Patient-specific iPSC-derived cardiomyocytes reveal variable phenotypic severity of Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is a cardiac channelopathy that can result in sudden cardiac death (SCD). SCN5A is the most frequent gene linked to BrS, but the genotype-phenotype correlations are not completely matched. Clinical phenotypes of a particular SCN5A variant may range from asymptomatic to SCD. Here, we used comparison of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from a SCN5A mutation-positive (D356Y) BrS family with severely affected proband, asymptomatic mutation carriers (AMCs) and healthy controls to investigate this variation. METHODS: 26 iPSC lines were generated from skin fibroblasts using nonintegrated Sendai virus. The generated iPSCs were differentiated into cardiomyocytes using a monolayer-based differentiation protocol. FINDINGS: D356Y iPSC-CMs exhibited increased beat interval variability, slower depolarization, cardiac arrhythmias, defects of Na+ channel function and irregular Ca2+ signaling, when compared to controls. Importantly, the phenotype severity observed in AMC iPSC-CMs was milder than that of proband iPSC-CMs, an observation exacerbated by flecainide. Interestingly, the iPSC-CMs of the proband exhibited markedly decreased Ca2+ currents in comparison with control and AMC iPSC-CMs. CRISPR/Cas9-mediated genome editing to correct D356Y in proband iPSC-CMs effectively rescued the arrhythmic phenotype and restored Na+ and Ca2+ currents. Moreover, drug screening using established BrS iPSC-CM models demonstrated that quinidine and sotalol possessed antiarrhythmic effects in an individual-dependent manner. Clinically, venous and oral administration of calcium partially reduced the malignant arrhythmic events of the proband in mid-term follow-up. INTERPRETATION: Patient-specific and genome-edited iPSC-CMs can recapitulate the varying phenotypic severity of BrS. Our findings suggest that preservation of the Ca2+ currents might be a compensatory mechanism to resist arrhythmogenesis in BrS AMCs. FUNDING: National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81922006, 81870175), Natural Science Foundation of Zhejiang Province (LD21H020001, LR15H020001), National Natural Science Foundation of China (81970269), Key Research and Development Program of Zhejiang Province (2019C03022) and Natural Science Foundation of Zhejiang Province (LY16H020002).

Clinical and Molecular Data Define a Diagnosis of Arrhythmogenic Cardiomyopathy in a Carrier of a Brugada-Syndrome-Associated PKP2 Mutation.

Plakophilin-2 (PKP2) is the most frequently mutated desmosomal gene in arrhythmogenic cardiomyopathy (ACM), a disease characterized by structural and electrical alterations predominantly affecting the right ventricular myocardium. Notably, ACM cases without overt structural alterations are frequently reported, mainly in the early phases of the disease. Recently, the PKP2 p.S183N mutation was found in a patient affected by Brugada syndrome (BS), an inherited arrhythmic channelopathy most commonly caused by sodium channel gene mutations. We here describe a case of a patient carrier of the same BS-related PKP2 p.S183N mutation but with a clear diagnosis of ACM. Specifically, we report how clinical and molecular investigations can be integrated for diagnostic purposes, distinguishing between ACM and BS, which are increasingly recognized as syndromes with clinical and genetic overlaps. This observation is fundamentally relevant in redefining the role of genetics in the approach to the arrhythmic patient, progressing beyond the concept of "one mutation, one disease", and raising concerns about the most appropriate approach to patients affected by structural/electrical cardiomyopathy. The merging of genetics, electroanatomical mapping, and tissue and cell characterization summarized in our patient seems to be the most complete diagnostic algorithm, favoring a reliable diagnosis.

Putative role of Brugada syndrome genes in familial atrial fibrillation.

OBJECTIVE: Familial atrial fibrillation (FAF), a not uncommon arrhythmia of the atrium, is characterized by heritability, early onset and absence of other heart defects. The molecular and genetic basis is still not completely clear and genetic diagnosis cannot be achieved in about 90% of patients. In this study, we present the results of genetic screening by next generation sequencing in affected Russian families. PATIENTS AND METHODS: Sixty subjects (18 probands and 42 relatives) with a clinical diagnosis of FAF were enrolled in the study. Since AF frequently associates with other cardiomyopathies, we included all genes that were known to be associated with these disorders at the time of our study. All probands were therefore systematically screened for 47 genes selected from the literature. RESULTS: Our study revealed that seven variants co-segregated with the clinical phenotype in seven families. Interestingly, four out of six genes and three out of seven variants have already been associated with Brugada syndrome in the literature. CONCLUSIONS: To our knowledge, this is the first report of association of the CACNA1C, CTNNA3, PKP2, ANK2 and SCN10A genes with FAF; it is also the first study in Russian families.

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.

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.

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.

Vulnerability of fourth ventricle choroid plexus in sudden unexplained fetal and infant death syndromes related to smoking mothers.

The human choroid plexuses in the ventricular system represent the main source of cerebrospinal fluid secretion and constitute a major barrier interface that controls the brain's environment. The present study focused on the choroid plexus of the fourth ventricle, the main cavity of the brainstem containing important nuclei and/or structures mediating autonomic vital functions. In serial sections of 84 brainstems of subjects aged from 17 gestational weeks to 8 postnatal months of life, the deaths due to both known and unknown causes, we examined the cytoarchitecture and the developmental steps of the fourth ventricle choroid plexus to determine whether this structure shows morphological and/or functional alterations in unexplained perinatal deaths (Sudden Infant Death Syndrome and Sudden Intrauterine Unexplained Death Syndrome). High incidence of histological and immunohistochemical alterations (prevalence of epithelial dark cells, the presence of cystic cells in the stroma, decreased number of blood capillaries, hyperexpression of Substance P and apoptosis) were prevalently observed in unexplained death victims (p<0.05 vs. controls). A significant correlation was found between maternal smoking in pregnancy and choroidal neuropathological parameters (p<0.01). This work underscores the negative effects of prenatal exposure to nicotine on the development of the autonomic nervous system, and in particular of the fourth ventricle choroid plexus that is a very vulnerable structure in the developing CSF-brain system.

Neuronal nuclear antigen (NeuN): a useful marker of neuronal immaturity in sudden unexplained perinatal death.

INTRODUCTION: In the developing brain neuronal differentiation is associated with permanent exit from the mitotic cycle. Neuronal nuclear antigen (NeuN) is a nuclear protein widely expressed in the mature postmitotic neurons. METHODS: We applied NeuN immunocytochemistry in 65 cases of perinatal death (16 victims of sudden intrauterine unexplained death syndrome/SIUDS, 19 of sudden infant death syndrome/SIDS and 30 controls) to test the physiological status of the brain neurons. In addition we applied both TUNEL and Caspase 3 immunohistochemical methods in order to highlight a possible relation between decreased NeuN expression and apoptotic outcome. We also attempted to see whether or not NeuN pathological changes can be related to cigarette smoke absorption in pregnancy. RESULTS: NeuN staining was considerably reduced or lost in SIUDS/SIDS compared to controls. However neurons with decreased NeuN-labeling showed no sign of apoptosis. A significant association was found between NeuN depletion and maternal smoking. CONCLUSION: Altered NeuN expression can be a marker of immature and/or suffering neurons. The exclusive presence of this pattern of expression in SIUDS/SIDS victims, leads us to recommend the NeuN immunohistochemistry as a routine method in neuropathological protocols to convalidate a diagnosis of sudden perinatal death.

A novel strategy using cardiac sodium channel polymorphic fragments to rescue trafficking-deficient SCN5A mutations.

BACKGROUND: Brugada syndrome (BrS) is associated with mutations in the cardiac sodium channel (Na(v)1.5). We previously reported that the function of a trafficking-deficient BrS Na(v)1.5 mutation, R282H, could be restored by coexpression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Na(v)1.5, spanning the H558R polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations. METHODS AND RESULTS: Whole-cell patch clamping revealed that cotransfection in human embryonic kidney (HEK293) cells of the R282H channel with either the 40- or 20-amino acid cDNA fragments of Na(v)1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence resonance energy transfer suggested that the trafficking-deficient R282H channel was misfolded, and this was corrected on coexpression with R558-containing peptides that restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R polymorphism with 8 additional BrS Na(v)1.5 mutations with reduced currents and demonstrated that the peptide was able to restore significant sodium currents in 4 of them. CONCLUSIONS: In the present study, we demonstrate that small peptides, spanning the H558R polymorphism, are sufficient to restore the trafficking defect of BrS-associated Na(v)1.5 mutations. Our findings suggest that it might be possible to use short cDNA constructs as a novel strategy tailored to specific disease-causing mutants of BrS.

Prevalence of HCM and long QT syndrome mutations in young sudden cardiac death-related cases.

Cardiomyopathies and channelopathies are major causes of sudden cardiac death. The genetic study of these diseases is difficult because of their heterogenic nature not only in their genetic traits but also in their phenotypic expression. The purpose of the present study is the analysis of a wide spectrum of previously known genetic mutations in key genes related to hypertrophic cardiomyopathy (HCM), long QT syndrome (LQTS), and Brugada syndrome (BrS) development. The samples studied include cases of sudden cardiac death (SCD) in young adults and their relatives in order to identify the real impact of genetic screening of SCD in forensic cases. Genetic screening of described variation in 16 genes implicated in the development of HCM and three more genes implicated in LQTS and BrS was performed by using MassARRAY technology. In addition, direct sequencing of the two most prevalent genes implicated in the development of SQTL type 1 and 2 was also carried out. Genetic screening allowed us to unmask four possibly pathogenic mutation carriers in the 49 SCD cases considered; carriers of mutation represent 9% (2/23) of the probands with structural anomalies found after autopsy and 7% (1/14) of the probands with structurally normal hearts after in depth autopsy protocol. One mutation was found among 12 of the recovered SCD cases considered. In people with direct family history of sudden cardiac death, but not themselves, 11 additional mutation carriers were found. Three different mutations were found in six of the 19 LQTS patients, representing three families and two different mutations were found among six patients with previous syncope. Genetic analysis in sudden cardiac death cases could help to elucidate the cause of death, but it also can help in the prevention of future deaths in families at risk. The study presented here shows the importance and relevance of genetic screening in patients with signs of cardiac hypertrophy and in family cases with more than one relative affected.

Elevated oxidative stress is associated with ventricular fibrillation episodes in patients with Brugada-type electrocardiogram without SCN5A mutation.

BACKGROUND: Brugada syndrome is a disease known to cause ventricular fibrillation with a structurally normal heart and is linked to SCN5A gene mutation. However, the mechanism by which ventricular fibrillation develops in cases of Brugada-type electrocardiogram without SCN5A mutation has remained unclear. Recently, oxidative stress has been implicated in the pathophysiology of cardiac arrhythmia. We also investigated oxidative stress levels in the myocardia of patients with Brugada-type electrocardiogram. METHODS: Endomyocardial biopsy samples were obtained from 68 patients with Brugada-type electrocardiogram (66 males and two females). We performed histological and immunohistochemical analyses for CD45, CD68, and 4-hydroxy-2-nonenal-modified protein, which is a major lipid peroxidation product. RESULTS: SCN5A mutation was detected in 14 patients. Ventricular fibrillation was documented in three patients with SCN5A mutation and in 11 without SCN5A mutation. In patients with SCN5A mutation, 4-hydroxy-2-nonenal-modified protein-positive area was not significantly different between the documented ventricular fibrillation (VF) group (VF+ group) and the group without documented VF (VF- group). However, in patients without SCN5A, the area was significantly larger in the VF+ group than that in the VF- group (P<.05). All other parameters (fibrosis area, CD45, and CD68) were not different between the VF+ and VF- group in both SCN5A+ and SCN5A- patients. CONCLUSION: Oxidative stress is elevated in the myocardium of patients with Brugada-type electrocardiogram who have VF episodes and do not have SCN5A gene mutations. Oxidative stress may be associated with the occurrence of VF in patients with Brugada-type electrocardiogram without SCN5A mutation.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND: Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE: This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS: In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS: Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION: This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Right ventricular histological substrate and conduction delay in patients with Brugada syndrome.

The reported pathogenesis of Brugada syndrome is phase 2 reentry resulting from shortening of the epicardial action potential duration at the right ventricular outflow tract (RVOT). However, several studies have revealed a high incidence of ventricular late potentials and high rate of ventricular fibrillation (VF) induced by programmed ventricular stimulation (PVS). The aim of the present study was to evaluate the role of slow conduction at the RVOT for the initiation of VF by PVS and any underlying pathological conditions in Brugada syndrome. Endocardial mapping of the RVOT and endomyocardial biopsy of the right ventricle were performed in 25 patients with Brugada syndrome with inducible VF. Late potentials were positive in 11 of the 25 (44%) patients. Low-amplitude fragmented and delayed electrograms were recorded at the RVOT in 13 of 18 (72.2%) patients. Histologic examination of the biopsy samples revealed fatty tissue infiltration, interstitial fibrosis, lymphocyte infiltration, and/or myocyte disorganization in 13 patients. Slow conduction at the RVOT may contribute to the induction of VF by PVS in Brugada syndrome. Various pathomorphologic changes may contribute to slow conduction at the RVOT.

Absence of pathognomonic or inflammatory patterns in cardiac biopsies from patients with Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is characterized by the presence of coved ST-segment elevations in the right precordial leads (so-called type I ECG) and additional clinical features. Caused by cardiac ion channel gene mutations, BrS may be associated with ventricular and atrial conduction disturbances as well as ventricular fibrillation. Recent studies have discussed whether BrS is merely a primary electric disorder or whether inflammatory or other histopathologic abnormalities in the right ventricle (RV) underlie the ECG phenotype. METHODS AND RESULTS: We retrospectively analyzed BrS biopsy samples from 21 unrelated patients for histopathologic abnormalities (hypertrophy, fibrosis, inflammation, fatty tissue) together with the patients' clinical, genetic, and imaging data. Eleven patients (52%) had normal RV imaging (by angiography, echocardiography, or cardiac MRI). Results of myocardial biopsies were normal in 3 patients (14%) and revealed mostly moderate abnormalities in the others. Four patients (19%) had predominant fatty tissue in the RV myocardium. Using immunohistochemistry and conventional tissue staining, we could not detect inflammatory tissue changes, an observation compatible with the clinical absence of signs for myocarditis. CONCLUSIONS: Imaging and histopathologic evaluation may detect moderate but uncharacteristic cardiac abnormalities in patients with BrS. None of the patients had arrhythmogenic RV cardiomyopathy or overt myocarditis. Only in a small subset did predominant histopathologic abnormalities in the biopsy samples of the RV outflow tract occur that could provide a link to the ECG phenotype. A variety of mechanisms, including genetic and structural RV alterations, may underlie the Brugada ECG phenotype.