Genetic background determines the severity of age-dependent cardiac structural abnormalities and arrhythmia susceptibility in Scn5a-1798insD mice.

AIMS: Patients with mutations in SCN5A encoding NaV1.5 often display variable severity of electrical and structural alterations, but the underlying mechanisms are not fully elucidated. We here investigate the combined modulatory effect of genetic background and age on disease severity in the Scn5a1798insD/+ mouse model. METHODS AND RESULTS: In vivo electrocardiogram and echocardiograms, ex vivo electrical and optical mapping, and histological analyses were performed in adult (2-7 months) and aged (8-28 months) wild-type (WT) and Scn5a1798insD/+ (mutant, MUT) mice from the FVB/N and 129P2 inbred strains. Atrio-ventricular (AV) conduction, ventricular conduction, and ventricular repolarization are modulated by strain, genotype, and age. An aging effect was present in MUT mice, with aged MUT mice of both strains showing prolonged QRS interval and right ventricular (RV) conduction slowing. 129P2-MUT mice were severely affected, with adult and aged 129P2-MUT mice displaying AV and ventricular conduction slowing, prolonged repolarization, and spontaneous arrhythmias. In addition, the 129P2 strain appeared particularly susceptible to age-dependent electrical, functional, and structural alterations including RV conduction slowing, reduced left ventricular (LV) ejection fraction, RV dilatation, and myocardial fibrosis as compared to FVB/N mice. Overall, aged 129P2-MUT mice displayed the most severe conduction defects, RV dilatation, and myocardial fibrosis, in addition to the highest frequency of spontaneous arrhythmia and inducible arrhythmias. CONCLUSION: Genetic background and age both modulate disease severity in Scn5a1798insD/+ mice and hence may explain, at least in part, the variable disease expressivity observed in patients with SCN5A mutations. Age- and genetic background-dependent development of cardiac structural alterations furthermore impacts arrhythmia risk. Our findings therefore emphasize the importance of continued assessment of cardiac structure and function in patients carrying SCN5A mutations.

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5. In both lines, we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in Tbx5RE(int)KO cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in Tbx5RE(int)KO mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

Secretome of atrial epicardial adipose tissue facilitates reentrant arrhythmias by myocardial remodeling.

BACKGROUND: Epicardial adipose tissue (EAT) accumulation is associated with cardiac arrhythmias. The effect of EAT secretome (EATs) on cardiac electrophysiology remains largely unknown. OBJECTIVE: The purpose of this study was to investigate the arrhythmogenicity of EATs and its underlying molecular and electrophysiological mechanisms. METHODS: We collected atrial EAT and subcutaneous adipose tissue (SAT) from 30 patients with atrial fibrillation (AF), and EAT from 3 donors without AF. The secretome was collected after a 24-hour incubation of the adipose tissue explants. We cultured neonatal rat ventricular myocytes (NRVMs) with EATs, subcutaneous adipose tissue secretome (SATs), and cardiomyocytes conditioned medium (CCM) for 72 hours. We implemented the electrophysiological changes observed after EATs incubation into a model of human left atrium and tested arrhythmia inducibility. RESULTS: Incubation of NRVMs with EATs decreased expression of the potassium channel subunit Kcnj2 by 26% and correspondingly reduced the inward rectifier K+ current IK1 by 35% compared to incubation with CCM, resulting in a depolarized resting membrane of cardiomyocytes. EATs decreased expression of connexin43 (29% mRNA, 46% protein) in comparison to CCM. Cells incubated with SATs showed no significant differences in Kcnj2 or Gja1 expression in comparison to CCM, and their resting potential was not depolarized. Cardiomyocytes incubated with EATs showed reduced conduction velocity and increased conduction heterogeneity compared to SATs and CCM. Computer modeling of human left atrium revealed that the electrophysiological changes induced by EATs promote sustained reentrant arrhythmias if EAT partially covers the myocardium. CONCLUSION: EAT slows conduction, depolarizes the resting potential, alters electrical cell-cell coupling, and facilitates reentrant arrhythmias.

Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation.

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

How Cardiac Embryology Translates into Clinical Arrhythmias.

The electrophysiological signatures of the myocardium in cardiac structures, such as the atrioventricular node, pulmonary veins or the right ventricular outflow tract, are established during development by the spatial and temporal expression of transcription factors that guide expression of specific ion channels. Genome-wide association studies have shown that small variations in genetic regions are key to the expression of these transcription factors and thereby modulate the electrical function of the heart. Moreover, mutations in these factors are found in arrhythmogenic pathologies such as congenital atrioventricular block, as well as in specific forms of atrial fibrillation and ventricular tachycardia. In this review, we discuss the developmental origin of distinct electrophysiological structures in the heart and their involvement in cardiac arrhythmias.

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias.

[Figure: see text].

Why Ablation of Sites With Purkinje Activation Is Antiarrhythmic: The Interplay Between Fast Activation and Arrhythmogenesis.

Ablation of sites showing Purkinje activity is antiarrhythmic in some patients with idiopathic ventricular fibrillation (iVF). The mechanism for the therapeutic success of ablation is not fully understood. We propose that deeper penetrance of the Purkinje network allows faster activation of the ventricles and is proarrhythmic in the presence of steep repolarization gradients. Reduction of Purkinje penetrance, or its indirect reducing effect on apparent propagation velocity may be a therapeutic target in patients with iVF.

Critical repolarization gradients determine the induction of reentry-based torsades de pointes arrhythmia in models of long QT syndrome.

BACKGROUND: Torsades de pointes arrhythmia is a potentially lethal polymorphic ventricular tachyarrhythmia (pVT) in the setting of long QT syndrome. Arrhythmia susceptibility is influenced by risk factors modifying repolarization. OBJECTIVE: The purpose of this article was to characterize repolarization duration and heterogeneity in relation to pVT inducibility and maintenance. METHODS: Sotalol was infused regionally or globally in isolated Langendorff blood-perfused pig hearts (N = 7) to create repolarization time (RT) heterogeneities. Programmed stimulation and epicardial activation and repolarization mapping were performed. The role of RT (heterogeneities) was studied in more detail using a computer model of the human heart. RESULTS: pVTs (n = 11) were inducible at a critical combination of RT and RT heterogeneities. The pVT cycle lengths were similar in the short and long RT regions. Short-lasting pVTs were maintained by focal activity while longer-lasting pVTs by reentry wandering along the interface between the 2 regions. Local restitution curves from the long and short RT regions crossed. This was associated with T-wave inversion at coupling intervals at either side of the crossing point. These experimental observations were confirmed by the computer simulations. CONCLUSION: pVTs are inducible within a critical range of RT and RT heterogeneities and are maintained by reentry wandering along the repolarization gradient. Double potentials localize at the core of the reentrant circuit and reflect phase singularities. RT gradient and T waves invert with short-coupled premature beats in the long RT region as a result of the crossing of the restitution curves allowing reentry initiation.

Functional modulation of atrio-ventricular conduction by enhanced late sodium current and calcium-dependent mechanisms in Scn5a1798insD/+ mice.

AIMS: SCN5A mutations are associated with arrhythmia syndromes, including Brugada syndrome, long QT syndrome type 3 (LQT3), and cardiac conduction disease. Long QT syndrome type 3 patients display atrio-ventricular (AV) conduction slowing which may contribute to arrhythmogenesis. We here investigated the as yet unknown underlying mechanisms. METHODS AND RESULTS: We assessed electrophysiological and molecular alterations underlying AV-conduction abnormalities in mice carrying the Scn5a1798insD/+ mutation. Langendorff-perfused Scn5a1798insD/+ hearts showed prolonged AV-conduction compared to wild type (WT) without changes in atrial and His-ventricular (HV) conduction. The late sodium current (INa,L) inhibitor ranolazine (RAN) normalized AV-conduction in Scn5a1798insD/+ mice, likely by preventing the mutation-induced increase in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) concentrations. Indeed, further enhancement of [Na+]i and [Ca2+]i by the Na+/K+-ATPase inhibitor ouabain caused excessive increase in AV-conduction time in Scn5a1798insD/+ hearts. Scn5a1798insD/+ mice from the 129P2 strain displayed more severe AV-conduction abnormalities than FVB/N-Scn5a1798insD/+ mice, in line with their larger mutation-induced INa,L. Transverse aortic constriction (TAC) caused excessive prolongation of AV-conduction in FVB/N-Scn5a1798insD/+ mice (while HV-intervals remained unchanged), which was prevented by chronic RAN treatment. Scn5a1798insD/+-TAC hearts showed decreased mRNA levels of conduction genes in the AV-nodal region, but no structural changes in the AV-node or His bundle. In Scn5a1798insD/+-TAC mice deficient for the transcription factor Nfatc2 (effector of the calcium-calcineurin pathway), AV-conduction and conduction gene expression were restored to WT levels. CONCLUSIONS: Our findings indicate a detrimental role for enhanced INa,L and consequent calcium dysregulation on AV-conduction in Scn5a1798insD/+ mice, providing evidence for a functional mechanism underlying AV-conduction disturbances secondary to gain-of-function SCN5A mutations.

Heritable arrhythmia syndromes associated with abnormal cardiac sodium channel function: ionic and non-ionic mechanisms.

The cardiac sodium channel NaV1.5, encoded by the SCN5A gene, is responsible for the fast upstroke of the action potential. Mutations in SCN5A may cause sodium channel dysfunction by decreasing peak sodium current, which slows conduction and facilitates reentry-based arrhythmias, and by enhancing late sodium current, which prolongs the action potential and sets the stage for early afterdepolarization and arrhythmias. Yet, some NaV1.5-related disorders, in particular structural abnormalities, cannot be directly or solely explained on the basis of defective NaV1.5 expression or biophysics. An emerging concept that may explain the large disease spectrum associated with SCN5A mutations centres around the multifunctionality of the NaV1.5 complex. In this alternative view, alterations in NaV1.5 affect processes that are independent of its canonical ion-conducting role. We here propose a novel classification of NaV1.5 (dys)function, categorized into (i) direct ionic effects of sodium influx through NaV1.5 on membrane potential and consequent action potential generation, (ii) indirect ionic effects of sodium influx on intracellular homeostasis and signalling, and (iii) non-ionic effects of NaV1.5, independent of sodium influx, through interactions with macromolecular complexes within the different microdomains of the cardiomyocyte. These indirect ionic and non-ionic processes may, acting alone or in concert, contribute significantly to arrhythmogenesis. Hence, further exploration of these multifunctional effects of NaV1.5 is essential for the development of novel preventive and therapeutic strategies.

Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease.

BACKGROUND: Cardiovascular diseases (CVDs) culminating into heart failure (HF) are major causes of death in men and women. Prevalence and manifestation, however, differ between sexes, since men mainly present with coronary artery disease (CAD) and myocardial infarction (MI), and post-menopausal women predominantly present with hypertension. These discrepancies are probably influenced by underlying genetic and molecular differences in structural remodeling pathways involved in hypertrophy, inflammation, fibrosis, and apoptosis. In general, men mainly develop eccentric forms, while women develop concentric forms of hypertrophy. Besides that, women show less inflammation, fibrosis, and apoptosis upon HF. This seems to emerge, at least partially, from the fact that the underlying pathways might be modulated by estrogen, which changes after menopause due to declining of the estrogen levels. CONCLUSION: In this review, sex-dependent alterations in adverse cardiac remodeling are discussed for various CVDs. Moreover, potential therapeutic options, like estrogen treatment, are reviewed.

Flotillins in the intercalated disc are potential modulators of cardiac excitability.

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Enhanced late sodium current underlies pro-arrhythmic intracellular sodium and calcium dysregulation in murine sodium channelopathy.

BACKGROUND: Long QT syndrome mutations in the SCN5A gene are associated with an enhanced late sodium current (INa,L) which may lead to pro-arrhythmic action potential prolongation and intracellular calcium dysregulation. We here investigated the dynamic relation between INa,L, intracellular sodium ([Na+]i) and calcium ([Ca2+]i) homeostasis and pro-arrhythmic events in the setting of a SCN5A mutation. METHODS AND RESULTS: Wild-type (WT) and Scn5a1798insD/+ (MUT) mice (age 3-5 months) carrying the murine homolog of the SCN5A-1795insD mutation on two distinct genetic backgrounds (FVB/N and 129P2) were studied. [Na+]i, [Ca2+]i and Ca2+ transient amplitude were significantly increased in 129P2-MUT myocytes as compared to WT, but not in FVB/N-MUT. Accordingly, INa,L wassignificantly more enhanced in 129P2-MUT than in FVB/N-MUT myocytes, consistent with a dose-dependent correlation. Quantitative RT-PCR analysis revealed intrinsic differences in mRNA expression levels of the sodium/potassium pump, the sodium/hydrogen exchanger, and sodium­calcium exchanger between the two mouse strains. The rate of increase in [Na+]i, [Ca2+]i and Ca2+ transient amplitude following the application of the Na+/K+-ATPase inhibitor ouabain was significantly greater in 129P2-MUT than in 129P2-WT myocytes and was normalized by the INa,L inhibitor ranolazine. Furthermore, ranolazine decreased the incidence of pro-arrhythmic calcium after-transients elicited in 129P2-MUT myocytes. CONCLUSIONS: In this study we established a causal link between the magnitude of INa,L, extent of Na+ and Ca2+ dysregulation, and incidence of pro-arrhythmic events in murine Scn5a1798insD/+ myocytes. Furthermore, our findings provide mechanistic insight into the anti-arrhythmic potential of pharmacological inhibition of INa,L in patients with LQT3 syndrome.

A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac sodium channelopathy.

Aims: Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results: Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion: This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals.

Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes.

BACKGROUND: Cardiac sodium channel (NaV1.5) dysfunction contributes to arrhythmogenesis during pathophysiological conditions. Nav1.5 localizes to distinct subcellular microdomains within the cardiomyocyte, where it associates with region-specific proteins, yielding complexes whose function is location specific. We herein investigated sodium channel remodeling within distinct cardiomyocyte microdomains during heart failure. METHODS AND RESULTS: Mice were subjected to 6 weeks of transverse aortic constriction (TAC; n=32) to induce heart failure. Sham-operated on mice were used as controls (n=20). TAC led to reduced left ventricular ejection fraction, QRS prolongation, increased heart mass, and upregulation of prohypertrophic genes. Whole-cell sodium current (INa) density was decreased by 30% in TAC versus sham-operated on cardiomyocytes. On macropatch analysis, INa in TAC cardiomyocytes was reduced by 50% at the lateral membrane (LM) and by 40% at the intercalated disc. Electron microscopy and scanning ion conductance microscopy revealed remodeling of the intercalated disc (replacement of [inter-]plicate regions by large foldings) and LM (less identifiable T tubules and reduced Z-groove ratios). Using scanning ion conductance microscopy, cell-attached recordings in LM subdomains revealed decreased INa and increased late openings specifically at the crest of TAC cardiomyocytes, but not in groove/T tubules. Failing cardiomyocytes displayed a denser, but more stable, microtubule network (demonstrated by increased α-tubulin and Glu-tubulin expression). Superresolution microscopy showed reduced average NaV1.5 cluster size at the LM of TAC cells, in line with reduced INa. CONCLUSIONS: Heart failure induces structural remodeling of the intercalated disc, LM, and microtubule network in cardiomyocytes. These adaptations are accompanied by alterations in NaV1.5 clustering and INa within distinct subcellular microdomains of failing cardiomyocytes.

Misinterpretation of the mouse ECG: 'musing the waves of Mus musculus'.

The ECG is a primary diagnostic tool in patients suffering from heart disease, underscoring the importance of understanding factors contributing to normal and abnormal electrical patterns. Over the past few decades, transgenic mouse models have been increasingly used to study pathophysiological mechanisms of human heart diseases. In order to allow extrapolation of insights gained from murine models to the human condition, knowledge of the similarities and differences between the mouse and human ECG is of crucial importance. In this review, we briefly discuss the physiological mechanisms underlying differences between the baseline ECG of humans and mice, and provide a framework for understanding how these inherent differences are relevant to the interpretation of the mouse ECG during pathology and to the translation of the results from the mouse to man.