Mutation in nuclear pore component NUP155 leads to atrial fibrillation and early sudden cardiac death.

Atrial fibrillation (AF) is the most common form of sustained clinical arrhythmia. We previously mapped an AF locus to chromosome 5p13 in an AF family with sudden death in early childhood. Here we show that the specific AF gene underlying this linkage is NUP155, which encodes a member of the nucleoporins, the components of the nuclear pore complex (NPC). We have identified a homozygous mutation, R391H, in NUP155 that cosegregates with AF, affects nuclear localization of NUP155, and reduces nuclear envelope permeability. Homozygous NUP155(-/-) knockout mice die before E8.5, but heterozygous NUP155(+/-) mice show the AF phenotype. The R391H mutation and reduction of NUP155 are associated with inhibition of both export of Hsp70 mRNA and nuclear import of Hsp70 protein. These human and mouse studies indicate that loss of NUP155 function causes AF by altering mRNA and protein transport and link the NPC to cardiovascular disease.

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4.

Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.

Optical mapping of cryoinjured rat myocardium grafted with mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.

Molecular charge associated with antiarrhythmic actions in a series of amino-2-cyclohexyl ester derivatives.

A series of amino-2-cyclohexyl ester derivatives were studied for their ion channel blocking and antiarrhythmic actions in the rat and a structure-activity analysis was conducted. The compounds are similar in chemical structure except for ionizable amine groups (pK values 6.1-8.9) and the positional arrangements of aromatic naphthyl moieties. Ventricular arrhythmias were produced in rats by coronary-artery occlusion or electrical stimulation. The electrophysiological effects of these compounds on rat heart sodium channels (Nav1.5) expressed in Xenopus laevis oocytes and transient outward potassium currents (Kv4.3) from isolated rat ventricular myocytes were examined. The compounds reduced the incidence of ischemia-related arrhythmias and increased current threshold for induction of ventricular fibrillo-flutter (VFt) dose-dependently. As pK increased compounds showed a diminished effectiveness against ischemia-induced arrhythmias, and were less selective for ischemia- versus electrically-induced arrhythmias. Where tested, compounds produced a concentration-dependent tonic block of Nav1.5 channels. An increased potency for inhibition of Nav1.5 occurred when the external pH (pHo) was reduced to 6.5. Some compounds inhibited Kv4.3 in a pH-independent manner. Overall, the differences in antiarrhythmic and ion channel blocking properties in this series of compounds can be explained by differences in chemical structure. Antiarrhythmic activity for the amino-2-cyclohexyl ester derivatives is likely a function of mixed ion channel blockade in ischemic myocardium. These studies show that drug inhibition of Nav1.5 occurred at lower concentrations than Kv4.3 and was more sensitive to changes in the ionizable amine groups rather than on positional arrangements of the naphthyl constituents. These results offer insight into antiarrhythmic mechanisms of drug-ion channel interactions.

Beta 1-adrenergic receptor autoantibodies mediate dilated cardiomyopathy by agonistically inducing cardiomyocyte apoptosis.

BACKGROUND: Antibodies to the beta1-adrenergic receptor (beta1AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby beta1AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM. METHODS AND RESULTS: We used the beta1AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the beta1AR. After transfer into naive male hosts, beta1AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of beta1AR IgG before transfer and by selective pharmacological antagonism of host beta1AR but not beta2AR. We found that beta1AR autoantibodies shifted the beta1AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by beta1AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK. CONCLUSIONS: Our data show how beta1AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Animal models for cardiac arrhythmias.

Transgenic and gene-targeted mice are now frequently used to expand the study of cardiac physiology and pathophysiology owing to the ease with which the mouse genome can be manipulated. There are many measures by which an assessment of the phenotypical expression of the transgenic mouse can be made. In the case of cardiac channelopathies and how they relate to cardiac function, telemetry is a technology that utilizes transmitters that are surgically implanted in animals for the purpose of acquiring biopotentials or physiological parameters. Electrophysiological techniques have also been used to assess cardiac function at the cellular level, by measuring whole-cell ionic currents and/or transmembrane potentials. This chapter will discuss the surgical procedures involved in successfully implanting the transmitter device in a mouse, as well as highlight the recording of and analysis of electrocardiograms. This chapter will also outline the procedures involved in isolating single-ventricular myocytes from a mouse heart. It is a protocol that was developed in our laboratory for which we have routinely and successfully isolated myocytes from both transgenic and nontransgenic mouse hearts. Although no one isolation protocol is alike, we also present our own observations that have assisted in maximizing myocyte bioavailability and yield.

Correction: Interaction of G-Protein ßγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation.

[This corrects the article DOI: 10.1371/journal.pone.0052689.].

Mechanisms by which SCN5A mutation N1325S causes cardiac arrhythmias and sudden death in vivo.

OBJECTIVE: Mutations in the cardiac sodium channel gene SCN5A are responsible for type-3 long QT disease (LQT3). The genesis of cardiac arrhythmias in LQT3 is multifaceted, and the aim of this study was to further explore mechanisms by which SCN5A mutations lead to arrhythmogenesis in vivo. METHODS: We engineered selective cardiac expression of a long QT syndrome (LQTS) mutation (N1325S) in human SCN5A and generated a transgenic mouse model, TGM(NS31). RESULTS: Conscious and unrestrained TGM(NS31)L12 mice demonstrated a significant prolongation of the QT-interval and a high incidence of spontaneous polymorphic ventricular tachycardia (VT) and fibrillation (VF), often resulting in sudden cardiac death (n=52:156). Arrhythmias were suppressed by mexiletine, a sodium channel blocker for the late persistent sodium current. Action potentials (APs) from TGM(NS31)L12 ventricular myocytes exhibited early afterdepolarizations and longer 90% AP durations (APD90=69 +/- 5.9 ms) than control (APD90=46.7 +/- 4.8 ms). Voltage-clamp experiments in transgenic myocytes revealed a peak inward sodium current (INa) followed by a slow recovery from inactivation. After mexiletine application, transgenic ventricular APDs were shortened, and recovery from inactivation of INa was enhanced. These suggest that the N1325S transgene is responsible for the abnormal signals present in transgenic cells as well as the genesis of lethal arrhythmias in mice. Interestingly, transgenic but not wild-type myocytes displayed longer APDs with a shortening of CLs. CONCLUSIONS: Our findings show that a new model for LQTS has been established, and we report on an arrhythmogenic mechanism that, unlike other SCN5A mutations, results in poor restitution of APD with increasing rate as a possible substrate for arrhythmogenesis.

MOG1 rescues defective trafficking of Na(v)1.5 mutations in Brugada syndrome and sick sinus syndrome.

BACKGROUND: Loss-of-function mutations in Na(v)1.5 cause sodium channelopathies, including Brugada syndrome, dilated cardiomyopathy, and sick sinus syndrome; however, no effective therapy exists. MOG1 increases plasma membrane (PM) expression of Na(v)1.5 and sodium current (I(Na)) density, thus we hypothesize that MOG1 can serve as a therapeutic target for sodium channelopathies. METHODS AND RESULTS: Knockdown of MOG1 expression using small interfering RNAs reduced Na(v)1.5 PM expression, decreased I(Na) densities by 2-fold in HEK/Na(v)1.5 cells and nearly abolished I(Na) in mouse cardiomyocytes. MOG1 did not affect Na(v)1.5 PM turnover. MOG1 small interfering RNAs caused retention of Na(v)1.5 in endoplasmic reticulum, disrupted the distribution of Na(v)1.5 into caveolin-3-enriched microdomains, and led to redistribution of Na(v)1.5 to noncaveolin-rich domains. MOG1 fully rescued the reduced PM expression and I(Na) densities by Na(v)1.5 trafficking-defective mutation D1275N associated with sick sinus syndrome/dilated cardiomyopathy/atrial arrhythmias. For Brugada syndrome mutation G1743R, MOG1 restored the impaired PM expression of the mutant protein and restored I(Na) in a heterozygous state (mixture of wild type and mutant Na(v)1.5) to a full level of a homozygous wild-type state. CONCLUSIONS: Use of MOG1 to enhance Na(v)1.5 trafficking to PM may be a potential personalized therapeutic approach for some patients with Brugada syndrome, dilated cardiomyopathy, and sick sinus syndrome in the future.

Interaction of G-protein ßγ complex with chromatin modulates GPCR-dependent gene regulation.

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gßγ in the cytosol. In this study, we show novel nuclear functions of Gßγ through demonstrating interaction of Gß(2) with integral components of chromatin and effects of Gß(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gß(2) levels in the nucleus and Gß(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gß(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gß(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gß(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gßγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.

LQTS mutation N1325S in cardiac sodium channel gene SCN5A causes cardiomyocyte apoptosis, cardiac fibrosis and contractile dysfunction in mice.

OBJECTIVE: Mutations in the cardiac sodium channel gene SCN5A cause long QT syndrome (LQTS). We previously generated an LQTS mouse model (TG-NS) that overexpresses the LQTS mutation N1325S in SCN5A. The TG-NS mice manifested the clinical features of LQTS including spontaneous VT, syncope and sudden death. However, the long-term prognosis of LQTS on the structure of the heart has not been investigated in this or any other LQTS models and human patients. METHODS AND RESULTS: Impaired systolic function and reduced left ventricular fractional shortening were detected by echocardiography, morphological and histological examination in two lines of adult mutant transgenic mice. Histological and TUNEL analyses of heart sections revealed fibrosis lesions and increased apoptosis in an age-dependent manner. Cardiomyocyte apoptosis was associated with the increased activation of caspases 3 and 9 in TG-NS hearts. Western blot analysis showed a significantly increased expression of the key Ca(2+) handling proteins L-type Ca(2+) channel, RYR2 and NCX in TG-NS hearts. Increased apoptosis and an altered expression of Ca(2+) handling proteins could be detected as early as 3months of age when echocardiography showed little or no alterations in TG-NS mice. CONCLUSIONS: Our findings revealed for the first time that the LQTS mutation N1325S in SCN5A causes cardiac fibrosis and contractile dysfunction in mice, possibly through cellular mechanisms involving aberrant cardiomyocyte apoptosis. Therefore, we provide the experimental evidence supporting the notion that some LQTS patients have an increased risk of structural and functional cardiac damage in a prolonged disease course.

Prevention of cardiac hypertrophy and heart failure by silencing of NF-kappaB.

Activation of the nuclear factor (NF)-kappaB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-kappaB activity. Using this mouse model and an NF-kappaB-targeted gene array, we first determined the components of NF-kappaB signaling cascade and the NF-kappaB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-kappaB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-kappaB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-kappaB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-kappaB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-kappaB as a therapeutic target to prevent hypertrophy/HF.

Identification of a new co-factor, MOG1, required for the full function of cardiac sodium channel Nav 1.5.

The cardiac sodium channel Nav 1.5 is essential for the physiological function of the heart and contributes to lethal cardiac arrhythmias and sudden death when mutated. Here, we report that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and modulates the physiological function of Nav 1.5. The yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domains DII and DIII) of Nav 1.5. The interaction was further demonstrated by both in vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation assays in both HEK293 cells with co-expression of MOG1 and Nav1.5 and native cardiac cells. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities. In neonatal myocytes, overexpression of MOG1 increased current densities nearly 2-fold. Western blot analysis revealed that MOG1 increased cell surface expression of Nav1.5, which may be the underlying mechanism by which MOG1 increased sodium current densities. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues with predominant localization at the intercalated discs. In cardiomyocytes, MOG1 is mostly localized in the cell membrane and co-localized with Nav1.5. These results indicate that MOG1 is a critical regulator of sodium channel function in the heart and reveal a new cellular function for MOG1. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which serve important functions for Nav1.5 under different cellular conditions.

Cardiac-specific overexpression of SCN5A gene leads to shorter P wave duration and PR interval in transgenic mice.

The Cardiac sodium channel gene SCN5A plays a critical role in cardiac electrophysiology and its mutations, either gain- or loss-of-functions, are associated with lethal arrhythmias. In this study, we investigated the effect of overexpression of SCN5A on the cardiac phenotype in a transgenic mouse model (TG-WT L10). Compared to NTG mice, heart rate, QRS duration, and QT intervals remained unchanged in TG-WT mice. Moreover, no spontaneous ventricular arrhythmias were detected in TG-WT hearts. Despite these results, a mild, irregular cardiac phenotype was observed in TG-WT mice. The P wave and PR interval were significantly shorter in TG-WT compared with NTG mice (P, 8.8+/-0.8 ms vs. 12.6+/-0.9 ms; PR, 12.5+/-2 ms vs. 33.5+/-0.7 ms). Furthermore, spontaneous premature atrial contractions were often detected in TG-WT mice. These results suggest that the expression level of the SCN5A gene is a determinant for the length of the P wave duration and PR interval on electrocardiograms (ECG).

Optical mapping of ventricular arrhythmias in LQTS mice with SCN5A mutation N1325S.

Transgenic expression of SCN5A mutation N1325S creates a mouse model for type-3 long QT syndrome (LQT3), TG-NS/LQT3. Optical mapping is a high temporal and spatial resolution fluorescence mapping system that records 256 action potentials simultaneously in a Langendorff-perfused heart. Here for the first-time, we provide a spatial view of VT in a genetic LQT3 model using optical mapping. Spontaneous VT was detected in TG-NS/LQT3 hearts, but not in littermate control hearts. VT was initiated primarily by activation of a new firing focus as well as functional conduction block of new activation waves. New firing was initiated at many different Loci in the heart, suggesting that "increased automaticity" is a key mechanism for initiation of VT. The sustained VT was maintained by a reentry mechanism. Nifedipine, an L-type calcium channel blocker, decreased the frequency of VT, indicating the involvement of abnormalities of the calcium homeostasis in the genesis of VT in TG-NS/LQT3 mice.

Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes.

The N(1325)S mutation in the cardiac sodium channel gene SCN5A causes the type-3 long-QT syndrome but the arrhythmogenic trigger associated with N(1325)S has not been characterized. In this study, we investigated the triggers for cardiac events in the expanded N(1325)S family. Among 11 symptomatic patients with document triggers, six died suddenly during sleep or while sitting (bradycardia-induced trigger), three died suddenly, and two developed syncope due to stress and excitement (non-bradycardia-induced). Patch-clamping studies revealed that the late sodium current (I(Na,L)) generated by mutation N(1325)S in ventricular myocytes from TG-NS/LQT3 mice was reduced with increased pacing, which explains bradycardia-induced mortalities in the family. The non-bradycardic triggers are related to the finding that APD became prolonged and unstable at increasing rates, often with alternating repolarization phases which was corrected with verapamil. This implies that Ca2+ influx and intracellular Ca2+ ([Ca2+]i) ions are involved and that [Ca2+]i inhomogeneity may be the underlying mechanisms behind non-bradycardia LQT3 arrhythmogenesis associated with mutation N(1325)S.

Droperidol inhibits intracellular Ca2+, myofilament Ca2+ sensitivity, and contraction in rat ventricular myocytes.

BACKGROUND: Droperidol has recently been associated with cardiac arrhythmias and sudden cardiac death. Changes in action potential duration seem to be the cause of the arrhythmic behavior, which can lead to alterations in intracellular free Ca concentration ([Ca]i). Because [Ca]i and myofilament Ca sensitivity are key regulators of myocardial contractility, the authors' objective was to identify whether droperidol alters [Ca]i or myofilament Ca sensitivity in rat ventricular myocytes and to identify the cellular mechanisms responsible for these effects. METHODS: Freshly isolated rat ventricular myocytes were obtained from adult rat hearts. Myocyte shortening, [Ca]i, nitric oxide production, intracellular pH, and action potentials were monitored in cardiomyocytes exposed to droperidol. Langendorff perfused hearts were used to assess overall cardiac function. RESULTS: Droperidol (0.03-1 mum) caused concentration-dependent decreases in peak [Ca]i and shortening. Droperidol inhibited 35 mm KCl-induced increase in [Ca]i, with little direct effect on sarcoplasmic reticulum Ca stores. Droperidol had no effect on action potential duration but caused a rightward shift in the concentration-response curve to extracellular Ca for shortening, with no concomitant effect on peak [Ca]i. Droperidol decreased pHi and increased nitric oxide production. Droperidol exerted a negative inotropic effect in Langendorff perfused hearts. CONCLUSION: These data demonstrate that droperidol decreases cardiomyocyte function, which is mediated by a decrease in [Ca]i and a decrease in myofilament Ca sensitivity. The decrease in [Ca]i is mediated by decreased sarcolemmal Ca influx. The decrease in myofilament Ca sensitivity is likely mediated by a decrease in pHi and an increase in nitric oxide production.

LQT4 gene: the "missing" ankyrin.

Mutations in ion channels have been implicated in the formation of long QT syndrome (LQTS). However, Mohler et al. have recently uncovered a role for ankyrin-B, a non-ion channel protein, in type IV LQTS. Calcium signalling is altered, and the functions of several channels and pumps that normally interact with wild-type ankyrin-B are impaired in the presence of mutant ankyrin-B. The authors suggest that by disrupting the functions of these channels, a new mechanism has been uncovered that can lead to cardiac myopathy.