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Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare cardiac disease characterized by myocardial fibrofatty replacement, which can lead to sudden death. Previous studies have described a reduction of plakoglobin (PKG) protein at the level of intercalated disks as the hallmark of ARVC. The main objective of this study was to investigate the involvement of desmosome mutations in the histological phenotype of ARVC. We performed a genetic analysis of ARVC cases, and histological characterization of ARVC heart tissue samples. We genetically analyzed 48 ARVC cases distributed into two groups: 42 human tissue heart samples with conclusive diagnoses of ARVC after post-mortem examination; and six DNA samples from peripheral blood of living patients who were clinically diagnosed. Sequenom MassARRAY analysis revealed three ARVC-associated variants in three patients in 42 tissue samples (7.14 %). Three individuals carried one single pathogenic mutation, p.R811S _ PKP2, p.S824L_ DSC2, and p.T526M_ PKP2 in postmortem samples. In the living patients group, Sequenom MassARRAY revealed no mutation, however, later Sanger sequencing analysis identified three ARVC mutations in 2/6 patients not included in the Sequenom design. In post-mortem tissue samples we performed immunohistochemical labeling for desmosomal proteins and Connexin 43. This study revealed that PKP2 carriers present either absent or clearly reduced PKG immunolabeling, while the DSC2 carrier showed PKG immunolabeling similar to control samples. Immunolabeling for Cx43 did not show any differences compared to controls. The present Sequenom MassARRAY design is a useful tool for post-mortem genetic diagnosis of ARVC. Plakoglobin reduction occurs at intercalated disks, while other desmosome proteins and Cx43 remain unaltered. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Obra Social "La Caixa Foundation" Introduction Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a heritable cardiac disease characterized by a progressive loss of myocardium and its replacement by fibrofatty tissue that predominantly affects the right ventricle (1,2). Worldwide prevalence of ARVC is ranging from 1:1000 to 1:5000 (3,4), and it is responsible for around 20% of all cases of sudden cardiac death (SCD) among people under 35 years of age (5). The main genetic cause are mutations in desmosomal genes (4). It is known that mutations in those genes could alter important molecular functions such as calcium homeostasis (6), and they also could cause important structural and electrical alterations such as fibrosis, adipogenesis, inflammation and arrhythmogenesis (7). We lack better understanding of those pathophysiological mechanisms and their relationship with the causal gene. Purpose This study aims to elucidate molecular pathways triggered by genetic defects in three of the main genes in ARVC and determine common and gene-specific molecular features. Methods Cardiac HL-1 cell line was edited by CRISPR/Cas9 to generate Knock Outs (KO) of three of the most frequent causal genes in ARVC: PKP2, DSG2 and DSC2. Expression levels of ARVC-related genes were tested by RT-PCR: desmosome, calcium handling and connexome related genes. Changes in the total amount of protein were checked by western blot using total protein lysate. Moreover, calcium transients were performed on all clones. Results HL1 KO-PKP2 showed downregulation of SCN5A, CX43, RyR2, Atp2a2, Slc8a1, Casq2, ANK2, DSC2 DSG2 and DSP. HL1 KO-DSG2 showed a downregulation of ANK2, DSC2, CX43 and PG and HL1 KO-DSC2 clones presented a downregulation of CASQ2 and ANK2. Functionally, calcium transients showed significant differences in 10mM peak of caffeine in PKP2 but still not conclusive for DSC2 and DSG2 clones. Conclusions Results suggest that pathophysiological mechanisms of ARVC are partially depending on the causal gene. Loss of function of PKP2 and DSC2 may be related to a dysregulation of calcium homeostasis and loss of function of DSG2 may cause alterations in connexome genes expression related to electrical dysfunction. Moreover, the absence of ANK2 expression in all HL1 cell lines might suggest a common molecular hallmark of ARVC in the presented cell model. [ABSTRACT FROM AUTHOR]

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Fatty acid synthase (FASN) expression and activity has emerged as a common phenotype in most human carcinomas, including breast cancer, and its expression is tightly linked to HER2 signaling pathways. The development of inhibitors of FASN activity has consequently appeared as a novel antitarget modality for treating cancer. However, the clinical use of FASN inhibitors, such as cerulenin, C75, and epigallocatechin 3-gallate (EGCG), is limited by anorexia and induced body weight loss or by its low in vivo potency and stability. Here, we summarize the design and development of G28UCM, the lead-compound of a novel family of synthetic FASN inhibitors, with both in vitro and in vivo activity in a human breast cancer model of FASN and HER2. [ABSTRACT FROM AUTHOR]

Barahona-Dussault C, Benito B, Campuzano O, Iglesias A, Leung TL, Robb L, Talajic M, Brugada R. Role of genetic testing in arrhythmogenic right ventricular cardiomyopathy/dysplasia. In a cohort of patients with confirmed or suspected arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), genetic testing is useful in confirming the diagnosis, particularly in individuals who do not completely fulfil Task Force criteria for the disease, thereby also enabling the adoption of preventive measures in family members. Due to the high percentage of novel mutations that are expected to be identified in ARVC/D, the use of genetic screening technology based on the identification of known mutations seems to have very restricted value. Our results support that the presence of certain genetic variations could play a role in the final phenotype of patients with ARVC/D, where single and compound mutation carriers would have more symptomatic forms of the disease and the polymorphism P366L could be associated to a more benign phenotype. [ABSTRACT FROM AUTHOR]

Objective: Short QT syndrome (SQTS) is characterized by ventricular arrhythmias and sudden death. One form of SQTS is caused by mutation N588K in human ether-a-go-go-related gene (HERG). In this study we sought to determine the potential role of N588K in arrhythmias. Methods: We measured the characteristics of HERG current generated by wild-type (WT) KCNH2 and the N588K mutant channel expressed in mammalian TSA201 cells. Results: Whole-cell patch-clamp recordings of WT HERG currents showed the usual rapid onset of inactivation (rectification) at potentials more positive than +10 mV. In contrast, N588K currents rectified at potentials over +80 mV. Over the physiological range of potentials, N588K currents do not inactivate. During an action potential clamp, WT currents displayed a “hump” like waveform with slow activation kinetics and a rapid increase during phase 3 repolarization. In contrast, N588K currents were proportional to the amplitude of the action potential and displayed a dome-like configuration and a much larger current during the initial phases in the ventricle. Purkinje cell action potentials display a more negative phase 2 repolarization than the ventricle and elicited much smaller WT and N588K currents of similar amplitudes. Conclusions: Physiologically the N588K mutation abolishes rectification of HERG currents and specifically increases I Kr in the ventricle with minimal effects on the Purkinje fiber action potential duration. Such preferential prolongation may explain the separation of the T and U waves observed in the ECG of SQTS patients and lead to re-excitation of the ventricle endocardium. [Copyright &y& Elsevier]

Background The syndrome with an electrocardiographic pattern of right bundle branch block, ST segment elevation in leads V1to V3and sudden death is genetically determined and caused by mutations in the cardiac sodium channel. The inheritance of the disease is autosomal dominant. Sudden death may, however, occur from a variety of causes in relatives and patients with this syndrome.Patients and Methods Twenty-five Flemish families with this syndrome with a total of 334 members were studied. Affected members were recognized by means of a typical electrocardiogram either occurring spontaneously or after the intravenous administration of antiarrhythmic drugs. Sudden deaths in these families were classified as related or not to the syndrome by analysis of the data at the time of the event, mode of inheritance of the disease, and data provided by survivors.Results Of the 25 families with the syndrome, 18 were symptomatic (at least one sudden death related to the syndrome) and seven were asymptomatic (no sudden deaths related to the syndrome). In total, there were 42 sudden cardiac deaths (12% incidence). Twenty-four sudden deaths were related to the syndrome and all occurred in symptomatic families. Eighteen sudden deaths (43% of total sudden deaths) were not related to the syndrome (nine cases) or were of unclear cause (nine cases). Three of them occurred in two asymptomatic families and the remaining 15 in five symptomatic families. Twenty-four of the 50 affected members (47%) suffered (aborted) sudden death and 18 of the 284 unaffected members (6%). This difference in the incidence of sudden death was statistically significant (P<0·0001). Patients with (aborted) sudden death caused by the syndrome were younger than patients with sudden death of other or unclear causes (38±4 years vs 59±3 years respectively,P =0·0003).Conclusions In families at high risk of sudden death because of genetically determined diseases, the main cause of sudden death remains the disease. However, almost the half of sudden deaths are caused by unrelated diseases or are of unclear cause. Accurate classification of the causes of sudden death is mandatory for appropriate analysis of the causes of death when designing preventive treatments. [ABSTRACT FROM PUBLISHER]

In 1992 a new syndrome was described consisting of syncopal episodes and/or sudden death in patients with a structurally normal heart and an electrocardiogram (ECG) characteristic of right bundle branch block with ST segment elevation in leads V1to V3. The disease is genetically determined, with an autosomal dominant pattern of transmission. Three different mutations that affect the structure and function of the cardiac sodium channel gene SCN5A have been identified. Two mutations result in total loss of function of the sodium channel. The other mutation results in acceleration of the recovery of the sodium channel from inactivation. The incidence of the disease is difficult to estimate, but it causes 4 to 10 sudden deaths per 10 000 inhabitants per year in areas like Thailand and Laos. In these countries, the disease represents the most frequent cause of death in young adults. Up to 50% of the yearly sudden deaths in patients with a structurally normal heart are caused by this syndrome. The diagnosis is easily made by means of the ECG. The presence of concealed and intermittent forms, however, make the diagnosis difficult in some patients. The ECG can be modulated by changes in autonomic balance and the administration of antiarrhythmic drugs. Beta-adrenergic stimulation normalizes the ECG, while intravenous ajmaline, flecainide or procainamide accentuate ST segment elevation and are capable of unmasking concealed and intermittent forms of the disease. Recent data suggest that loss of the action potential dome in the right ventricular epicardium but not the endocardium underlies ST segment elevation seen in the Brugada syndrome. Also, electrical heterogeneity within the right ventricular epicardium leads to the development of closely coupled extrasystoles via a phase 2 reentrant mechanism, which then precipitates ventricular tachycardia–ventricular fibrillation. Right ventricular epicardium is preferentially affected because of the predominance of transient outward current in this tissue. Antiarrhythmic drugs like amiodarone and beta-blockers do not prevent sudden death in symptomatic or asymptomatic individuals. Gene therapy may offer a cure in future years. Implantation of an automatic cardioverter–defibrillator is the only currently proven effective therapy. [ABSTRACT FROM PUBLISHER]

Brugada Syndrome (BrS) is a life-threating arrhythmogenic disease associated with a high risk of Sudden Cardiac Death. Genetic alterations in SCN5A, which encodes the alpha subunit of the cardiac voltage-gated sodium channel (Nav1.5), are the most common cause of BrS. Nav1.5 channel is responsible for the rapid influx of sodium ions that initiate the propagation of the action potential in cardiomyocytes. However, mutations in SCN5A gene only explain 20-25% of the cases with BrS. Our working hypothesis is that a deregulation of SCN5A expression could cause BrS and explain, at least in part, some cases of BrS with no mutation identified. Nevertheless, little is known regarding the regulation of SCN5A expression. Therefore, it is the goal of this study to gain insight into the molecular mechanisms that regulate SCN5A expression at the transcriptional level, and how alterations of these mechanisms contribute to cardiac arrhythmias.The human SCN5A promoter contains putative binding sites for the zinc finger transcription factor GATA-4. We performed GATA-4 overexpression and knockdown studies in cardiac H9c2 cells, and analyzed the effect on the SCN5A promoter by luciferase reporter experiments and real-time PCR. Our data showed that GATA-4 is a novel transcriptional activator of the SCN5A promoter. We also analyzed the effect of other GATA family members, GATA-5 and -6. Whereas GATA-6 did not have any effect on the SCN5A promoter, GATA-5 increased SCN5A transcriptional activity although to a less extent than GATA-4. Moreover, we observed that GATA-4 significantly synergizes with GATA-5 on SCN5A transcriptional activation. Chromatin Immunoprecipitation (ChIP) experiments from human cardiac tissue showed that GATA-4 and GATA-5 bind to the SCN5A proximal promoter region, confirming a critical role of these factors in regulating SCN5A expression in vivo. Our current experiments are also focused on studying the interaction between GATA-4 and GATA-5 transcription factors.We performed co-transfection experiments of GATA-4 with p300 acetyltransferase and observed a further increase on SCN5A promoter activity. These results suggest that GATA-4 transcriptional activity on the SCN5A promoter is modulated by acetylation. We are now investigating which histones acetyltransferases (HDACs) could participate in the activity of GATA-4 on the SCN5A promoter.Our findings provide novel insights on the regulation of Nav1.5 expression, which will ultimately contribute to further understanding unexplored causes of BrS and other type of cardiac arrhythmias. [ABSTRACT FROM AUTHOR]

Long QT Syndrome (LQTS) is a rare inherited cardiac disorder with a high risk of sudden cardiac death in a structurally normal heart. To date, pathogenic mutations have been described as responsible for approximately 70-75% of LQTS patients, mainly in ion channel genes. Mutations in genes encoding the sodium channel α subunit or other regulatory proteins, affecting cardiac sodium current, have been previously related to LQTS. Five sodium channel β subunits have been identified, which are encoded by four genes (SCN1B-4B). Pathogenic mutations in the SCN4B gene, but not in other β subunits, have been reported in LQTS. We tested whether mutations in SCN1B-4B could be responsible for LQTS in patients without mutations in the common LQTS-related genes.We screened for mutations in SCN1B-4B genes in 30 non-related patients clinically diagnosed with LQTS carrying no mutations in the major LQTS-related genes. The screening revealed a novel mutation in the SCN1B gene in an 8-year-old boy. The base change resulted in an amino acid variation from proline to threonine in the alternative C-terminus of the sodium channel β1 subunit (β1b).Using the patch clamp technique, we measured sodium current density, and Nav1.5 gaiting properties, in HEK cells transiently transfected with Nav1.5 and β1b subunits. Our electrophysiological analysis revealed that the mutant β1b altered Nav1.5 function by shifting the window current to negative potentials, increasing recovery from inactivation, decreasing slow inactivation, and increasing late sodium current. In addition, we recorded action potentials from mouse atrial cardiomyocytes, HL-1 cells, transfected with β1b subunits. These experiments revealed that the action potential duration significantly increased when the mutant β1b was overexpressed compared to β1bWT.These findings suggest that the mutation in β1b could explain the LQTS in our patient, revealing SCN1Bb as a new susceptibility gene for LQTS. Our results confirm the importance of sodium channel β subunits in the modulation of cardiac sodium channel. In addition, they highlight the need for further investigation to detect new candidate genes underlying the LQTS patients that currently remain without genetic diagnosis. [ABSTRACT FROM AUTHOR]

Objective: Plakophilin-2 (PKP2) mutations represent 50% of desmosomal mutations associated to Arrythmogenic Right Ventricular Cardiomyopathy (ARVC) in humans. Many of these mutations cause a truncated protein PKP2. However, the mechanism whereby truncated PKP2 causes disease is unclear. The aim of this study is to unravel the molecular and cellular mechanism of truncated PKP2 in ARVD pathogenesis.Methods: For this study, we developed a transgenic mouse model containing a truncated PKP2 (329 of 837aa native protein) under specific cardiac promoter (α-MHC). We have studied alterations in the expression and the localization of different desmosomal proteins. In addition, we have studied alterations in cardiac morphology and size. We have also analyzed the histological phenotype using hematoxylin-eosin and Masson trichrome staining and oil red and Sirius red methods to detect the fatty and fibrous tissue. The level of cardiomyocytes early apoptosis has been analyzed by TUNEL. Finally, the ultrastructure of the intercalated disc has been analyzed using a transmission electronic microscopy.Results: The truncated PKP2 was localized in the intercalated disc and a small fraction in the cytoplasm of cardiomyocytes. Interestingly, an intense signal of truncated protein was found in the nucleus of some cardiomyocytes. The expression and localization of distinct desmosomal proteins was similar in hearts from wildtype and transgenic mice. We do not detect any cardiac hypertrophy in the transgenic mice when it was compared with wildtype mice (0.65 ± 0.11 vs 0.71 ± 0.15; respectively). We have observed fibrosis in cardiac tissue in 2 of 30 total transgenic mice analyzed (<7%). This is characterized by the presence of fibrous tissue with absence of adipocytes and apoptosis. Ultrastructure of desmosome reveals micro-breakages in the intercalated discs of cardiomyocytes from transgenic mice including the ones without fibrous tissue replacement.Conclusions: Although a low frequency of fibrosis was found, the presence of micro-breakages suggests that the truncated PKP2 reduces the cardiomyocytes adhesion/viability and increases the predisposition to the development of fibrosis. We can hypothesize that presence of truncated PKP2 in the intercalated disc could compete with the endogenous PKP2 binding sites, affecting to the cardiac tissue integrity. [ABSTRACT FROM PUBLISHER]

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare cardiac disease characterized by fibrofatty replacement of the right ventricular myocardium, which may cause ventricular arrhythmias and sudden cardiac death (SCD).Mutations in genes encoding desmosome proteins have been associated with ARVC: plakophilin-2 (PKP2), desmoplakin (DSP), desmocolin-2 (DSC2), desmoglein-2 (DSG2) and plakoglobin (PG). Plakophilin-2 (PKP2) is the most prevalent gene associated with ARVC, especially stop-gained mutations, which lead to truncated PKP2 (PKP2TR).Our recent genetic studies have shown that PKP2TR is associated to a later age of ARVC onset (37 years old) compared to missense carriers (27 years old). This raised an alternative hypothesis as to the pathogenicity of missense and truncated proteins, and their role in phenotype. We hypothesize that PKP2TR is associated with trafficking to the plasma membrane, which can be compensated by the normal allele; while missense variations may act through a dominant negative effect, disrupting the function of the wild type protein since they are mostly incorporated to desmosome.To explore this hypothesis, we performed an in vitro study with the three following aims: (1) to examine the subcellular location of PKP2TR forms in the in HL-1 cardiac-like cells, (2) how PKP2TR length affect to their proper traffic to plasma membrane and (3) PKP2TR effect on desmosome assembly. Based on mutations associated to ARVC in our genetic study, we selected four PKP2TR versions (at amino acids 91, 412, 637 and 734), which were transiently co-transfected in HL-1 cells with major desmosome proteins (DSP,DSC2, DSG2 and PG).We observed that these PKP2TR forms are not localized efficiently to the plasma membrane; PKP2TR-91 remained diffuse in the cytoplasm; PKP2TR-412 was retained in the endoplasmic reticulum displaying a dotted pattern. In contrast PKP2TR-637 and PKP2TR-734 were only partially located to the plasma membrane with a major fraction still cytosolic. We also observed that all PKP2TR caused deficient localization of other desmosome proteins, especially DSP, which requires PKP2 for its proper trafficking to the plasma membrane.Thus, these data suggest that the degree of the truncation in PKP2 does not correlate with a clear effect in desmosome assembly. Results clearly show that PKP2TR forms are not efficiently incorporated to the plasma membrane, and they affect desmosome assembly. These data favour the idea that PKP2TR may be associated to trafficking defect, while missense mutations may act in a dominant negative way disrupting function of the wild-type protein. [ABSTRACT FROM PUBLISHER]

Purpose: Cardiovascular pathologies are the main cause of sudden death in developed countries. In population older than 50 years old, most deaths are due to coronary disease alterations, in the young deaths may be caused by "monogenetic disorders", channelopathies, or cardiomyopathies. Genetic analysis is not routinely performed in autopsy protocol when a cause of death is not identified in the young. In recent years the development of next generation sequencing (NGS) technology allows the investigation of high number of genes in a short period of time at low cost. We launched the MOSCAT project, a prospective post-mortem study that aims to identify the genetic cause of death in cases that remains without a conclusive diagnosis after a complete autopsy. Methods: Our cohort included a total of 88 cases: <50 years old, and no-conclusive cause of death after complete autopsy investigation. We divided the cohort into two groups, depending on DNA quality for NGS analysis. The first group included 59 samples with low quality. The main seven arrhythmogenic genes (KCNQ1, KCNH2, KCNE1, KCNE2, KCNE3, RYR2 and SCN5A) were analyzed using Sanger method. The second group included 43 samples analyzed by NGS, a total of 55 genes associated with SCD were analyzed. Potentially pathogenic role was determined when minor allele frequency (MAF) was less than 1% and at least one in silico prediction. A total of 14 samples were analyzed using both methods as internal control. Results: Our cohort of 88 samples showed a gender ratio of 2:1 (men: women respectively). In group 1 (sanger, 59 samples) we identified 30% arrythmogenic mutation carriers. In group 2 (NGS, 43 samples) we identified 15% arrythmogenic mutations and 85% associated with structural cardiomyopathies. Acording to age in group 1 we identified 27% mutations in patients younger than 35 and 34% in older than 35. By NGS we identified 50% arrythmogenic mutations and 50% structural mutations in less than 35 and 40% arrythmogenic mutations and 60% structural mutations in older than 35. Conclusions: In patients younger than 50, we identified arrythmogenic mutations in 25% and structural mutations in 70% of cases studied. Nearly 30% of post-mortem cases minor than 50 years with a no-conclusive cause of death carry a potential pathogenic variation that could explain the death. NGS technology adds additional information and may be useful in the identification of genetic causality. We believe that genetic analysis should be included in routine forensic guidelines when a no-conclusive cause of death arises, especially in cases under 35 years old. [ABSTRACT FROM PUBLISHER]