Identification of transmembrane protein 168 mutation in familial Brugada syndrome.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.

Fibrosis and Conduction Abnormalities as Basis for Overlap of Brugada Syndrome and Early Repolarization Syndrome.

Brugada syndrome and early repolarization syndrome are both classified as J-wave syndromes, with a similar mechanism of arrhythmogenesis and with the same basis for genesis of the characteristic electrocardiographic features. The Brugada syndrome is now considered a conduction disorder based on subtle structural abnormalities in the right ventricular outflow tract. Recent evidence suggests structural substrate in patients with the early repolarization syndrome as well. We propose a unifying mechanism based on these structural abnormalities explaining both arrhythmogenesis and the electrocardiographic changes. In addition, we speculate that, with increasing technical advances in imaging techniques and their spatial resolution, these syndromes will be reclassified as structural heart diseases or cardiomyopathies.

iPSC-Cardiomyocyte Models of Brugada Syndrome-Achievements, Challenges and Future Perspectives.

Brugada syndrome (BrS) is an inherited cardiac arrhythmia that predisposes to ventricular fibrillation and sudden cardiac death. It originates from oligogenic alterations that affect cardiac ion channels or their accessory proteins. The main hurdle for the study of the functional effects of those variants is the need for a specific model that mimics the complex environment of human cardiomyocytes. Traditionally, animal models or transient heterologous expression systems are applied for electrophysiological investigations, each of these models having their limitations. The ability to create induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), providing a source of human patient-specific cells, offers new opportunities in the field of cardiac disease modelling. Contemporary iPSC-CMs constitute the best possible in vitro model to study complex cardiac arrhythmia syndromes such as BrS. To date, thirteen reports on iPSC-CM models for BrS have been published and with this review we provide an overview of the current findings, with a focus on the electrophysiological parameters. We also discuss the methods that are used for cell derivation and data acquisition. In the end, we critically evaluate the knowledge gained by the use of these iPSC-CM models and discuss challenges and future perspectives for iPSC-CMs in the study of BrS and other arrhythmias.

Relationship between sodium channel function and clinical phenotype in SCN5A variants associated with Brugada syndrome.

The identification of a pathogenic SCN5A variant confers an increased risk of conduction defects and ventricular arrhythmias (VA) in Brugada syndrome (BrS). However, specific aspects of sodium channel function that influence clinical phenotype have not been defined. A systematic literature search identified SCN5A variants associated with BrS. Sodium current (INa ) functional parameters (peak current, decay, steady-state activation and inactivation, and recovery from inactivation) and clinical features (conduction abnormalities [CA], spontaneous VA or family history of sudden cardiac death [SCD], and spontaneous BrS electrocardiogram [ECG]) were extracted. A total of 561 SCN5A variants associated with BrS were identified, for which data on channel function and clinical phenotype were available in 142. In the primary analysis, no relationship was found between any aspect of channel function and CA, VA/SCD, or spontaneous BrS ECG pattern. Sensitivity analyses including only variants graded pathogenic or likely pathogenic suggested that reduction in peak current and positive shift in steady-state activation were weakly associated with CA and VA/SCD, although sensitivity and specificity remained low. The relationship between in vitro assessment of channel function and BrS clinical phenotype is weak. The assessment of channel function does not enhance risk stratification. Caution is needed when extrapolating functional testing to the likelihood of variant pathogenicity.

Brugada syndrome with SCN5A mutations exhibits more pronounced electrophysiological defects and more severe prognosis: A meta-analysis.

Whether the presence of SCN5A mutation is a predictor of BrS risk remains controversial, and patient selection bias may have weakened previous findings. Therefore, we performed this study to clarify the clinical characteristics and outcomes of BrS probands with SCN5A mutations. We systematically retrieved eligible studies published through October 2018. A total of 17 studies enrolling 1780 BrS patients were included. Overall, our results found that compared with BrS patients without SCN5A mutations, patients with SCN5A mutations exhibited a younger age at the onset of symptoms and higher rate of the spontaneous type-1 electrocardiogram pattern, more pronounced conduction or repolarization abnormalities, and increased atrial vulnerability. In addition, the presence of SCN5A mutations was associated with an elevated risk of major arrhythmic events in both Asian (odds ratio [OR] = 1.82, 95% confidence interval [CI] 1.07-3.11; P = .03) and Caucasian (OR = 2.24, 95% CI 1.02-4.90; P = .04) populations. In conclusions, patients with SCN5A mutations exhibit more pronounced electrophysiological defects and more severe prognosis. Clinicians should be cautious when utilizing genetic testing for risk stratification or treatment guidance before determining whether the causal relationship regarding SCN5A mutation status is an independent predictor of risk.

HSPB7 prevents cardiac conduction system defect through maintaining intercalated disc integrity.

HSPB7 is a member of the small heat-shock protein (HSPB) family and is expressed in the cardiomyocytes from cardiogenesis onwards. A dramatic increase in HSPB7 is detected in the heart and blood plasma immediately after myocardial infarction. Additionally, several single-nucleotide polymorphisms of HSPB7 have been identified to be associated with heart failure caused by cardiomyopathy in human patients. Although a recent study has shown that HSPB7 is required for maintaining myofiber structure in skeletal muscle, its molecular and physiological functions in the heart remain unclear. In the present study, we generated a cardiac-specific inducible HSPB7 knockout mouse and demonstrated that the loss of HSPB7 in cardiomyocytes results in rapid heart failure and sudden death. The electrocardiogram showed cardiac arrhythmia with abnormal conduction in the HSPB7 mutant mice before death. In HSPB7 CKO cardiomyocytes, no significant defect was detected in the organization of contractile proteins in sarcomeres, but a severe structural disruption was observed in the intercalated discs. The expression of connexin 43, a gap-junction protein located at the intercalated discs, was downregulated in HSPB7 knockout cardiomyocytes. Mislocalization of desmoplakin, and N-cadherin, the intercalated disc proteins, was also observed in the HSPB7 CKO hearts. Furthermore, filamin C, the interaction protein of HSPB7, was upregulated and aggregated in HSPB7 mutant cardiomyocytes. In conclusion, our findings characterize HSPB7 as an intercalated disc protein and suggest it has an essential role in maintaining intercalated disc integrity and conduction function in the adult heart.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome.

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

Mechanisms of Arrhythmias in the Brugada Syndrome.

Arrhythmias in Brugada syndrome patients originate in the right ventricular outflow tract (RVOT). Over the past few decades, the characterization of the unique anatomy and electrophysiology of the RVOT has revealed the arrhythmogenic nature of this region. However, the mechanisms that drive arrhythmias in Brugada syndrome patients remain debated as well as the exact site of their occurrence in the RVOT. Identifying the site of origin and mechanism of Brugada syndrome would greatly benefit the development of mechanism-driven treatment strategies.

Enhanced closed-state inactivation of mutant cardiac sodium channels (SCN5A N1541D and R1632C) through different mechanisms.

BACKGROUND: SCN5A variants can be associated with overlapping phenotypes such as Brugada syndrome (BrS), sinus node dysfunction and supraventricular tachyarrhythmias. Our genetic screening of SCN5A in 65 consecutive BrS probands revealed two patients with overlapping phenotypes: one carried an SCN5A R1632C (in domain IV-segment 4), which we have previously reported, the other carried a novel SCN5A N1541D (in domain IV-segment 1). OBJECTIVE: We sought to reveal whether or not these variants are associated with the same biophysical defects. METHODS: Wild-type (WT) or mutant SCN5A was expressed in tsA201-cells, and whole-cell sodium currents (hNav1.5/INa) were recorded using patch-clamp techniques. RESULTS: The N1541D-INa density, when assessed from a holding potential of -150 mV, was not different from WT-INa as with R1632C-INa, indicating that SCN5A N1541D did not cause trafficking defects. The steady-state inactivation curve of N1541D-INa was markedly shifted to hyperpolarizing potentials in comparison to WT-INa (V1/2-WT: -82.3 ±â€¯0.9 mV, n = 15; N1541D: -108.8 ±â€¯1.6 mV, n = 26, P < .01) as with R1632C-INa. Closed-state inactivation (CSI) was evaluated using prepulses of -90 mV for 1460 ms. Residual N1541D-INa and R1632C-INa were markedly reduced in comparison to WT-INa (WT: 63.8 ±â€¯4.6%, n = 18; N1541D: 15.1 ±â€¯2.3%, n = 19, P < .01 vs WT; R1632C: 5.3 ±â€¯0.5%, n = 15, P < .01 vs WT). Entry into CSI of N1541D-INa was markedly accelerated, and that of R1632C-INa was weakly accelerated in comparison to WT-INa (tau-WT: 65.8 ±â€¯7.4 ms, n = 18; N1541D: 13.7 ±â€¯1.1 ms, n = 19, P < .01 vs WT; R1632C: 39.5 ±â€¯2.9 ms, n = 15, P < .01 vs WT and N1541D). Although N1541D-INa recovered from closed-state fast inactivation at the same rate as WT-INa, R1632C-INa recovered very slowly (tau-WT: 1.90 ±â€¯0.16 ms, n = 10; N1541D: 1.72 ±â€¯0.12 ms, n = 10, P = .41 vs WT; R1632C: 53.0 ±â€¯2.5 ms, n = 14, P < .01 vs WT and N1541D). CONCLUSIONS: Both N1541D-INa and R1632C-INa exhibited marked enhancement of CSI, but through different mechanisms. The data provided a novel understanding of the mechanisms of CSI of INa. Clinically, the enhanced CSI of N1541D-INa leads to a severe loss-of-function of INa at voltages near the physiological resting membrane potential (~-90 mV) of cardiac myocytes; this can be attributable to the patient's phenotypic manifestations.

Novel SCN5A p.W697X Nonsense Mutation Segregation in a Family with Brugada Syndrome.

Brugada syndrome (BrS) is marked by an elevated ST-segment elevation and increased risk of sudden cardiac death. Variants in the SCN5A gene are considered to be molecular confirmation of the syndrome in about one third of cases, while the genetics remain a mystery in about half of the cases, with the remaining cases being attributed to variants in any of a number of genes. Before research models can be developed, it is imperative to understand the genetics in patients. Even data from humans is complicated, since variants in the most common gene in BrS, SCN5A, are associated with a number of pathologies, or could even be considered benign, depending on the variant. Here, we provide crucial human data on a novel NM_198056.2:c.2091G>A (p.Trp697X) point-nonsense heterozygous variant in the SCN5A gene, as well as its segregation with BrS. The results herein suggest a pathogenic effect of this variant. These results could be used as a stepping stone for functional studies to better understand the molecular effects of this variant in BrS.

Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.

Trafficking and localisation to the plasma membrane of Nav 1.5 promoted by the ß2 subunit is defective due to a ß2 mutation associated with Brugada syndrome.

BACKGROUND INFORMATION: Cardiac channelopathies arise by mutations in genes encoding ion channel subunits. One example is Brugada Syndrome (BrS), which causes arrhythmias and sudden death. BrS is often associated with mutations in SCN5A, encoding Nav 1.5, the α subunit of the major cardiac voltage-gated sodium channel. This channel forms a protein complex including one or two associated ß subunits as well as other proteins. RESULTS: We analysed regulation of Nav 1.5 localisation and trafficking by ß2, specifically, Nav 1.5 arrival to the cell surface. We used polarised Madin-Darby canine kidney (MDCK) cells and mouse atria-derived HL-1 cells, which retain phenotypic features of adult cardiomyocytes. In both, Nav 1.5 was found essentially intracellular, mainly in the endoplasmic reticulum, whereas ß2 localised to the plasma membrane, and was restricted to the apical surface in MDCK cells. A fraction of ß2 interacted with Nav 1.5, despite their limited overlap. Importantly, ß2 promoted Nav 1.5 localisation to the cell surface. Both ß2 WT and the BrS-associated mutation D211G (substitution of Asp for Gly) effectively reached the plasma membrane. Strikingly, however, ß2 D211G was defective in promoting Nav 1.5 surface localisation. CONCLUSIONS: Our data sustain that ß2 promotes surface localisation of Nav 1.5, which can be affected due to ß2 mutations associated with channelopathies. SIGNIFICANCE: Our findings add to the understanding of ß2 role in Nav 1.5 trafficking and localisation, which must influence cell excitability and electrical coupling in the heart. This study will contribute to knowledge on development of arrhythmias.

[Research Progress of the Relationship between SUNDS and OSAHS].

Sudden unexplained nocturnal death syndrome （SUNDS） is always a difficulty in forensic medicine researches. Although the development of molecular genetics promotes the etiologic study of SUNDS, the pathogenesis of most such cases is still unclear. Sleep apnea syndrome （SAS） is one of the common forms of sleep disorders, and obstructive sleep apnea hypopnea syndrome （OSAHS） is the most common. In recent years, some domestic and international researches show that OSAHS is related to the development of cardiovascular disease, which may cause cardiac arrhythmia, even sudden death. This article reviews the relationship between SUNDS and OSAHS and aims to provide new ideas for the pathogenesis of SUNDS.

High-fat diet increases vulnerability to atrial arrhythmia by conduction disturbance via miR-27b.

BACKGROUND: Lifestyle-related diseases, such as obesity and dyslipidemia are important risk factors for atrial fibrillation (AF). However, the underlying mechanism linking these diseases and AF has not been fully investigated. METHODS: Adult male mice were fed a high-fat diet (HFD) or vehicle (NC) for 2 months. Electrocardiography and in vivo electrophysiological study were performed. Mice were then sacrificed for quantification of mRNA, microRNA, and protein in atria, in addition to histological analysis. Conduction velocity (CV) in right atrium was measured by optical mapping in Langendorff perfused hearts. Cultured atrial cardiomyocytes were treated with palmitate with or without a specific microRNA inhibitor. Twelve hours after stimulation, cells were lysed, and subjected to analysis with qPCR and Western blotting. RESULTS: HFD mice showed prolonged P wave duration, increased inducibility of sustained atrial tachycardia, and reduced atrial CV than NC mice. HFD mice also showed increased expression in inflammatory cytokines, whereas fibrotic area and signals relating fibrosis were not changed. HFD mice demonstrated reduced expression of Cx40 in mRNA and protein levels, and its lateralized expression in atria. MicroRNA array analysis revealed that miR-27b expression was up-regulated in HFD mice, and luciferase assay confirmed the direct interaction between miR-27b and Cx40 3'UTR. In palmitate-stimulated atrial cardiomyocytes, miR-27b up-regulation and Cx40 down-regulation were observed, while expression of inflammatory cytokines was not altered. Inhibition of miR-27b with antisense oligonucleotides reversed the alteration caused by palmitate stimulation. CONCLUSION: HFD may increase the vulnerability to atrial arrhythmia by down-regulation of Cx40 via miR-27b, rather than fibrosis, which is independent of inflammation.

Clinical impact of myocardial mTORC1 activation in nonischemic dilated cardiomyopathy.

BACKGROUND: Activity of mTOR complex 1 (mTORC1) has been shown to be up-regulated in animal models of heart failure. Here, we investigated the change and role of mTORC1 in human nonischemic dilated cardiomyopathy (NICM). METHODS: Endomyocardial biopsy specimens were obtained from patients with NICM (n=52) and from Brugada syndrome patients with normal LVEF as controls (n=10). The specimens were stained for phospho-ribosomal protein S6 (p-Rps6) and phospho-p70S6K (p-p70S6K), and the area with p-Rps6 signal was used as an index of mTORC1 activity. Using median mTORC1 activity, patients were divided into a high mTORC1 activity (H-mTOR) group and a low mTORC1 activity (L-mTOR) group. RESULTS: The ratio of p-Rps6-positive area in biopsy samples was 10-fold larger in patients with NICM than in controls (2.0±2.2% vs. 0.2±0.2%, p<0.01). p-p70S6K signal level was higher in the H-mTOR group than in the L-mTOR group. The proportion of patients with a family history of cardiomyopathy was higher and the proportion of patients on ACE inhibitors or angiotensin receptor blockers was lower in the H-mTOR group than in the L-mTOR group. The p-Rps6-positive area was correlated with extent of myocardial fibrosis (r=0.46, p<0.01). The cardiac event-free survival rate during a 5-year follow-up period tended to be lower in the H-mTOR group than in the L-mTOR group (52.9% vs. 81.6%, P=0.10). CONCLUSION: Aberrant activation of mTORC1 in cardiomyocytes was associated with myocardial fibrosis and a trend for worse prognosis in patients with NICM, indicating that persistently activated mTORC1 contributes to progression of human heart failure.

Brugada syndrome: clinical and genetic findings.

Brugada syndrome is a rare, inherited cardiac disease leading to ventricular fibrillation and sudden cardiac death in structurally normal hearts. Clinical diagnosis requires a Brugada type I electrocardiographic pattern in combination with other clinical features. The most effective approach to unmasking this diagnostic pattern is the use of ajmaline and flecainide tests, and the most effective intervention to reducing the risk of death is the implantation of a cardioverter defibrillator. To date, 18 genes have been associated with the disease, with the voltage-gated sodium channel α type V gene (SCN5A) being the most common one to date. However, only 30-35% of diagnosed cases are attributable to pathogenic variants in known genes, emphasizing the need for further genetic studies. Despite recent advances in clinical diagnoses and genetic testing, risk stratification and clinical management of patients with Brugada syndrome remain challenging.Genet Med 18 1, 3-12.

Mosaic mutations in early-onset genetic diseases.

PURPOSE: An emerging approach in medical genetics is to identify de novo mutations in patients with severe early-onset genetic disease that are absent in population controls and in the patient's parents. This approach, however, frequently misses post-zygotic "mosaic" mutations that are present in only a portion of the healthy parents' cells and are transmitted to offspring. METHODS: We constructed a mosaic transmission screen for variants that have an ~50% alternative allele ratio in the proband but are significantly less than 50% in the transmitting parent. We applied it to two family-based genetic disease cohorts consisting of 9 cases of sudden unexplained death in childhood (SUDC) and 338 previously published cases of epileptic encephalopathy. RESULTS: The screen identified six parental-mosaic transmissions across the two cohorts. The resultant rate of ~0.02 identified transmissions per trio is far lower than that of de novo mutations. Among these transmissions were two likely disease-causing mutations: an SCN1A mutation transmitted to an SUDC proband and her sibling with Dravet syndrome, as well as an SLC6A1 mutation in a proband with epileptic encephalopathy. CONCLUSION: These results highlight explicit screening for mosaic mutations as an important complement to the established approach of screening for de novo mutations.Genet Med 18 7, 746-749.

Subepicardial delayed gadolinium enhancement in asymptomatic athletes: let sleeping dogs lie?

BACKGROUND: Subepicardial delayed gadolinium enhancement (DGE) patches without underlying cardiomyopathy is poorly understood. It is often reported as the result of prior silent myocarditis. Its prognostic relevance in asymptomatic athletes is unknown; therefore, medical clearance for competitive sports participation is debated. This case series aims to relate this pattern of DGE in athletes to outcome. METHODS: We report on seven young asymptomatic athletes with isolated subepicardial DGE detected during workup of abnormalities on their regular screening examination, that is, pathological T-wave inversions on ECG (n=4) or ventricular arrhythmias on exercise test (n=3). All underwent a comprehensive initial investigation in order to assess left ventricular (LV) function at rest and exercise (exercise cardiac MRI and/or exercise echocardiography) and occurrence of arrhythmias (exercise test, 24 h-ECG Holter, electrophysiological study). All underwent a careful follow-up with biannual evaluation. RESULTS: All athletes had extensive subepicardial DGE (12.0±4.8% of LV mass), predominantly in the lateral wall. Three athletes had non-sustained ventricular arrhythmias, whereas two of them had LV ejection fraction <50% at rest with no contractile reserve at exercise. During a follow-up of 3.0±1.5 years in the four remaining athletes, two had symptomatic ventricular tachycardia and one demonstrated progressive LV dysfunction. Hence, six of seven athletes had to be excluded from competitive sports participation. CONCLUSIONS: Isolated large areas of subepicardial DGE in an asymptomatic athlete are not benign and require a careful evaluation at exercise and a strict follow-up. These findings question whether extreme exercise during silent myocarditis may facilitate fibrosis generation and adverse remodelling.

In silico screening of the impact of hERG channel kinetic abnormalities on channel block and susceptibility to acquired long QT syndrome.

Accurate diagnosis of predisposition to long QT syndrome is crucial for reducing the risk of cardiac arrhythmias. In recent years, drug-induced provocative tests have proved useful to unmask some latent mutations linked to cardiac arrhythmias. In this study we expanded this concept by developing a prototype for a computational provocative screening test to reveal genetic predisposition to acquired long-QT syndrome (aLQTS). We developed a computational approach to reveal the pharmacological properties of I(Kr) blocking drugs that are most likely to cause aLQTS in the setting of subtle alterations in I(Kr) channel gating that would be expected to result from benign genetic variants.Weused themodel to predict themost potentially lethal combinations of kinetic anomalies and drug properties. In doing so, we also implicitly predicted ideal inverse therapeutic properties of K channel openers that would be expected to remedy a specific defect. We systematically performed "in silico mutagenesis" by altering discrete kinetic transition rates of the Fink et al. Markov model of human I(Kr) channels, corresponding to activation, inactivation, deactivation and recovery from inactivation of I(Kr) channels. We then screened and identified the properties of I(Kr) blockers that caused acquired long QT and therefore unmasked mutant phenotypes formild,moderate and severe variants. Mutant I(Kr) channels were incorporated into the O'Hara et al. human ventricular action potential (AP) model and subjected to simulated application of a wide variety of I(Kr)-drug interactions in order to identify the characteristics that selectively exacerbate the AP duration (APD) differences between wild-type and I(Kr) mutated cells. Our results show that drugs with disparate affinities to conformation states of the I(Kr) channel are key to amplify variants underlying susceptibility to acquired long QT syndrome, an effect that is especially pronounced at slow frequencies. Finally, we developed a mathematical formulation of the M54T MiRP1 latent mutation and simulated a provocative test. In this setting, application of dofetilide dramatically amplified the predicted QT interval duration in the M54T hMiRP1 mutation compared to wild-type.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload.

BACKGROUND: One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS: A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION: Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.