hERG Research Articles

Cellular impedance assay for prediction of QT prolongation induced by hERG and non-hERG channel modulators using human stem cell derived cardiomyocytes

Torsades de pointes (TdP) arrhythmia is a potentially fatal form of ventricular arrhythmia that occurs under conditions where cardiac repolarization is delayed (as indicated by prolonged QT intervals from electrocardiographic recordings). A likely mechanism for QT interval prolongation and TdP arrhythmias is blockade of the rapid component of the cardiac delayed rectifier K+ current (IKr), which is encoded by human ether-a-go-go-related gene (HERG). Over 100 non-cardiovascular drugs have the potential to induce QT interval prolongations in the electrocardiogram (ECG) or TdP arrhythmias. The binding site of most HERG channel blockers is located inside the central cavity of the channel. An evaluation of possible effects on HERG channels during the development of novel drugs is recommended by international guidelines. During cardiac ischaemia activation of ATP-sensitive K+ (KATP) channels contributes to action potential (AP) shortening which is either cardiotoxic by inducing re-entrant ventricular arrhythmias or cardioprotective by inducing energy-sparing effects or ischaemic preconditioning (IPC). KATP channels are formed by an inward-rectifier K+ channel (Kir6.0) and a sulfonylurea receptor (SUR) subunit: Kir6.2 and SUR2A in cardiac myocytes, Kir6.2 and SUR1 in pancreatic β-cells. Sulfonylureas and glinides stimulate insulin secretion via blockade of the pancreatic β-cell KATP channel. Clinical studies about cardiotoxic effects of sulfonylureas are contradictory. Sulfonylureas and glinides differ in their selectivity for pancreatic over cardiovascular KATP channels, being either selective (tolbutamide, glibenclamide) or non-selective (repaglinide). The possibility exists that non-selective KATP channel inhibitors might have cardiovascular side effects. Blockers of the pore-forming Kir6.2 subunit are insulin secretagogues and might have cardioprotective or cardiotoxic effects during cardiac ischaemia.

Construction of a HERG mutant L539fs/47-*558W pEGFP vector and the expression of the fusion protein in HEK293 cells

ObjectiveTo construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs/47-*558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 (HEK293) cells. MethodsThe mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 I and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-*558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. ResultsThe about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery. pEGFP-C2-L539fs/47*-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. ConclusionpEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-*558W.

Abstract 142: Modeling Drug-Induced Long QT Syndrome with Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Long QT syndrome (LQTS) is characterized by prolonged cardiac repolarization time and increased risk of ventricular arrhythmia. LQTS can be either inherited or induced notably after drugs intake. Mutations in genes encoding cardiac ion channels have been reported to underlie inherited LQTS. In contrast, drug-induced LQTS (diLQTS) most frequently arises from altered function of the hERG channel; the risk of developing diLQTS varies largely between subjects and most people who have life-threatening diLQTS have no known genetic risk factors. We investigated whether the susceptibility to develop diLQTS observed in vivo can be recapitulated in vitro using patient-specific induced pluripotent stem cell (iPSC) technology. We collected skin fibroblasts from ten subjects who developed significant diLQTS after administration of Sotalol and/or Erythromycin. Ten other individuals who displayed no changes in QT interval after administration of the same drugs, were selected. iPSC were generated by retroviral delivery of Oct4, Sox2, Nanog and Klf4 in 17 of the 20 individuals. We report preliminary results obtained from iPSC-derived cardiomyocytes (iPSC-CMs) of two subjects. All experiments were performed in a blinded fashion without knowledge of the associated clinical phenotype. Cardiac differentiation of iPSC resulted in the generation of spontaneously beating embryoid bodies. iPSC-CMs showed positive staining for TNNT2, ACTN2 and Cx43. Gene expression analysis confirmed the expression of NKX2.5, MLC2v, MYH6 and MYH7, and of the relevant KCNH2 gene. The two lines had similar basal electrophysiological properties as assessed by measurements of action potential (AP) by patch-clamp technique and extracellular field potentials (FP) using micro-electrode array (MEA). E4031, a classical HERG blocker, significantly prolonged the FP duration (FPD) in a dose-dependent manner in both lines (EC50: 30.19 and 51.57 respectively). When both Sotalol and Erythromicin were used, FPD was prolonged in one of the two samples in a dose-dependent manner (EC50Sotalol: 100; EC50Erythr: 9.64) while drug response was blunted in the other cell line. This study suggests that patient-specific iPSC can be used to model the functional abnormalities observed in acquired diLQTS.

Loss‐of‐function KCNH2 mutation in a family with long QT syndrome, epilepsy, and sudden death

There has been increased interest in a possible association between epilepsy channelopathies and cardiac arrhythmias, such as long QT syndrome (LQTS). We report a kindred that features LQTS, idiopathic epilepsy, and increased risk of sudden death. Genetic study showed a previously unreported heterozygous point mutation (c.246T>C) in the KCNH2 gene. Functional studies showed that the mutation induces severe loss of function. This observation provides further evidence for a possible link between idiopathic epilepsy and LQTS.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression

Human ether-a-go-go-related gene (HERG) K+ channel underlies the rapidly activating delayed rectifier K+ conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

Mutation analysis of ion channel genes promoters in ventricular fibrillation survivors with coronary artery disease

IntroductionStrong evidence suggests that sudden cardiac death (SCD) is genetically determined. In our previous study we found that the prevalence of selected, rare coding variants in 5 long QT genes was significantly higher in ventricular fibrillation (VF) survivors with coronary artery disease (CAD) than in controls. In the present study we performed mutational analysis of the promoters of 5 LQTS-related myocardial ion channel genes in the same group of patients and in control populations. MethodsThe promoters of KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 genes were analyzed in 45 CAD individuals – survivors of documented VF. The allelic frequencies were compared either to data from the 1000 Genomes Project or from a local DNA bank of patients with coronary artery disease and no malignant arrhythmia (141 individuals). ResultsIn 34 (75.5%) of 45 VF survivors 9 different promoter variants were found: 2 in KCNQ1 gene promoter, 1 in KCNE1 promoter, and 6 in SCN5A promoter. Statistically significant differences were found in the allelic frequencies of both KCNQ1 gene promoter variants: 1-182C>T (P=0.008), 1-119G>A (P=0.007). Nevertheless, these variants did not segregate with long QT phenotype in a previous study. While the allelic frequency of the SCN5A gene promoter variant 225-1072T>C significantly differed in VF survivors compared to the 1000 Genomes Project (P=0.001), this allelic frequency was not different when compared to the group of local CAD controls. ConclusionsOur findings demonstrated that variants of ion channel gene promoters are common, both in VF survivors and control groups. These results suggest that promoter variants are geographically-specific and are not a common cause of SCD.

Postmortem molecular analysis of KCNQ1, KCNH2, KCNE1 and KCNE2 genes in sudden unexplained nocturnal death syndrome in the Chinese Han population

The etiology of sudden unexplained nocturnal death syndrome (SUNDS) remains unclear. Previous studies have implicated that SUNDS is probably allelic to cardiac sodium channel diseases such as Brugada syndrome. The variation in cardiac potassium channels is the main genetic cause of inherited long QT syndrome (LQTS), which may manifest as syncope and sudden cardiac death without structural disease. We hypothesized that cardiac potassium channel disease may be responsible for certain Chinese SUNDS cases. Genotyping of 4 main LQTS-susceptibility genes (KCNQ1, KCNH2, KCNE1, and KCNE2) was performed here for the first time in SUNDS victims from the Chinese Han population to address the pathogenic cause of some SUNDS using polymerase chain reaction and direct DNA sequencing. 120 sporadic SUNDS cases were enrolled. Genomic DNA was extracted from blood samples. A total of 2 novel non-synonymous mutations and 3 previously reported arrhythmia susceptibility polymorphisms were identified in KCNQ1, KCNH2, KCNE1, and KCNE2. We concluded that the variants in KCNQ1, KCNH2, KCNE1 and KCNE2 genes may be correlated with the occurrence of part of SUNDS cases in southern China.

Sequence and structure‐specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5' sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.

Negating the dominant-negative allele: a new treatment paradigm for arrhythmias explored in human induced pluripotent stem cell-derived cardiomyocytes

This editorial refers to ‘Allele-specific RNA interference rescues the long-QT syndrome phenotype in human induced pluripotent stem cell cardiomyocytes’[†][1], by E. Matsa et al. , on page 1078 Congenital long-QT syndrome (LQTS) is a disorder of cardiac repolarization that manifests typically with ventricular arrhythmias such as torsades de pointes in patients with prolonged QT intervals in the ECG and structurally normal hearts. The known mutations affect genes encoding either subunits of cardiac ion channels or proteins involved in their regulation. Matsa and colleagues1 describe how they succeeded in using human induced pluripotent stem cell (hiPSC) technology not only to model a subtype of LQTS (LQT2), but also to evaluate a novel therapeutic approach using human cardiomyocytes derived from patient-specific hiPSCs. The authors generated cardiomyocytes from hiPSCs derived from a patient carrying a heterozygous mutation in the KCNH2 gene, corresponding to an A561T amino acid exchange in the KCNH2 -encoded protein hERG. Four hERG subunits co-assemble to form the pore of the ion channel generating the delayed rectifier potassium current IKr, an important determinant of the cardiac action potential duration. In accordance with the clinical phenotype of the patient, the hiPSC-derived cardiomyocytes displayed prolonged action potentials. The authors further demonstrated that, similar to other LQT2-causing ion channel mutations,2 the A561T mutation results in impaired ion channel trafficking: mutated subunits were not glycosylated properly and thus were retained in a perinuclear compartment instead of being integrated into the plasma membrane. Presumably due to co-assembly, the mutation also interfered with normal trafficking of the wild-type subunits in … [1]: #fn-2

Identification of a novel KCNQ1 mutation in a large Saudi family with long QT syndrome: clinical consequences and preventive implications

Congenital long QT syndrome (LQTS) is an inherited potentially fatal arrhythmogenic disorder that is characterized by prolonged corrected QT (QTc) interval. Mutations in three genes (KCNQ1, KCNH2, and SCN5A) account for the majority of the cases. However, 10 other genes are now known to be implicated in LQTS. In this work, we describe the clinical and molecular analysis in a large Saudi family with LQTS. Screening KCNQ1, KCNH2, and SCN5A genes in the proband, who presented with syncope, led to the identification of a heterozygous mutation (p.H258P) in KCNQ1. An extended clinical and genetic screening of the family identified 11 other members who were carriers for this mutation. All identified carriers had prolonged QTc intervals; yet, only two were symptomatic. Screening the family members for three LQTS modifiers (rs4657139 and rs16847548 in NOS1AP and KCNE1-D85N) did not reveal a correlation with symptoms or QTc intervals. The electrocardiographic and molecular analysis stratified seven carriers at high risk of a cardiac event as they had a QTc of ≥500 ms and were carriers of a KCNQ1 mutation. Our work illustrates the importance of extended family screening in LQTS to identify silent carriers and hence adopt the most appropriate therapeutic and preventive intervention.

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Behavioural and functional characterization of Kv10.1 (Eag1) knockout mice

Kv10.1 (Eag1), member of the Kv10 family of voltage-gated potassium channels, is preferentially expressed in adult brain. The aim of the present study was to unravel the functional role of Kv10.1 in the brain by generating knockout mice, where the voltage sensor and pore region of Kv10.1 were removed to render non-functional proteins through deletion of exon 7 of the KCNH1 gene using the ‘3 Lox P strategy’. Kv10.1-deficient mice show no obvious alterations during embryogenesis and develop normally to adulthood; cortex, hippocampus and cerebellum appear anatomically normal. Other tests, including general health screen, sensorimotor functioning and gating, anxiety, social behaviour, learning and memory did not show any functional aberrations in Kv10.1 null mice. Kv10.1 null mice display mild hyperactivity and longer-lasting haloperidol-induced catalepsy, but there was no difference between genotypes in amphetamine sensitization and withdrawal, reactivity to apomorphine and haloperidol in the prepulse inhibition tests or to antidepressants in the haloperidol-induced catalepsy. Furthermore, electrical properties of Kv10.1 in cerebellar Purkinje cells did not show any difference between genotypes. Bearing in mind that Kv10.1 is overexpressed in over 70% of all human tumours and that its inhibition leads to a reduced tumour cell proliferation, the fact that deletion of Kv10.1 does not show a marked phenotype is a prerequisite for utilizing Kv10.1 blocking and/or reduction techniques, such as siRNA, to treat cancer.

Novel mutation in the KCNH2 gene associated with long QT syndrome

Novel mutation in the KCNH2 gene associated with long QT syndrome

Novel mutation in the KCNH2 gene associated with long QT syndrome

Novel mutation in the KCNH2 gene associated with long QT syndrome

Modulation of HERG K+ Channels by Chronic Exposure to Activators and Inhibitors of PKA and PKC: Actions Independent of PKA and PKC Phosphorylation

Background: Human ether-a-go-go-related gene (HERG) channel is the major molecular component of the native rapid delayed rectifier K⁺ current (I_Kr) that is a crucial determinant of cardiac repolarization. Impairment of I_Kr/HERG function is commonly believed to be a mechanism causing long QT syndromes (LQTS), a lethal ventricular tachyarrhythmia. The cAMP-dependent protein kinase A (PKA) and PKC activities are markedly increased in some pathological conditions of the heart such as heart failure. This study was designed to investigate the effects of acute and chronic exposure to PKA or PKC activators and inhibitors on HERG channel activities and to provide insight into the mechanisms for the modulations. Methods: Channel activity was measured in HEK293 cells stably expressing HERG using whole-cell patch-clamp techniques. Intracellular reactive oxygen species (ROS) were measured by CM-H2DFDA. Mitochondrial membrane potential (ΔΨ_m) was measured using JC-1 dye. HERG channel phosphorylation was assayed by [³²P]orthophosphate methods. Results: Acute exposure of cells to PKA or PKC activators by bath superfusion minimally affected I_HERG, and so did the PKA or PKC inhibitor. By comparison, prolonged exposure (chronic incubation) of cells to PKA or PKC activators significantly impaired HERG K⁺ channel function as reflected by reduced I_HERG density and positive shift of the steady-state activation curve. Antioxidants vitamin E and MnTBAP both abolished the depressive effects of PKA or PKC activators on HERG function. Further, both PKA and PKC activators stimulated production of intracellular reactive oxygen species (ROS), an effect efficiently prevented by antioxidants or by PKA and PKC inhibitors. Conclusions: HERG function is insensitive to PKA or PKC phosphorylation modulation per se, but can be impaired by the activators of PKA or PKC with long exposure likely via generation of ROS. In view of the critical role of HERG K⁺ channel in regulating cardiac repolarization and the sustained activation of both PKA and PKC in many pathological conditions of the heart such as heart failure, it is conceivable that HERG impairment by ROS accumulation induced by PKA and PKC contributes to the impaired cardiac repolarization.

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

Blocking of the Human Ether-à-go-go-Related Gene Channel by Imatinib Mesylate

Imatinib mesylate (IM), a widely prescribed powerful tyrosine kinase inhibitor, has been associated with increased risk of heart failure and is known to induce cell apoptosis and death in isolated cardiomyocytes. In addition to acquired long QT syndrome, pharmacological inhibition of human ether-à-go-go-related gene (HERG) channel has been reported to involve in apoptosis. The present study was undertaken to characterize the biophysical properties of IM on HERG and the molecular determinants of HERG blockade using mutant channels (Y652A and F656A). Wild type (WT) and mutant HERG channels were expressed in HEK-293 cells and Xenopus oocytes and the currents (I(HERG)) were measured using patch-clamp and two-microelectrode voltage-clamp techniques. IM inhibited WT I(HERG) in a concentration-dependent manner with an IC(50) of 19.51±2.50 µmol/L and 44.76±1.54 µmol/L in HEK-293 cells and Xenopus oocytes, respectively. The IM-induced inhibition of WT I(HERG) followed a voltage- and time-dependent manner. The blockade was enhanced by further activation of currents, which were in accordance with an open-channel blockade. The V(1/2) for steady-state activation shifted from -15.48±1.21 to -26.66±2.98 mV (p<0.05, n=6). The inactivation kinetics and voltage dependence of steady-state inactivation of the WT HERG channel were not significantly altered by IM. Two S6 domain mutants, F652A and Y656A, attenuated IM-induced inhibition of WT I(HERG). Therefore, IM preferentially blocked the open HERG channel through F652 and Y656, providing a molecular mechanism for the cardiac side effects during the clinical administration of IM.

Genetic Mutation in Korean Patients of Sudden Cardiac Arrest as a Surrogating Marker of Idiopathic Ventricular Arrhythmia

Mutation or common intronic variants in cardiac ion channel genes have been suggested to be associated with sudden cardiac death caused by idiopathic ventricular tachyarrhythmia. This study aimed to find mutations in cardiac ion channel genes of Korean sudden cardiac arrest patients with structurally normal heart and to verify association between common genetic variation in cardiac ion channel and sudden cardiac arrest by idiopathic ventricular tachyarrhythmia in Koreans. Study participants were Korean survivors of sudden cardiac arrest caused by idiopathic ventricular tachycardia or fibrillation. All coding exons of the SCN5A, KCNQ1, and KCNH2 genes were analyzed by Sanger sequencing. Fifteen survivors of sudden cardiac arrest were included. Three male patients had mutations in SCN5A gene and none in KCNQ1 and KCNH2 genes. Intronic variant (rs2283222) in KCNQ1 gene showed significant association with sudden cardiac arrest (OR 4.05). Four male sudden cardiac arrest survivors had intronic variant (rs11720524) in SCN5A gene. None of female survivors of sudden cardiac arrest had SCN5A gene mutations despite similar frequencies of intronic variants between males and females in 55 normal controls. Common intronic variant in KCNQ1 gene is associated with sudden cardiac arrest caused by idiopathic ventricular tachyarrhythmia in Koreans.

Importance of post-mortem genetic testing in SUDEP patients

There has been increased interest in a possible association between epilepsy channelopathies and cardiac arrhythmias, such as long QT syndrome (LQTS). We report a kindred that features LQTS, idiopathic epilepsy, and increased risk of sudden death. Genetic study showed a previously unreported heterozygous point mutation (c.246T>C) in the KCNH2 gene. Functional studies showed that the mutation induces severe loss of function. This observation provides further evidence for a possible link between idiopathic epilepsy and LQTS.

078: Application of Next Generation Sequencing (NGS) technologies to the molecular diagnosis of inherited cardiac diseases

Inherited cardiomyopathies and arrhythmias are autosomal dominant diseases with an important genetic heterogeneity. Their overall pre-valence, associated with the risk of sudden death, stress the need for high throughput and sensitive genetic diagnosis in order to improve genetic counseling and medical management. We evaluated the strength of Next Gene-ration Sequencing (NGS) technologies to fulfill these criteria through simultaneous sequencing of the major known causative genes described as responsible for these phenotypes. We designed a custom target capture system (Agilent) of 64 genes followed by high-throughput sequencing on HiSeq2000 (Illumina). Ten DNAs with mutations (in MYH7, MYBPC3, LMNA, FHL1, SCN5A, KCNH2, KCNQ1, DSG2, DSP and PKP2 genes) were included as control. Validation was done by evaluating capture efficiency, coverage deep, sequencing sensitivity and reproducibility. More than 90% of targeted sequences were fully covered (> 50X). After variants filtering, each DNA sample presented ~20 variations. Variants were classified according to multiple criteria such as allelic frequency, mutation type and functional effects. All 10 control mutations were identified. However 2 to 4 additional potential pathogenic variants were found in each individual. NGS based diagnostic is reliable in cardiac disorders. This technique should allow a sensitive, comprehensive, cost-effective and rapid molecular diagnosis of patients. However, our results underlined the complexity of the genetic profile of patients that should be taken into account into clinical practice.