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Heart failure has reached epidemic proportions in a progressively ageing population. The molecular mechanisms underlying heart failure remain elusive, but evidence indicates that DNA damage is enhanced in failing hearts. Here, we tested the hypothesis that endogenous DNA repair in cardiomyocytes is critical for maintaining normal cardiac function, so that perturbed repair of spontaneous DNA damage drives early onset of heart failure. To increase the burden of spontaneous DNA damage, we knocked out the DNA repair endonucleases xeroderma pigmentosum complementation group G (XPG) and excision repair cross-complementation group 1 (ERCC1), either systemically or cardiomyocyte-restricted, and studied the effects on cardiac function and structure. Loss of DNA repair permitted normal heart development but subsequently caused progressive deterioration of cardiac function, resulting in overt congestive heart failure and premature death within 6 months. Cardiac biopsies revealed increased oxidative stress associated with increased fibrosis and apoptosis. Moreover, gene set enrichment analysis showed enrichment of pathways associated with impaired DNA repair and apoptosis, and identified TP53 as one of the top active upstream transcription regulators. In support of the observed cardiac phenotype in mutant mice, several genetic variants in the ERCC1 and XPG gene in human GWAS data were found to be associated with cardiac remodelling and dysfunction. In conclusion, unrepaired spontaneous DNA damage in differentiated cardiomyocytes drives early onset of cardiac failure. These observations implicate DNA damage as a potential novel therapeutic target and highlight systemic and cardiomyocyte-restricted DNA repair-deficient mouse mutants as bona fide models of heart failure.

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.

Aim The voltage-gated sodium channel NaV 1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5amutations. We investigated when sodium current (INa ) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV 1.5 dysfunction impacts on cardiac development. Methods Involvement of NaV 1.5-generated INa in murine cardiac electrical function was assessed by optical mapping in wild type (WT) embryos (embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µmol/L). INa was assessed by patch-clamp analysis in cardiomyocytes isolated from WT embryos (E9.5-17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a-1798insD-/- embryos (E9.5-10.5) and their WT littermates. Results In WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch-clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a-1798insD-/- embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a-1798insD-/- E9.5 embryos before the onset of INa , in addition to reduced cardiac tissue volume compared to WT littermates. Conclusion We here demonstrate that NaV 1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a-1798insD-/- embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV 1.5 dysfunction impacts on embryonic cardiac development in a non-electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV 1.5 dysfunction.

The voltage-gated sodium channel NaInvolvement of NaIn WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch-clamp measurements revealed that IWe here demonstrate that Na

Background Electrocardiographic ( ECG ) parameters are regarded as intermediate phenotypes of cardiac arrhythmias. Insight into the genetic underpinnings of these parameters is expected to contribute to the understanding of cardiac arrhythmia mechanisms. Here we used HXB / BXH recombinant inbred rat strains to uncover genetic loci and candidate genes modulating ECG parameters. Methods and Results RR interval, PR interval, QRS duration, and QT c interval were measured from ECG s obtained in 6 male rats from each of the 29 available HXB / BXH recombinant inbred strains. Genes at loci displaying significant quantitative trait loci (QTL) effects were prioritized by assessing the presence of protein‐altering variants, and by assessment of cis expression QTL ( eQTL ) effects and correlation of transcript abundance to the respective trait in the heart. Cardiac RNA ‐seq data were additionally used to generate gene co‐expression networks. QTL analysis of ECG parameters identified 2 QTL for PR interval, respectively, on chromosomes 10 and 17. At the chromosome 10 QTL , cis ‐ eQTL effects were identified for Acbd4 , Cd300lg , Fam171a2 , and Arhgap27 ; the transcript abundance in the heart of these 4 genes was correlated with PR interval. At the chromosome 17 QTL , a cis ‐ eQTL was uncovered for Nhlrc1 candidate gene; the transcript abundance of this gene was also correlated with PR interval. Co‐expression analysis furthermore identified 50 gene networks, 6 of which were correlated with PR interval or QRS duration, both parameters of cardiac conduction. Conclusions These newly identified genetic loci and gene networks associated with the ECG parameters of cardiac conduction provide a starting point for future studies with the potential of identifying novel mechanisms underlying cardiac electrical function.

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.

Background— Sodium channel Na V 1.5 underlies cardiac excitability and conduction. The last 3 residues of Na V 1.5 (Ser-Ile-Val) constitute a PDZ domain–binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of Na V 1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results— To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced Na V 1.5 expression and sodium current ( I Na ), specifically at the lateral myocyte membrane, whereas Na V 1.5 expression and I Na at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV I Na , suggesting that the SIV motif is important for regulation of Na V 1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased Na V 1.5 cell surface expression and I Na when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of Na V 1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral Na V 1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10(-68); rs9388451, P = 5.1 × 10(-17)) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10(-14)). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10(-81)). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Distinct stressors may induce heart failure. As compensation, β-adrenergic stimulation enhances myocardial contractility by elevating cardiomyocyte intracellular Ca2+ ([Ca2+]i). However, chronic β-adrenergic stimulation promotes adverse cardiac remodelling. Cardiac expression of nuclear receptor Nur77 is enhanced by β-adrenergic stimulation, but its role in cardiac remodelling is still unclear. We show high and rapid Nur77 upregulation in cardiomyocytes stimulated with β-adrenergic agonist isoproterenol. Nur77 knockdown in culture resulted in hypertrophic cardiomyocytes. Ventricular cardiomyocytes from Nur77-deficient (Nur77-KO) mice exhibited elevated diastolic and systolic [Ca2+]i and prolonged action potentials compared to wild type (WT). In vivo, these differences resulted in larger cardiomyocytes, increased expression of hypertrophic genes and more cardiac fibrosis in Nur77-KO mice upon chronic isoproterenol stimulation. In line with the observed elevated [Ca2+]i, Ca2+-activated phosphatase calcineurin was more active in Nur77-KO mice compared to WT. In contrast, after cardiac pressure overload by aortic constriction, Nur77-KO mice exhibited attenuated remodelling compared to WT. Concluding, Nur77-deficiency results in significantly altered cardiac Ca2+ homeostasis and distinct remodelling outcome depending on the type of insult. Detailed knowledge on the role of Nur77 in maintaining cardiomyocyte Ca2+ homeostasis and the dual role Nur77 plays in cardiac remodelling will aid in developing personalized therapies against heart failure.

Aims Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, the consequences of these mutations in early disease stages are unknown. We investigated whether mutation-induced intercalated disc remodelling impacts on electrophysiological properties before the onset of cell death and replacement fibrosis. Methods and results Transgenic mice with cardiac overexpression of mutant Desmoglein2 ( Dsg2 ) Dsg2 -N271S (Tg-NS/L) were studied before and after the onset of cell death and replacement fibrosis. Mice with cardiac overexpression of wild-type Dsg2 and wild-type mice served as controls. Assessment by electron microscopy established that intercellular space widening at the desmosomes/adherens junctions occurred in Tg-NS/L mice before the onset of necrosis and fibrosis. At this stage, epicardial mapping in Langendorff-perfused hearts demonstrated prolonged ventricular activation time, reduced longitudinal and transversal conduction velocities, and increased arrhythmia inducibility. A reduced action potential (AP) upstroke velocity due to a lower Na+ current density was also observed at this stage of the disease. Furthermore, co-immunoprecipitation demonstrated an in vivo interaction between Dsg2 and the Na+ channel protein NaV1.5. Conclusion Intercellular space widening at the level of the intercalated disc (desmosomes/adherens junctions) and a concomitant reduction in AP upstroke velocity as a consequence of lower Na+ current density lead to slowed conduction and increased arrhythmia susceptibility at disease stages preceding the onset of necrosis and replacement fibrosis. The demonstration of an in vivo interaction between Dsg2 and NaV1.5 provides a molecular pathway for the observed electrical disturbances during the early ARVC stages.

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform Na V 1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for Na V 1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense Na V 1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest Na V 1.8 expression within the myocardium. Immunocytochemistry further revealed substantial Na V 1.8 staining in isolated neurons from murine intracardiac ganglia but no Na V 1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the Na V 1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (I Na ) density or I Na gating kinetics in isolated myocytes but significantly reduced I Na density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of Na V 1.8-based I Na . In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for Na V 1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /Na V 1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH 4 Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current–voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli . In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p -coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).

HEY2, A NOVEL SUSCEPTIBILITY GENE FOR BRUGADA SYNDROME, CONTROLS DEPOLARIZATION AND REPOLARIZATION GRADIENTS IN THE RVOT AND ACROSS THE VENTRICULAR WALL S. Podliesna, A.O. Verkerk, R. Wolswinkel, L. Beekman, J. Barc, M. Gessler, V.M. Christoffels, A.A. Wilde, C.A. Remme, C.R. Bezzina Academic Medical Center, Amsterdam, Netherlands, Theodor-Boveri-Institute, Biocenter, University of Wurzburg, Wurzburg, Germany.

Introduction: Transgenic Scn5a-1798insD+/- mice display atrio-ventricular conduction disturbances and develop susceptibility to arrhythmias with age. Isolated cardiomyocytes from Scn5a-1798insD+/- mouse hearts exhibit increased sustained inward sodium current which is associated with elevated intracellular sodium (Na+), intracellular diastolic calcium ([Ca2+]i) levels and Ca2+ transient amplitudes. We investigated whether the Scn5a-1798insD+/- mutation leads to activation of Ca2+-dependent calcineurin/Nfat signaling pathway, and assessed its impact on atrio-ventricular conduction. Results: Despite increased [Ca2+]i, activation of the calcineurin/Nfat pathway was not observed in Scn5a-1798insD+/- hearts at baseline (as evidenced by unaltered nuclear levels of Nfat protein as compared to wild type hearts). Wild-type and Scn5a-1798insD+/- mice displayed a similar hypertrophic response to 2 weeks of TAC (as evidenced by similar increases in heart weight and Anf mRNA levels), indicating equal activation of the calcineurin/Nfat pathway. However, Scn5a-1798insD+/–TAC mice displayed increased incidence of sudden death preceded by progressive bradycardia and AV-block. Furthermore, isolated Scn5a-1798insD+/–TAC hearts showed AV-block and significantly prolonged AV-delay as compared to wild-type-TAC hearts. Since Nfatc2 is a well-described downstream effector of the calcineurin pathway, we next investigated whether the observed electrical abnormalities after TAC could be rescued by crossing Scn5a-1798insD+/- with Nfatc2-KO mice. Scn5a-1798insD+/-/Nfatc2-KO mice showed a lower response to TAC as Scn5a-1798insD+/–TAC mice (as evidenced by lower increase in heart weight and Anf mRNA levels). The ex vivo AV-delay after TAC was attenuated in Scn5a-1798insD+/-/Nfatc2-KO TAC hearts, indicating rescue of the AV-conduction disturbances in Scn5a-1798insD+/–TAC mice by Nfatc2 deficiency. Moreover, His-ventricular conduction was unaffected by TAC, indicating that the increase in AV-delay in Scn5a-1798insD+/–TAC mice occurs proximal to the His bundle. Conclusions: Scn5a-1798insD+/- mice show intracellular Na+/Ca2+-dysregulation at baseline and severe AV-conduction abnormalities and sudden death upon increased activation of the calcineurin/Nfat-pathway by TAC challenge. Nfatc2 deficiency prevents these electrical disturbances secondary to TAC in Scn5a-1798insD+/-/Nfatc2-KO mice, indicating a role of the calcineurin/Nfat pathway on atrio-ventricular conduction.

Background. Recently we observed differential myocardial expression of the brain-type sodium channel isoform Scn10a between two inbred mouse strains, both harboring the Scn5a-1798insD+/- mutation and displaying different severity of conduction disease. The functional role of Scn10a in the heart is as yet unknown, and we therefore investigated expression and channel activity of NaV1.8 (encoded by Scn10a) in intracardiac neurons and myocardium of the murine heart.Methods. Immunocytochemistry was performed using anti-NaV1.8 antibody on mouse embryos and adult murine cardiac tissue sections. The effect of the NaV1.8 blocker A-803467 (500 nM) on action potentials (AP's) and sodium current (INa) properties was assessed in isolated intracardiac neurons and ventricular myocytes.Results. In embryonic and adult heart tissue sections, NaV1.8 staining was observed at the epicardial surface, and within the myocardium in between cardiomyocytes. The NaV1.8 blocker A-803467 had no effect on either mean INa density or INa kinetic properties in isolated myocytes, but clearly reduced INa density in intracardiac neurons (−344±51 pA/pF versus control −448±61; mean±SEM, n=11). In addition, the slow component of the current decay (τslow) at −20 mV was accelerated in the presence of A-803467 (2.8±0.3 ms versus control 3.4±0.4 ms; mean±SEM, n=5) and V1/2 of voltage-dependent inactivation was shifted by −9.6 mV (−73.6±2.0 mV versus control −64.0±1.6 mV; mean±SEM, n=5). This is consistent with a reduction in slowly inactivating brain-type sodium current with depolarized voltage-dependent inactivation. In AP measurements A-803467 did not affect cardiomyocyte upstroke velocity, but reduced AP firing frequency in intracardiac neurons by 50%.Conclusion. The sodium channel NaV1.8 is expressed in murine heart, and is functionally present in intracardiac neurons, but absent in cardiomyocytes. Thus, NaV1.8 may influence myocardial electrophysiological properties through its contribution to cardiac neuronal activity.