin A/C interact with higher affinity in the presence of I229T, thus may downregulate Nav1.5 channel expression.Our findings expanded the spectrum of mutations associated with CCD and were valuable in the genetic diagnosis and clinical screening for CCD. Molecular docking analysis provided useful information of increased binding affinity between mutant Lamin A/C and polycomb repressive complex 2. However, the concrete mechanism of LMNA mutation (I229T) remains undetermined in our study, future genetics and molecular studies are still needed.

Cardiac Emerinopathy: A Nonsyndromic Nuclear Envelopathy With Increased Risk of Thromboembolic Stroke Due to Progressive Atrial Standstill and Left Ventricular Noncompaction.

BACKGROUND: Mutations in the nuclear envelope genes encoding LMNA and EMD are responsible for Emery-Dreifuss muscular dystrophy. However, LMNA mutations often manifest dilated cardiomyopathy with conduction disturbance without obvious skeletal myopathic complications. On the contrary, the phenotypic spectrums of EMD mutations are less clear. Our aims were to determine the prevalence of nonsyndromic forms of emerinopathy, which may underlie genetically undefined isolated cardiac conduction disturbance, and the etiology of thromboembolic complications associated with EMD mutations. METHODS: Targeted exon sequencing was performed in 87 probands with familial sick sinus syndrome (n=36) and a progressive cardiac conduction defect (n=51). RESULTS: We identified 3 X-linked recessive EMD mutations (start-loss, splicing, missense) in families with cardiac conduction disease. All 3 probands shared a common clinical phenotype of progressive atrial arrhythmias that ultimately resulted in atrial standstill associated with left ventricular noncompaction (LVNC), but they lacked early contractures and progressive muscle wasting and weakness characteristic of Emery-Dreifuss muscular dystrophy. Because the association of LVNC with EMD has never been reported, we further genetically screened 102 LVNC patients and found a frameshift EMD mutation in a boy with progressive atrial standstill and LVNC without complications of muscular dystrophy. All 6 male EMD mutation carriers of 4 families underwent pacemaker or defibrillator implantation, whereas 2 female carriers were asymptomatic. Notably, a strong family history of stroke observed in these families was probably due to the increased risk of thromboembolism attributable to both atrial standstill and LVNC. CONCLUSIONS: Cardiac emerinopathy is a novel nonsyndromic X-linked progressive atrial standstill associated with LVNC and increased risk of thromboembolism.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Impact of the DSP-H1684R Genetic Variant on Ion Channels Activity in iPSC-Derived Cardiomyocytes.

BACKGROUND/AIMS: Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. METHODS: We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. RESULTS: In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. CONCLUSION: Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.

POPDC2 a novel susceptibility gene for conduction disorders.

Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188⁎), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188⁎ causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188⁎ knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188⁎ loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.

Identification of a nonsense mutation in TNNI3K associated with cardiac conduction disease.

BACKGROUND: Cardiac conduction disease (CCD) is a common cardiovascular disease which can lead to life-threatening conditions. The importance of heredity in CCD has been realized in recent years. Several causal genes have been found to be implicated in CCD such as SCN5A, TRPM4, SCN1B, TNNI3K, LMNA, and NKX2.5. To date, only four genetic mutations in TNNI3K have been identified related to CCD. METHODS: Whole-exome sequencing (WES) was carried out in order to identify the underlying disease-causing mutation in a Chinese family with CCD. The potential mutations were confirmed by Sanger sequencing. Real-time qPCR was used to detect the level of TNNI3K mRNA expression. RESULTS: A nonsense mutation in TNNI3K (NM_015978.2: g.170891C > T, c.1441C > T) was identified in this family and validated by Sanger sequencing. Real-time qPCR confirmed that the level of TNNI3K mRNA expression was decreased compared with the controls. CONCLUSIONS: This study found the first nonsense TNNI3K mutation associated with CCD in a Chinese family. TNNI3K harboring the mutation (c.1441C > T) implicated a loss-of-function pathogenic mechanism with an autosomal dominant inheritance pattern. This research enriches the phenotypic spectrum of TNNI3K mutations, casting a new light upon the genotype-phenotype correlations between TNNI3K mutations and CCD and indicating the importance of TNNI3K screening in CCD patients.