Cardiac Electrical Disorder Research Articles

Systematic analysis of SCN5A variants associated with inherited cardiac diseases

BackgroundSCN5A variants are associated with a spectrum of cardiac electrical disorders with clear phenotypes. However, they may also be associated with complex phenotypic traits like overlap syndromes or pleiotropy, which have not been systematically described. In addition, the involvement of SCN5A in dilated cardiomyopathies (DCMs) remains controversial. ObjectiveWe aimed to evaluate the different phenotypes associated with pathogenic (P)/likely pathogenic (LP) SCN5A variants and to determine the prevalence of pleiotropy in a large multicentric cohort of P/LP SCN5A variant carriers. MethodsThe DNA of 13,510 consecutive probands (9960 with cardiomyopathies) was sequenced with a custom panel of genes. Individuals carrying a heterozygous single P/LP SCN5A variant were selected and phenotyped. ResultsThe study included 170 P/LP variants found in 495 patients. Of them, 119 (70%) were exclusively associated with a single well-established phenotype: 91 with Brugada syndrome, 15 with type 3 long QT syndrome, 6 with progressive cardiac conduction disease, 4 with multifocal ectopic Purkinje-related premature contractions, and 3 with sick sinus syndrome. Thirty-two variants (19%) were associated with overlap syndromes or pleiotropy. The 19 remaining variants (11%) were associated with atypical or unclear phenotypes. Of those, 8 were carried by 8 patients presenting with DCM with a debatable causative genotype/phenotype link. ConclusionMost P/LP SCN5A variants were found in patients with primary electrical disorders, mainly Brugada syndrome. Nearly 20% were associated with overlap syndromes or pleiotropy, underscoring the need for comprehensive phenotypic evaluation. The concept of SCN5A variants causing DCM is extremely rare (8/9960) if not questionable.

PO-01-157 MULTIELECTRODE ARRAY RECORDINGS OF IPSC CARDIOMYOCYTES SHOW GREATER VARIABILITY BETWEEN REPLICATE DIFFERENTIATIONS THAN BETWEEN DIFFERENT IPSC LINES FROM HEALTHY INDIVIDUALS AND CORRELATE WITH EXPRESSION OF SARCOMERIC GENES

Induced pluripotent stem cell (iPSC) derived cardiomyocytes are an important model system for cardiac electrical disorders. They offer the potential to detect disease-specific differences in cell lines as well as between healthy individuals of differing genetic backgrounds. However, variability of iPSC cardiomyocyte differentiations makes these studies prone to type 1 errors and optimal experimental designs and sample sizes remain undefined. We aimed to characterise the variability in electrical properties of cardiac myocytes derived from three iPSC lines obtained from healthy individuals, at baseline, in response to varying pacing rates and in the presence of two pharmacological challenges to repolarisation reserve: inhibition of IKr and augmentation of INaL. We aimed to explore the mechanisms underlying the variability in iPSC-derived cardiomyocyte phenotypes by analysing transcriptional profiles. Four replicate cardiomyocyte differentiations from three iPSC lines were analysed on 48-well multielectrode arrays (MEA). Field potential recordings were made from spontaneous beating monolayers as well as during pacing at 0.5, 1.0 and 1.5 Hz at baseline and in the presence of dofetilide (10 nM) and ATX II (25 nM). Field potential parameters were correlated with mRNA levels for a panel of 48 transcripts of key cardiac ion channel, calcium handling, sarcomeric and metabolic genes, assayed using the NanoStrings platform. Significant variability in baseline field potential parameters was observed between replicate differentiations of each line, whereas differences between iPSC lines were not statistically significant in nested ANOVA and mixed effects model analyses after taking into account within line variability. The rate-dependence of repolarisation and repolarisation reserve also showed significant intra-line differences but again interline differences were not significant. Field potential durations showed the strongest correlation with expression of two sarcomeric genes, MYH2 and MYH7. Significant variability in phenotypes between replicate differentiations of the same iPSC line indicates that a large number of replicates are needed in studies designed to detect differences between lines derived from healthy individuals. One should also take this into account when comparing diseased lines with isogenic controls. Maturation state and cardiomyocyte purity of differentiations are an important cause of variability in MEA field potential recordings.

Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

Interleukin-17A (IL-17), a potent proinflammatory cytokine, has been shown to participate in cardiac electrical disorders. Diabetes mellitus is an independent risk factor for ventricular arrhythmia. In this study, we investigated the role of IL-17 in ventricular arrhythmia of diabetic mice. Diabetes was induced in both wild-type and IL-17 knockout mice by intraperitoneal injection of streptozotocin (STZ). High-frequency electrical stimuli were delivered into the right ventricle to induce ventricular arrhythmias. We showed that the occurrence rate of ventricular tachycardia was significantly increased in diabetic mice, which was attenuated by IL-17 knockout. We conducted optical mapping on perfused mouse hearts and found that cardiac conduction velocity (CV) was significantly decreased, and action potential duration (APD) wasprolonged in diabetic mice, which were mitigated by IL-17 knockout. We performed whole-cell patch clamp recordings from isolated ventricular myocytes, and found that the densities of Ito, INa and ICa,L were reduced, the APDs at 50% and 90% repolarization were increased, and early afterdepolarization (EAD) was markedly increased in diabetic mice. These alterations were alleviated by the knockout of IL-17. Moreover, knockout of IL-17 alleviated the downregulation of Nav1.5 (the pore forming subunit of INa), Cav1.2 (the main component subunit of ICa,L) and KChIP2 (potassium voltage-gated channel interacting protein 2, the regulatory subunit of Ito) in the hearts of diabetic mice. The expression of NF-κB was significantly upregulated in the hearts of diabetic mice, which was suppressed by IL-17 knockout. In neonatal mouse ventricular myocytes, knockdown of NF-κB significantly increased the expression of Nav1.5, Cav1.2 and KChIP2. These results imply that IL-17 may represent a potential target for the development of agents against diabetes-related ventricular arrhythmias.

Exposure to nanoparticles derived from diesel particulate filter equipped engine increases vulnerability to arrhythmia in rat hearts

Air pollution is well recognized as a central player in cardiovascular disease. Exhaust particulate from diesel engines (DEP) is rich in nanoparticles and may contribute to the health effects of particulate matter in the environment. Moreover, diesel soot emitted by modern engines denotes defective surfaces alongside chemically-reactive sites increasing soot cytotoxicity. We recently demonstrated that engineered nanoparticles can cross the air/blood barrier and are capable to reach the heart. We hypothesize that DEP nanoparticles are pro-arrhythmogenic by direct interaction with cardiac cells. We evaluated the internalization kinetics and the effects of DEP, collected from Euro III (DEPe3, in the absence of Diesel Particulate Filter, DPF) and Euro IV (DEPe4, in the presence of DPF) engines, on alveolar and cardiac cell lines and on in situ rat hearts following DEP tracheal instillation. We observed significant differences in DEP size, metal and organic compositions derived from both engines. DEPe4 comprised ultrafine particles (<100 nm) and denoted a more pronounced toxicological outcome compared to DEPe3. In cardiomyocytes, particle internalization is fastened for DEPe4 compared to DEPe3. The in-vivo epicardial recording shows significant alteration of EGs parameters in both groups. However, the DEPe4-instilled group showed, compared to DEPe3, a significant increment of the effective refractory period, cardiac conduction velocity, and likelihood of arrhythmic events, with a significant increment of membrane lipid peroxidation but no increment in inflammation biomarkers. Our data suggest that DEPe4, possibly due to ultrafine nanoparticles, is rapidly internalized by cardiomyocytes resulting in an acute susceptibility to cardiac electrical disorder and arrhythmias that could accrue from cellular toxicity. Since the postulated transfer of nanoparticles from the lung to myocardial cells has not been investigated it remains open whether the effects on the cardiovascular function are the result of lung inflammatory reactions or due to particles that have reached the heart.

Utility of Zebrafish Models of Acquired and Inherited Long QT Syndrome.

Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.

UNUSUAL CASE OF SEIZURES DUE TO PROLONGED QT SYNDROME - A CASE REPORT

Long QT syndrome (LQTS) is a cardiac electrical disorder. One of the rare symptoms of long QT caused by ventricular arrhythmia is seizure. Patients with LQTS may develop seizures due to an acute hypoxic-ischemic event associated with a ventricular arrhythmia. We present a case of a 47-year-old male who came to the Emergency Department (ED) with seizures and was diagnosed as LQTS. The cardiac cause of seizures was suspected because the patient was pulseless during the episode of seizures. The patient developed refractory ventricular tachycardia in the ED and was cardioverted (synchronized) multiple times. He was also put on amiodarone infusions. The patient was then urgently shifted to the Coronary Cath Lab for temporary pacemaker insertion with overdrive pacing. He was advised for an automated implantable cardioverter-defibrillator. This case illustrates that prolonged QT syndrome can masquerade as seizure. Therefore, a careful examination should be done in the patient presenting with the same and a cardiac cause should be excluded. Delays in recognition and treatment may expose the patient to a high risk of sudden cardiac death.

Inhibition of rapid delayed rectifier potassium current (IKr) by ischemia/reperfusion and its recovery by vitamin E in ventricular myocytes

Ischemia/reperfusion (I/R) induces prolongation of QT interval and action potential duration (APD), which is a major cardiac electrical disorder in patients with arrhythmias. However, the mechanism of QT interval prolongation induced by I/R remains unclear. In the present study, we hypothesized that the rapid component of delayed rectifier potassium (IKr) channel plays an important role in I/R-induced QT interval prolongation. We observed a marked attenuation of IKr and a significant prolongation of action potential duration (APD) in a simulated I/R system with sodium dithionite (Na2S2O4) in ventricular myocytes of guinea pigs. The IKr current density was inhibited by 64% and APD increased by 87% respectively. Moreover, the inhibition of IKr is primarily ascribed to overproduction of reactive oxygen species (ROS) by I/R, which can be partly reversed by antioxidant vitamin E (100μmol/L). The value of IKr tail current density increased from 0.516±0.040 pA/pF in I/R to 0.939±0.091 pA/pF when treated with vitamin E. Moreover, we also demonstrated that QTc interval was increased by I/R and reversed by Vitamin E in isolated guinea pig hearts. In conclusion, the inhibition of IKr is one of the underlying mechanisms of prolongation of QT interval and APD in I/R. Vitamin E might have a benefit in coronary reperfusion therapy.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

Regulation of the human ether-a-go-go-related gene (hERG) potassium channel by Nedd4 family interacting proteins (Ndfips).

The cardiac electrical disorder long QT syndrome (LQTS) pre-disposes affected individuals to ventricular arrhythmias and sudden death. Dysfunction of the human ether-a-go-go-related gene (hERG)-encoded rapidly activating delayed rectifier K(+) channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by anterograde trafficking of newly synthesized channels to and retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin (Ub) ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2) targets the PY motif of hERG channels to initiate channel degradation. Although both immature and mature hERG channels contain the PY motif, Nedd4-2 selectively mediates the degradation of mature hERG channels. In the present study, we demonstrate that Nedd4-2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Nedd4 family-interacting protein 1 (Ndfip1) and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4-2 to mediate the degradation of mature hERG proteins during channel trafficking to the plasma membrane. Although Ndfip2 directs Nedd4-2 to the Golgi apparatus, it also recruits Nedd4-2 to the multivesicular bodies (MVBs), which may impair MVB function and impede the degradation of mature hERG proteins mediated by Nedd4-2. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.

Regulation of the Human Ether‐a‐go‐go Related Gene (hERG) Potassium Channel by Nedd4 Family Interacting Proteins (Ndfips)

The cardiac electrical disorder long QT syndrome (LQTS) predisposes affected individuals to high risks of ventricular arrhythmias and sudden death. Dysfunction of the human ether‐a‐go‐go related gene (hERG)‐encoded rapidly activating delayed rectifier K+ channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by the anterograde trafficking of newly synthesized channels to and the retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin ligase Nedd4‐2 (neuronal precursor cell expressed, developmentally down‐regulated protein 4 type 2) targets the PY motif of hERG channels to initiate channel degradation. Although both premature and mature hERG channels contain the PY motif, Nedd4‐2 selectively mediates the degradation of mature hERG channels. Here, using whole‐cell patch clamp, Western blot and immunocytochemical analyses, we demonstrate that Nedd4‐2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Ndfip1 and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4‐2 to target mature hERG proteins for degradation during channel trafficking to the plasma membrane. Although Ndfip2 also recruits Nedd4‐2 to the Golgi apparatus to some extent, it mainly recruits Nedd4‐2 to the multivesicular bodies (MVBs), which impedes the degradation of internalized hERG proteins. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.(Supported by the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of Ontario)

De Novo Mutation in the SCN5A Gene Associated with Brugada Syndrome

Background: Brugada syndrome (BrS) is a genetically determined cardiac electrical disorder, characterized by typical electrocardiography (ECG) alterations, and it is an arrhythmogenic syndrome that may lead to sudden cardiac death. The most common genotype found among BrS patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na⁺) channel (Na_v1.5) by different mechanisms. Methods: The assay of confocal laser microscopy and western blot were used to identify the expression and location of L812Q at the cell surface. Characterization of Nav1.5 L812Q mutant Na⁺ channels was text by patch-clamp recordings, and the PHYRE2 server was used to build a model for human Nav1.5 channel. Results: Here, we report that a novel missense SCN5A mutation, L812Q, localized in the DII-S4 transmembrane region of the Na_v1.5 channel protein, was identified in an index patient who showed a typical BrS type-1 ECG phenotype. The mutation was absent in the patient's parents and brother. Heterologous expression of the wild-type (WT) and L812Q mutant Na_v1.5 channels in human embryonic kidney cells (HEK293 cells) reveals that the mutation results in a reduction of Na⁺ current density as well as ∼20 mV hyperpolarizing shift of the voltage dependence of inactivation. The voltage dependence of activation and the time course for recovery from inactivation are not affected by the mutation. The hyperpolarizing shift of the voltage dependence of inactivation caused a reduction of the Na⁺ window current as well. In addition, western blot and confocal laser microscopy imaging experiments showed that the mutation causes fewer channel to be expressed at the membrane than WT channel. A large proportion of the mutant channels are retained in the cytoplasm, probably in the endoplasmic reticulum. Conclusion: The decrease of channel expression, hyperpolarizing shift of voltage dependence of inactivation, and a decline of Na⁺ window current caused by L812Q mutation lead to a reduction of Na⁺ current during the upstroke and the repolarization phases of cardiac action potential, which contribute to the development of BrS.

Long QT Syndrome in a 10-year Old Boy Presenting with Seizure

BACKGROUND: Arrhythmias in children may be benign or life threatening. Some may produce only mild, non-specific symptoms. Thus, specific documentations of arrhythmia, such as electrocardiogram and Holter monitoring, correlated with reported symptoms, are critical in establishing the diagnosis. Long QT syndrome (LQTS) is rare in pediatric patients compared with adults. It may either be inherited or acquired, and results in abnormalities in the ventricular repolarization leading to ventricular arrhythmia, which can be fatal. CASE: This is the case of a 10-year old male who presented for the first time with generalized seizure associated with appearance of arrhythmia during active seizure. Video electroencephalography showed no definite epileptiform discharges. However, there were episodes of ventricular tachycardia (VT) progressing to ventricular fibrillation, with sudden loss of body tone. Electrocardiography showed prolonged corrected QT interval of 0.68 s with T wave alternans. Lidocaine drip was started. Holter monitor showed frequent episodes of T wave alternans, fusion beats and non-sustained polymorphic VT and Torsade des Pointes. With this diagnostic tests pointing to LQTS, he was given propranolol at a dose of 0.5 mg/kg body weight every 6 hours, and phenytoin. There was no recurrence of VT, and seizure was controlled. He was sent home with propranolol and phenytoin as home medication. Genetic testing done on follow-up was consistent with a genetic form of LQTS at gene SCN5A (LQTS3). CONCLUSION: This is the first reported case of a diagnosed LQTS in a pediatric patient at Philippine Heart Center. LQTS is a cardiac electrical disorder. It is often difficult for the families of affected individuals to understand this rare cardiac disease. Seizure is one of the symptoms of LQTS, caused by ventricular arrhythmia and can be fatal. Thus, the importance of obtaining an electrocardiogram in patients with seizure is always emphasized. Genetic testing is recommended to determine the specific genotype as a guide to treatment. Once established, genetic counselling is also recommended to discuss to the family the implications of the disease, and the risk of recurrence.

A Novel Cardiac Nav1.5 Channel Mutation, L812Q, Leads to Brugada Syndrome

Brugada syndrome (BS) is a genetically determined cardiac electrical disorder, characterized by typical electrocardiography (ECG) alterations, and it is an arrhythmogenic syndrome that may lead to sudden cardiac death. The most common genotype found among Brugada syndrome patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+) channel (Nav1.5) by different mechanisms. Here, we report that a novel nonsense SCN5A mutation, L812Q, localized in the transmembrane region DII-S4 of the Nav1.5 channel, was identified in an index patient who showed a Brugada syndrome type 1 ECG phenotype. The mutation was absent in the patient's parents and brother. Expression of the wild-type (WT) and L812Q mutant Nav1.5 channels in human embryonic kidney cells (HEK293 cells) reveals that the mutation result in a reduction of Na+ current density and a ∼20 mV negative shift of the voltage dependence of inactivation. However, the voltage dependence of activation and the time course for recovery from inactivation are not altered by the mutation. The negative shift of the steady-state inactivation curve reduced the Na+ window current. In addition, western blot and confocal laser microscopyimaging experiments suggest that the mutant channels may be retained in the endoplasmic reticulum, resulting in less channel expression at the membrane surface. Decreased membrane surface expression, a negative shift of voltage dependence of inactivation, and a decline of Na+ window current in the L812Q mutant may lead to a reduction of Na+ current during the upstroke of a cardiac action potential and an inhibition of the reopening of inactivated Na+ channels during the repolarization phases, all of which may contribute to the development of Brugada syndrome.

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit of the rapidly activating delayed rectifier K(+) channel in the heart, which plays a critical role in cardiac action potential repolarization. Dysfunction of IKr causes long QT syndrome, a cardiac electrical disorder that predisposes affected individuals to fatal arrhythmias and sudden death. The homeostasis of hERG channels in the plasma membrane depends on a balance between protein synthesis and degradation. Our recent data indicate that hERG channels undergo enhanced endocytic degradation under low potassium (hypokalemia) conditions. The GTPase Rab4 is known to mediate rapid recycling of various internalized proteins to the plasma membrane. In the present study, we investigated the effect of Rab4 on the expression level of hERG channels. Our data revealed that overexpression of Rab4 decreases the expression level of hERG in the plasma membrane. Rab4 does not affect the expression level of the Kv1.5 or EAG K(+) channels. Mechanistically, our data demonstrate that overexpression of Rab4 increases the expression level of endogenous Nedd4-2, a ubiquitin ligase that targets hERG but not Kv1.5 or EAG channels for ubiquitination and degradation. Nedd4-2 undergoes self- ubiquitination and degradation. Rab4 interferes with Nedd4-2 degradation, resulting in an increased expression level of Nedd4-2, which targets hERG. In summary, the present study demonstrates a novel pathway for hERG regulation; Rab4 decreases the hERG density at the plasma membrane by increasing the endogenous Nedd4-2 expression.

Rab4 GTPase regulates cell surface expression of hERG channels through Nedd4–2

The human ether‐a‐go‐go‐related gene (hERG) encodes the pore‐forming subunits of the cardiac rapidly activating delayed rectifier K+ channel (IKr) which is important for cardiac repolarization. Dysfunction of IKr causes long QT syndrome (LQTS), a cardiac electrical disorder which can lead to cardiac arrhythmias and sudden death. Currently, trafficking mechanisms of the hERG channel are poorly understood. We investigated the effects of Rab4 GTPase on the function and expression of hERG channels using Western blot, patch clamp and immunofluorescence staining. Our data revealed that Rab4 selectively decreased the hERG expression on the plasma membrane. Mechanistically, Rab4 did not directly target the hERG protein. Instead, it increased the expression of ubiquitin ligase Nedd4–2, which mediates hERG ubiquitination and degradation. Mutations that disrupt the Nedd4–2 targeting site in hERG completely abolished the effect of Rab4. Our data further revealed that Rab4 inhibited Nedd4–2 self ubiquitination, leading to a decreased degradation rate of Nedd4–2. We conclude that Rab4 regulates the cell surface expression of hERG channels through Nedd4–2.Supported by the Canadian Institutes of Health Research – CIHR.

Upregulation of microRNA-1 and microRNA-133 contributes to arsenic-induced cardiac electrical remodeling

BackgroundA large body of evidence showed that arsenic trioxide (As2O3), a front-line drug for the treatment of acute promyelocytic leukemia, induced abnormal cardiac QT prolongation, which hampers its clinical use. The molecular mechanisms for this cardiotoxicity remained unclear. This study aimed to elucidate whether microRNAs (miRs) participate in As2O3-induced QT prolongation. MethodsA guinea pig model of As2O3-induced QT prolongation was established by intravenous injection with As2O3. Real-time PCR and Western blot were employed to determine the expression alterations of miRs and mRNAs, and their corresponding proteins. ResultsThe QT interval and QRS complex were significantly prolonged in a dose-dependent fashion after 7-day administration of As2O3. As2O3 induced a significant upregulation of the muscle-specific miR-1 and miR-133, as well as their transactivator serum response factor. As2O3 depressed the protein levels of ether-a-go-go related gene (ERG) and Kir2.1, the K+ channel subunits responsible for delayed rectifier K+ current IKr and inward rectifier K+ current IK1, respectively. In vivo transfer of miR-133 by direct intramuscular injection prolonged QTc interval and increased mortality rate, along with depression of ERG protein and IKr in guinea pig hearts. Similarly, forced expression of miR-1 widened QTc interval and QRS complex and increased mortality rate, accompanied by downregulation of Kir2.1 protein and IK1. Application of antisense inhibitors to knockdown miR-1 and miR-133 abolished the cardiac electrical disorders caused by As2O3. ConclusionsDeregulation of miR-133 and miR-1 underlies As2O3-induced cardiac electrical disorders and these miRs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

Polymorphisms in the cardiac sodium channel promoter displaying variant in vitro expression activity

Variable transcription of the cardiac sodium channel gene is a candidate mechanism determining arrhythmia susceptibility. We have previously cloned and characterized the core promoter and flanking region of SCN5A, encoding the cardiac sodium channel. Loss-of-function mutations in this gene have been reported in approximately 20% of patients with Brugada syndrome, an inherited cardiac electrical disorder associated with a high incidence of life-threatening arrhythmias. In this study, we identified DNA variants in the proximal 2.8 kb promoter region of SCN5A and determined their frequency in 1,121 subjects. This population consisted of 88 Brugada syndrome patients with no SCN5A coding region mutation, and 1,033 anonymized subjects from various ethnicities. Variant promoter activity was assayed in CHO cells and neonatal cardiomyocytes by transient transfection of promoter-reporter constructs. Single-nucleotide polymorphisms (SNPs) were identified at approximately 1/200 base pairs which are: 11 in the 5'-flanking region, 1 in exon 1, and 5 in intron 1. In addition, a haplotype consisting of two SNPs in complete linkage disequilibrium was identified. Minor allele frequencies were >5% in at least one ethnic panel at 5/19 polymorphic sites. In vitro functional analysis in cardiomyocytes identified four variants with significantly (P<0.05) reduced reporter activity (up to 63% reduction). The largest changes were seen with c.-225-1790 G>A, which reduced reporter activity by 62.8% in CHO cells and 55% in cardiomyocytes. From these results, we can conclude that the SCN5A core promoter includes multiple DNA polymorphisms with altered in vitro activity, further supporting the concept of interindividual variability in transcription of this cardiac ion channel gene.

Role of Sodium Channels in Propagation in Heart Muscle: How Subtle Genetic Alterations Result in Major Arrhythmic Disorders

Sodium channels play a crucial role in initiation, propagation, and maintenance of cardiac excitation throughout the heart. Indeed, dysfunctional sodium channels have been shown to be responsible for several inherited cardiac electrical disorders, such as Long QT and Brugada syndromes (BrS), potentially leading to fatal arrhythmic events. Genetic approaches and functional experiments using heterologous systems have enabled the characterization of the molecular determinants involved in these disorders and their consequences on ion channel function. The improved understanding of the mechanisms leading to these cardiac arrhythmic events represents a first step in the development of therapeutic treatments.

Exclusion of multiple candidate genes and large genomic rearrangements in SCN5A in a Dutch Brugada syndrome cohort

The Brugada syndrome is an inherited cardiac electrical disorder associated with a high incidence of life-threatening arrhythmias. Screening for mutations in the cardiac Na+ channel-encoding gene SCN5A uncovers a mutation in approximately 20% of Brugada syndrome cases. Genetic heterogeneity and/or undetected SCN5A mutations, such as exon duplications and deletions, could be involved in the remaining 80% mutation-negative patients. Thirty-eight SCN5A mutation-negative Dutch Brugada syndrome probands were studied. The SCN5A gene was investigated for exon duplication and deletion, and a number of candidate genes (Caveolin-3, Irx-3, Irx-4, Irx-5, Irx-6, Plakoglobin, Plakophilin-2, SCN1B, SCN2B, SCN3B, and SCN4B) were tested for the occurrence of point mutations and small insertions/deletions. We used a quantitative multiplex approach to determine SCN5A exon copy numbers. Mutation analysis of the candidate genes was performed by direct sequencing of polymerase chain reaction-amplified coding regions. No large genomic rearrangements in SCN5A were identified. No mutations were found in the candidate genes. Twenty novel polymorphisms were identified in these genes. Large genomic rearrangements in SCN5A are not a common cause of Brugada syndrome. Similarly, the studied candidate genes are unlikely to be major causal genes of Brugada syndrome. Further studies are required to identify other genes responsible for this syndrome.

Electrolyte disorders in horses with colic. Part 1: potassium and magnesium

Electrolyte disorders are common in horses with acute abdominal disease, and are a significant cause of morbidity. They are especially prevalent in horses treated surgically for colic, and may be present before, during and after surgery. In one study, 54% of horses admitted for surgical colic had preoperative serum ionised magnesium concentrations below the reference range and 86% subnormal serum ionised calcium concentrations (Garcia-Lopez et al. 2001). Electrolyte abnormalities are particularly prevalent in horses with strangulating obstructions of the intestine. These horses have lower preoperative serum concentrations of ionised magnesium and calcium than horses with nonstrangulating lesions (Dart et al. 1992; Garcia-Lopez et al. 2001). Horses with decreased preoperative ionised magnesium were also more likely to be subjected to euthanasia during surgery. The role of electrolyte derangements in contributing to adverse clinical events in horses with colic is still being investigated. The association between cardiac electrical disorders and blood calcium, magnesium and potassium concentrations is well established in the horse (Harrington 1974; Glazier et al. 1979, 1982; Reef 1999) (Fig 1). Derangements in blood electrolyte concentration might also be associated with a longer hospital stay. Martin et al. (1994) reported that dogs with low serum magnesium concentrations were hospitalised for twice as long as those with a normal serum magnesium concentration. Conversely, Garcia-Lopez et al. (2001) did not find any correlation between serum ionised magnesium or calcium concentrations and length of hospitalisation, incidence of complications or survival in horses treated surgically for colic. The association between serum concentrations of potassium, calcium and magnesium with post operative ileus is still unknown in the horse. Dart et al. (1992) speculated that decreased serum calcium concentrations might contribute to ileus. Garcia-Lopez et al. (2001) found lower serum concentrations of ionised magnesium and calcium in horses that developed post operative ileus, than in those that did not. These decreased electrolyte concentrations may be due to decreased intake and increased loss of electrolytes in gastric reflux. It is now possible to measure electrolytes using ‘patientside’ portable analysers. The introduction of these cartridgebased systems has enabled results to be obtained quickly and easily when laboratory techniques are unavailable (Grosenbaugh et al. 1998). Different sensors are available to measure different combinations of electrolytes, blood biochemistry, PCV and blood gas tensions. The price of these hand-held devices is rapidly decreasing and they are now affordable for many private practices. The normal range for any electrolyte may vary to some degree between analysers. Therefore, horse specific normal ranges should be obtained from the manufacturers, if possible. In Part 1, we discuss the importance of potassium and magnesium and Part 2 covers calcium, sodium, chloride and phosphorus.