Sparsely methylated mitochondrial cell free DNA released from cardiomyocytes contributes to systemic inflammatory response accompanied by atrial fibrillation.

Systemic inflammation is assumed to be the consequence and the cause of atrial fibrillation (AF); however, the underlying mechanism remains unclear. We aimed to evaluate the level of cell-free DNA (cfDNA) in patients with AF and AF mimicking models, and to illuminate its impact on inflammation. Peripheral blood was obtained from 54 patients with AF and 104 non-AF controls, and cfDNA was extracted. We extracted total cfDNA from conditioned medium after rapid pacing to HL-1 cells. Nuclear and mitochondrial DNA were separately extracted and fragmented to simulate nuclear-cfDNA (n-cfDNA) and mitochondrial-cfDNA (mt-cfDNA). The AF group showed higher cfDNA concentration than the non-AF group (12.6 [9.0-17.1] vs. 8.1 [5.3-10.8] [ng/mL], p < 0.001). The copy numbers of n-cfDNA and mt-cfDNA were higher in AF groups than in non-AF groups; the difference of mt-cfDNA was particularly apparent (p = 0.011 and p < 0.001, respectively). Administration of total cfDNA and mt-cfDNA to macrophages significantly promoted IL-1ß and IL-6 expression through TLR9, whereas n-cfDNA did not. Induction of cytokine expression by methylated mt-cfDNA was lower than that by unmethylated mt-cfDNA. Collectively, AF was associated with an increased cfDNA level, especially mt-cfDNA. Sparsely methylated mt-cfDNA released from cardiomyocytes may be involved in sterile systemic inflammation accompanied by AF.

Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT.

Glypican-5 (GPC5) is a heparan sulfate proteoglycan (HSPG) localized to the plasma membrane. We previously reported that in the human mesenchymal stem cell line UE6E7T-3, GPC5 is overexpressed in association with transformation and promotes cell proliferation by acting as a co-receptor for Sonic hedgehog signaling. In this study, we found using immunofluorescence microscopy that in transformed cells (U3DT), GPC5 localized not only at primary cilia on the cell surface, but also at the leading edge of migrating cells, at the intercellular bridge and blebs during cytokinesis, and in extracellular vesicles. In each subcellular region, GPC5 colocalized with fibroblast growth factor receptor (FGFR) and the small GTPases Rab11 and ARF6, indicating that GPC5 is delivered to these regions by Rab11-associated recycling endosomes. These colocalizations suggest that GPC5 plays an important role in FGF2 stimulation of cell migration, which was abrogated by knockdown of GPC5. Our findings indicate that GPC5 plays a role in regulation of U3DT cell migration and provides several insights into the functions of GPC5 that could be elucidated by future studies.

In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells.

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

Chromosome 4q25 variants and recurrence after second-generation cryoballoon ablation in patients with paroxysmal atrial fibrillation.

BACKGROUND: Chromosome 4q25 single-nucleotide polymorphisms (SNPs) are associated with atrial fibrillation (AF) recurrence after radiofrequency catheter ablation, however the underlying mechanism is unknown. Pulmonary vein (PV) reconnections are common post-radiofrequency ablation. We explored the pre-procedural parameters, including AF susceptibility SNPs, predicting the response to PV isolation (PVI) using second-generation cryoballoons. METHODS: One hundred fifty-seven paroxysmal AF patients undergoing PVI using second-generation cryoballoons and genetic testing were enrolled. The top 6 AF-associated Japanese ancestry SNPs were evaluated. Fourteen-day consecutive monitoring was performed to detect AF recurrences. RESULTS: Early recurrence of AF (ERAF) was detected in 74(47.1%) patients, and the AF-free survival at 12-months after single procedures was 72.1%. Cox's proportional models determined that higher pro-BNP values (hazard ratio [HR]=1.001; 95% confidence interval [CI]=1.000-1.001; p=0.003) and the rs1906617 risk allele (HR=2.440; 95% CI=1.062-5.605; p=0.035) were independently associated with ERAFs, and the rs1906617 risk allele (HR=4.339; 95% CI=1.044-18.028; p=0.043) was the sole factor significantly associated with AF recurrence. Second procedures were performed in 41 patients a median of 6.0[5.0-9.5] months later, and 42/162(25.9%) PVs were reconnected. Reconnections were similarly observed in rs1906617 risk allele carriers and wild-type patients. Risk allele carriers at rs1906617 were more likely to have non-PV foci, but did not reach statistical significance (10/35 vs. 0/6, p=0.132). CONCLUSIONS: AF risk alleles on chromosome 4q25 modulated the risk of AF recurrence after PVI using second-generation cryoballoons in patients with paroxysmal AF. Our study results suggested that non-PV foci might be the more likely mechanism of a high AF recurrence in chromosome 4q25 variant carriers.

Association of ZFHX3 gene variation with atrial fibrillation, cerebral infarction, and lung thromboembolism: An autopsy study.

AIM: We aimed to study a single nucleotide polymorphism (SNP), rs2106261, in the transcription factor gene, ZFHX3, in atrial fibrillation (AF) and other related phenotypes by phenome scanning in a Japanese population. METHOD: We retrieved consecutive autopsy data (n=2433, mean age=80 years) from the Japanese SNP database for geriatric diseases (JG-SNP). Clinical data, including an AF diagnosis, were collected from medical charts. Genotyping was performed with the DNA chip method. We also analyzed 42 pathological and 26 clinical phenotypes, including cerebral infarctions (CIs) and lung thromboembolisms (LTs), diagnosed by macroscopic inspection during the autopsy. RESULT: Among the 2433 patients with available data, 18.6% had AF, 29.4% had CI, and 4.9% had LT phenotypes. The A allele of the rs2106261 SNP was significantly associated with AF, after adjusting for age, sex, diabetes, hypertension, and smoking (AA+AG/GG, OR=1.51, 95%CI: 1.16-1.97, p=0.002). In the entire cohort, CI was not associated with rs2106261 (p=0.14). However, among patients under 80 years old, rs2106261 was significantly associated with CI (AA+AG/GG, OR=1.57, 95%CI: 1.09-2.26, p=0.01). LT was also associated with rs2106261 (AA+AG/GG, OR=1.99, 95%CI: 1.31-3.01, p=0.001). Associations between rs2106261 and CI and LT remained positive after adjusting for the presence of AF, which indicated that this SNP variant might serve as an independent risk marker. CONCLUSION: We showed that the ZFHX3 polymorphism, rs2106261 (A allele), was a risk marker for AF and AF-related phenotypes. The roles of this variant in the development of AF and its related phenotypes warrant further investigation.

Sex hormonal regulation of cardiac ion channels in drug-induced QT syndromes.

Female sex is an independent risk factor for development of torsade de pointes (TdP) arrhythmias not only in congenital long QT syndromes but also in acquired long QT syndromes. Clinical and experimental evidences suggest that the gender differences may be due to, at least in part, gender differences in regulation of rate-corrected QT (QTC) interval between men and women. In adult women, both QTC interval and arrhythmic risks in TdP alter cyclically during menstrual cycle, suggesting a critical role of female sex hormones in cardiac repolarization process. These gender differences in fundamental cardiac electrophysiology result from variable ion channel expression and diverse sex hormonal regulation via long term genomic and acute non-genomic actions, and sex differences in drug responses and metabolisms. In particular, non-genomic actions of testosterone and progesterone on cardiac ion channels are likely to contribute to the gender differences in cardiac repolarization processes. This review summarizes current knowledge on sex hormonal regulation of cardiac ion channels which contribute to cardiac repolarization processes and its implication for gender differences in drug-induced long QT syndromes.

Oxidative Stress Induced Ventricular Arrhythmia and Impairment of Cardiac Function in Nos1ap Deleted Mice.

Genome-wide association study has identified that the genetic variations at NOS1AP (neuronal nitric oxide synthase-1 adaptor protein) were associated with QT interval and sudden cardiac death (SCD). However, the mechanism linking a genetic variant of NOS1AP and SCD is poorly understood. We used Nos1ap knockout mice (Nos1ap(-/-)) to determine the involvement of Nos1ap in SCD, paying special attention to oxidative stress.At baseline, a surface electrocardiogram (ECG) and ultrasound echocardiography (UCG) showed no difference between Nos1ap(-/-) and wild-type (WT) mice. Oxidative stress was induced by a single injection of doxorubicin (Dox, 25 mg/kg). After Dox injection, Nos1ap(-/-) showed significantly higher mortality than WT (93.3 versus 16.0% at day 14, P < 0.01). ECG showed significantly longer QTc in Nos1ap(-/-) than WT, and UCG revealed significant reduction of fractional shortening (%FS) only in Nos1ap(-/-) after Dox injection. Spontaneous ventricular tachyarrhythmias were documented by telemetry recording after Dox injection only in Nos1ap(-/-). Ex vivo optical mapping revealed that the action potential duration (APD)90 was prolonged at baseline in Nos1ap(-/-), and administration of Dox lengthened APD90 more in Nos1ap(-/-) than in WT. The expression of Bnp and the H2O2 level were higher in Nos1ap(-/-) after Dox injection. Nos1ap(-/-) showed a reduced amplitude of calcium transient in isolated cardiomyocytes after Dox injection. Administration of the antioxidant N-acetyl-L-cysteine significantly reduced mortality of Nos1ap(-/-) by Dox injection, accompanied by prevention of QT prolongation and a reduction in %FS.Although Nos1ap(-/-) mice have apparently normal hearts, oxidative stress evokes ventricular tachyarrhythmia and heart failure, which may cause sudden cardiac death.

Intravital imaging of Ca(2+) signals in lymphocytes of Ca(2+) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset.

Calcium ion (Ca(2+)) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca(2+) indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60(flox)). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca(2+)) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22(-/-) and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca(2+) fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca(2+) signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals.

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming.

Aromatase knockout mice reveal an impact of estrogen on drug-induced alternation of murine electrocardiography parameters.

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology.

The generation of induced pluripotent stem cells from a patient with KCNH2 G603D, without LQT2 disease associated symptom.

The long QT syndrome type 2 (LQT2) is inheritable life threatening arrhythmic disorder and one of the most common genetic variants in long QT syndrome. There are some indications for treatment of the patients with LQT2 but it is impossible to completely prevent fatal arrhythmia. To develop novel therapy for the patients with LQT2, it has been desired to generate diseasespecific and patient-specific disease model. Human induced pluripotent stem (iPS) cells are somatic cell-derived pluripotent stem cells with infinite proliferation ability and multipotency. Patient-specific iPS cells can be derived from patient somatic cells, have all genomic information encoded in patient's genome including mutation and all SNPs, and can be ideal disease models of the patients. To generate disease model for LQT2 by iPS cells, we should firstly generate iPS cells from the patient with LQT2 and confirm the genomic mutation in iPS cells. In this study, we showed the successful generation of iPS cells from a patient with KCNH2 G603D mutation. The patient specific iPS cells properly expressed stem cell markers, such as NANOG and OCT3/4. We also confirmed that the KCNH2 G603D (G1808A) mutation was taken over in patient specific iPS cells. These patient-specific iPS cells may contribute to the future analysis for disease pathogenesis and drug innovation.

Effects of an hERG activator, ICA-105574, on electrophysiological properties of canine hearts.

In short QT syndrome, inherited gain-of-function mutations in the human ether a-gogo-related gene (hERG) K(+) channel have been associated with development of fatal arrhythmias. This implies that drugs that activate hERG as a side effect may likewise pose significant arrhythmia risk. hERG activators have been found to have diverse mechanisms of activation, which may reflect their distinct binding sites. Recently, the new hERG activator ICA-105574 was introduced, which disables inactivation of the hERG channel with very high potency. We explored characteristics of this new drug in several experimental models. Patch clamp experiments were used to verify activation of hERG channels by ICA-105574 in human embryonic kidney cells stably-expressing hERG channels. ICA-105574 significantly shortened QT and QTc intervals and monophasic action potential duration (MAP(90)) in Langendorff-perfused guinea-pig hearts. We also administered ICA-105574 to anesthetized dogs while recording ECG and drug plasma concentrations. ICA-105574 (10 mg/kg) significantly shortened QT and QTc intervals, with a free plasma concentration of approximately 1.7 µM at the point of maximal effect. Our data showed that unbound ICA-105574 caused QT shortening in dogs at concentrations comparable to the half maximal effective concentration (EC(50), 0.42 µM) of hERG activation in the patch clamp studies.

Stretch of atrial myocytes stimulates recruitment of macrophages via ATP released through gap-junction channels.

Atrial inflammation is critical to atrial fibrillation initiation and progression. Although left atrial dilatation is a risk factor for atrial fibrillation, the mechanism linking atrial dilatation and inflammation is unclear. We evaluated the mechanisms underlying infiltration of macrophages induced by stretch of atrial myocytes. Murine macrophages were co-cultured with HL-1 murine atrial myocyte-derived cells. Mechanical stretch applied to atrial myocytes induced transwell macrophage migration. The gap junction-channel blocker carbenoxolone and the non-specific ATP-signaling modifiers apyrase and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate inhibited the enhanced migration. Mechanical stretch of atrial myocytes induced transient increase in extracellular ATP concentration, which was inhibited by carbenoxolone. siRNA knockdown of pannexin-2 inhibited ATP release and macrophage migration. Mice underwent transverse aortic constriction or sham procedure. Transverse aortic constriction procedure induced macrophage infiltration. Daily carbenoxolone administration significantly inhibited macrophage infiltration in the atrium. Thus, mechanical stretch of atrial myocytes induces macrophage migration by ATP released through gap-junction channels, at least in part, in vitro. Administering a gap junction family-channel blocker inhibited this inflammatory change in vivo.

Disease characterization using LQTS-specific induced pluripotent stem cells.

AIMS: Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. CONCLUSIONS: This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

Circulating KCNH2 current-activating factor in patients with heart failure and ventricular tachyarrhythmia.

BACKGROUND: It is estimated that approximately half of the deaths in patients with HF are sudden and that the most likely causes of sudden death are lethal ventricular tachyarrhythmias such as ventricular tachycardia (VT) or fibrillation (VF). However, the precise mechanism of ventricular tachyarrhythmias remains unknown. The KCNH2 channel conducting the delayed rectifier K(+) current (I(Kr)) is recognized as the most susceptible channel in acquired long QT syndrome. Recent findings have revealed that not only suppression but also enhancement of I(Kr) increase vulnerability to major arrhythmic events, as seen in short QT syndrome. Therefore, we investigated the existence of a circulating KCNH2 current-modifying factor in patients with HF. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of serum of HF patients on recombinant I(Kr) recorded from HEK 293 cells stably expressing KCNH2 by using the whole-cell patch-clamp technique. Study subjects were 14 patients with non-ischemic HF and 6 normal controls. Seven patients had a history of documented ventricular tachyarrhythmias (VT: 7 and VF: 1). Overnight treatment with 2% serum obtained from HF patients with ventricular arrhythmia resulted in a significant enhancement in the peaks of I(Kr) tail currents compared to the serum from normal controls and HF patients without ventricular arrhythmia. CONCLUSIONS/SIGNIFICANCE: Here we provide the first evidence for the presence of a circulating KCNH2 channel activator in patients with HF and ventricular tachyarrhythmias. This factor may be responsible for arhythmogenesis in patients with HF.

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages.

There is considerable evidence indicating that intracellular Ca2+ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca2+ concentrations ([Ca2+]i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca2+ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFalpha and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFalpha and IL-6 production as well as IkappaBalpha degradation. Experiments using BAPTA/AM and EGTA, and Ca2+ imaging suggested that the LPS-induced increase in [Ca2+]i involves both the TRPV2-mediated intracellular and extracellular Ca2+ mobilizations. BAPTA/AM abolished LPS-induced TNFalpha and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFalpha production. These data indicate that TRPV2 is involved in the LPS-induced Ca2+ mobilization from intracellular Ca2+ store and extracellular Ca2+. In addition to Ca2+ mobilization through the IP3-receptor, TRPV2-mediated intracellular Ca2+ mobilization is involved in NFkappaB-dependent TNFalpha and IL-6 expression, while extracellular Ca2+ entry is involved in NFkappaB-independent IL-6 production.

Acute effects of sex steroid hormones on susceptibility to cardiac arrhythmias: a simulation study.

Acute effects of sex steroid hormones likely contribute to the observation that post-pubescent males have shorter QT intervals than females. However, the specific role for hormones in modulating cardiac electrophysiological parameters and arrhythmia vulnerability is unclear. Here we use a computational modeling approach to incorporate experimentally measured effects of physiological concentrations of testosterone, estrogen and progesterone on cardiac ion channel targets. We then study the hormone effects on ventricular cell and tissue dynamics comprised of Faber-Rudy computational models. The "female" model predicts changes in action potential duration (APD) at different stages of the menstrual cycle that are consistent with clinically observed QT interval fluctuations. The "male" model predicts shortening of APD and QT interval at physiological testosterone concentrations. The model suggests increased susceptibility to drug-induced arrhythmia when estradiol levels are high, while testosterone and progesterone are apparently protective. Simulations predict the effects of sex steroid hormones on clinically observed QT intervals and reveal mechanisms of estrogen-mediated susceptibility to prolongation of QT interval. The simulations also indicate that acute effects of estrogen are not alone sufficient to cause arrhythmia triggers and explain the increased risk of females to Torsades de Pointes. Our results suggest that acute effects of sex steroid hormones on cardiac ion channels are sufficient to account for some aspects of gender specific susceptibility to long-QT linked arrhythmias.

A genetically encoded bioluminescent indicator for the sodium channel activity in living cells.

We have developed a genetically encoded bioluminescent indicator for the Na(+) channel, in which Na(+)-sensitive Gaussia luciferase is fused with the voltage-gated Na(+) channel. This indicator is capable of detecting Na(+) flow through the pores of Na(+) channels. Because of high sensitivity and low background in luminescence assays, the absence of toxicity, and a wide linear dynamic range, this luciferase can be used to generate a novel, genetically encoded Na(+) channel indicator. It may provide a high-throughput screening system for drug discovery against Na(+) channels, which should be useful in controlling lethal cardiac arrhythmias, epileptic seizures, and intolerable pain associated with terminal stages of cancer. It may also offer a system for monitoring the Na(+) channel activity in living cells, which may be useful in illuminating neuronal activity in vivo.