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Reports that in ADR mice a transposon of the `ETn' family has inserted into the corresponding gene, destroying its coding potential for several membrane-spanning domains. Strongly suggests a lack of functional chloride channels as the primary cause of mouse myotonia. Methods; Results; Discussion.

Describes the cloning of the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl... channel from `Torpedo mamorata.' Experimental methods; Results; Discussion.

Presents the cloning of a complementary DNA encoding a voltage-gated chloride channel from `Torpedo marmorata' electric organ. Site of active mRNA; Channel expression; Protein structure.

In failing hearts, Na + /Ca 2+ exchanger (NCX) overactivity contributes to Ca 2+ depletion, leading to contractile dysfunction. Inhibition of NCX is expected to normalize Ca 2+ mishandling, to limit afterdepolarization-related arrhythmias, and to improve cardiac function in heart failure (HF). SAR340835/SAR296968 is a selective NCX inhibitor for all NCX isoforms across species, including human, with no effect on the native voltage-dependent calcium and sodium currents in vitro. Additionally, it showed in vitro and in vivo antiarrhythmic properties in several models of early and delayed afterdepolarization-related arrhythmias. Its effect on cardiac function was studied under intravenous infusion at 250,750 or 1500 µg/kg per hour in dogs, which were either normal or submitted to chronic ventricular pacing at 240 bpm (HF dogs). HF dogs were infused with the reference inotrope dobutamine (10 µg/kg per minute, i.v.). In normal dogs, NCX inhibitor increased cardiac contractility (dP/dt max ) and stroke volume (SV) and tended to reduce heart rate (HR). In HF dogs, NCX inhibitor significantly and dose-dependently increased SV from the first dose (+28.5%, +48.8%, and +62% at 250, 750, and 1500 µg/kg per hour, respectively) while significantly increasing dP/dt max only at 1500 (+33%). Furthermore, NCX inhibitor significantly restored sympathovagal balance and spontaneous baroreflex sensitivity (BRS) from the first dose and reduced HR at the highest dose. In HF dogs, dobutamine significantly increased dP/dt max and SV (+68.8%) but did not change HR, sympathovagal balance, or BRS. Overall, SAR340835, a selective potent NCX inhibitor, displayed a unique therapeutic profile, combining antiarrhythmic properties, capacity to restore systolic function, sympathovagal balance, and BRS in HF dogs. NCX inhibitors may offer new therapeutic options for acute HF treatment. SIGNIFICANCE STATEMENT: HF is facing growing health and economic burden. Moreover, patients hospitalized for acute heart failure are at high risk of decompensation recurrence, and no current acute decompensated HF therapy definitively improved outcomes. A new potent, Na + /Ca 2+ exchanger inhibitor SAR340835 with antiarrhythmic properties improved systolic function of failing hearts without creating hypotension, while reducing heart rate and restoring sympathovagal balance. SAR340835 may offer a unique and attractive pharmacological profile for patients with acute heart failure as compared with current inotrope, such as dobutamine., Competing Interests: No author has an actual or perceived conflict of interest with the contents of this article., (Copyright © 2021 by The Author(s).)

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

Study Objectives: Drug treatment for obstructive sleep apnea (OSA) is desirable because at least 30% of patients do not tolerate continuous positive airway pressure (CPAP) treatment. The negative pressure reflex (NPR) involving superficially located mechanoreceptors in the upper airway (UA) is an important mechanism for UA patency inhibitable by topical UA anesthesia (lidocaine). The NPR may serve as a target for pharmacological intervention for a topical treatment of OSA. The objective was to determine the effect of pharmacological augmentation of the NPR on UA collapsibility., Design: We developed a model of UA collapsibility in which application of negative pressures caused UA collapses in spontaneously breathing α-chloralose-urethane anesthetized pigs as indicated by characteristic tracheal pressure and air flow changes., Setting: N/A., Patients or Participants: N/A., Interventions: N/A., Measurements and Results: The potassium channel blocker AVE0118 administered topically to the UA in doses of 1, 3, and 10 mg per nostril sensitized the NPR, shifting the mechanoreceptor response threshold for the genioglossus muscle to more positive pressures (P < 0.001; n = 6 per group) and dose-dependently inhibited UA collapsibility. Ten mg of AVE0118 prevented UA collapses against negative pressures of -150 mbar (P < 0.01) for > 4 h in all pigs, while in control pigs the UA collapsed at -50 mbar or less negative pressures. The effect of AVE0118 was abolished by UA lidocaine anesthesia. Acute intravenous administration of naloxone or acetazolamide was ineffective; paroxetine and mirtazepine were weakly effective and fluoxetine was moderately effective in line with reported clinical efficacy., Conclusion: Topical administration of AVE0118 to the UA is a promising pharmacologic approach for the treatment of OSA.

Diet and its various components are consistently identified as among the most important 'risk factors' for cancer worldwide, yet great uncertainty remains regarding the relative contribution of nutritive (e.g., vitamins, calories) vs. non-nutritive (e.g., phytochemicals, fiber, contaminants) factors in both cancer induction and cancer prevention. Among the most potent known human dietary carcinogens is the mycotoxin, aflatoxin B(1) (AFB). AFB and related aflatoxins are produced as secondary metabolites by the molds Aspergillus flavus and Aspergillus parasiticus that commonly infect poorly stored foods including peanuts, pistachios, corn, and rice. AFB is a potent hepatocarcinogenic agent in numerous animal species, and has been implicated in the etiology of human hepatocellular carcinoma. Recent research has shown that many diet-derived factors have great potential to influence AFB biotransformation, and some efficiently protect from AFB-induced genotoxicity. One key mode of action for reducing AFB-induced carcinogenesis in experimental animals was shown to be the induction of detoxification enzymes such as certain glutathione-S-transferases that are regulated through the Keap1-Nrf2-ARE signaling pathway. Although initial studies utilized the dithiolthione drug, oltipraz, as a prototypical inducer of antioxidant response, dietary components such as suforaphane (SFN) are also effective inducers of this pathway in rodent models. However, human GSTs in general do not appear to be extensively induced by SFN, and GSTM1 - the only human GST with measurable catalytic activity toward aflatoxin B(1)-8,9-epoxide (AFBO; the genotoxic metabolite of AFB), does not appear to be induced by SFN, at least in human hepatocytes, even though its expression in human liver cells does appear to offer considerable protection against AFB-DNA damage. Although induction of detoxification pathways has served as the primary mechanistic focus of chemoprevention studies, protective effects of chemoprotective dietary components may also arise through a decrease in the rate of activation of AFB to AFBO. Dietary consumption of apiaceous vegetables inhibits CYP1A2 activity in humans, and it has been demonstrated that some compounds in those vegetables act as potent inhibitors of human CYP1A2 and cause reduced hCYP1A2-mediated mutagenicity of AFB. Other dietary compounds of different origin (e.g., constituents of brassica vegetables and hops) have been shown to modify expression of human hepatic enzymes involved in the oxidation of AFB. SFN has been shown to protect animals from AFB-induced tumors, to reduce AFB biomarkers in humans in vivo and to reduce efficiently AFB adduct formation in human hepatocytes, although it appears that this protective effect is the result of repression of human hepatic CYP3A4 expression, rather than induction of protective GSTs, at least in human hepatocytes. If this mechanism were to occur in vivo in humans, it would raise safety concerns for the use of SFN as a chemoprotective agent as it may have important implications for drug-drug interactions in humans. A dietary chemoprevention pathway that is independent of AFB biotransformation is represented by the potential for dietary components, such as chlorophyllin, to tightly bind to and reduce the bioavailability of aflatoxins. Chlorophyllin has been shown to significantly reduce genotoxic AFB biomarkers in humans, and it therefore holds promise as a practical means of reducing the incidence of AFB-induced liver cancer. Recent reports have demonstrated that DNA repair mechanisms are inducible in mammalian systems and some diet-derived compounds elevated significantly the gene expression of enzymes potentially involved in nucleotide excision repair of AFB-DNA adducts. However, these are initial observations and more research is needed to determine if dietary modulation of DNA repair is a safe and effective approach to chemoprevention of AFB-induced liver cancer., (Copyright © 2012. Published by Elsevier Ireland Ltd.)

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.

Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions.

This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol.

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K(+) channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K(+) channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K(+) channels.

The chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman) is a lead compound of potential class III antiarrhythmics that inhibit cardiac I(Ks) potassium channels. These channels are formed by the coassembly of KCNQ1 (Kv7.1, KvLQT1) and KCNE1 subunits. Although homomeric KCNQ1 channels are the principal molecular targets, entry of KCNE1 to the channel complex enhances the chromanol block. Because closely related neuronal KCNQ2 potassium channels are insensitive to the drug, we used KCNQ1/KCNQ2 chimeras to identify the binding site of the inhibitor. We localized the putative drug receptor to the H5 selectivity filter and the S6 transmembrane segment. Single residues affecting 293B inhibition were subsequently identified through systematic exchange of amino acids that were either different in KCNQ1 and KCNQ2 or predicted by a docking model of 293B in the open and closed conformation of KCNQ1. Mutant channel proteins T312S, I337V, and F340Y displayed dramatically lowered sensitivity to chromanol block. The predicted drug binding receptor lies in the inner pore vestibule containing the lower part of the selectivity filter, and the S6 transmembrane domain also reported to be important for binding of benzodiazepines. We propose that the block of the ion permeation pathway involves hydrophobic interactions with the S6 transmembrane residues Ile337 and Phe340, and stabilization of chromanol 293B binding through electrostatic interactions of its oxygen atoms with the most internal potassium ion within the selectivity filter.

The novel compound AVE1231 was investigated in order to elucidate its potential against atrial fibrillation. In CHO cells, the current generated by hKv1.5 or hKv4.3 + KChIP2.2b channels was blocked with IC50 values of 3.6 microM and 5.9 microM, respectively. In pig left atrial myocytes, a voltage-dependent outward current was blocked with an IC50 of 1.1 microM, mainly by accelerating the time constant of decay. Carbachol-activated IKACh was blocked by AVE1231 with an IC50 of 8.4 microM. Other ionic currents, like the IKr, IKs, IKATP, ICa, and INa were only mildly affected by 10 microM AVE1231. In guinea pig papillary muscle the APD90 and the upstroke velocity were not significantly altered by 30 microM AVE1231. In anesthetized pigs, oral doses of 0.3, 1, and 3 mg/kg AVE1231 caused a dose-dependent increase in left atrial refractoriness (LAERP), associated by inhibition of left atrial vulnerability to arrhythmia. There were no effects on the ECG intervals, ventricular monophasic action potentials, or ventricular refractory periods at 3 mg/kg AVE1231 applied intravenously. In conscious goats, both AVE1231 (3 mg/kg/h iv) and dofetilide (10 microg/kg/h iv) significantly prolonged LAERP. After 72 hours of tachypacing, when LAERP was shortened significantly (electrical remodelling), the prolongation of LAERP induced by AVE1231 was even more pronounced than in sinus rhythm. In contrast, the effect of dofetilide was strongly decreased. The present data demonstrate that AVE1231 blocks early atrial K channels and prolongs atrial refractoriness with no effects on ECG intervals and ventricular repolarisation, suggesting that it is suited for the prevention of atrial fibrillation in patients.

In this study, we analysed the inhibitory potency, blocking characteristics and putative binding sites of three structurally distinct Kv1.5 channel inhibitors on cloned human Kv1.5 channels. Obtained IC(50) values for S9947, MSD-D and ICAGEN-4 were 0.7 microM, 0.5 microM, and 1.6 microM, respectively. The Hill-coefficients were close to 1 for S9947 and approximately 2 for MSD-D and ICAGEN-4. All three compounds inhibited Kv1.5 channels preferentially in the open state, with Kv1.5 block displaying positive frequency dependence, but no clear voltage and potassium dependence. In contrast to slow on- and off-rates of apparent binding of MSD-D and ICAGEN-4, S9947 had fast on- and off-rates resulting in faster adaptation to changes in pulse frequency. Utilizing Alanine-scanning and in silico modeling we suggest binding of the compounds to the central cavity with crucial residues Ile508 and Val512 in the S6-segment. Residue Thr480 located at the base of the selectivity filter is important for ICAGEN-4 and S9947 inhibition, but less so for MSD-D binding. Our docking models suggest that the innermost potassium ion in the selectivity filter may form a tertiary complex with oxygens of S9947 and ICAGEN-4 and residue Thr480. This binding component is absent in the MSD-D block. As S9947 and ICAGEN-4 show faster block with proceeding channel openings, formation of this tertiary complex may increasingly stabilise binding of S9947 and ICAGEN-4, thereby explaining open channel block kinetics of these compounds.

In humans, apiaceous vegetables (carrots, parsnips, celery, parsley, etc.) inhibit cytochrome P-450 1A2, a biotransformation enzyme known to activate several procarcinogens, including aflatoxin B1 (AFB). We evaluated eight phytochemicals from apiaceous vegetables for effects on human cytochrome P-450 1A2 (hCYP1A2) activity using a methoxyresorufin O-demethylase (MROD) assay and a trp-recombination assay. Saccharomyces cerevisiae was used for heterologous CYP1A2 expression and this yeast strain is also diploid and auxotrophic for tryptophan due to mutations in the trp5 alleles. When these two alleles undergo AFB-induced mitotic recombination, gene conversion occurs, allowing yeast to grow in the absence of tryptophan. The apiaceous constituents psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and apigenin were potent inhibitors of hCYP1A2-mediated MROD activity in yeast microsomes, whereas quercetin was a modest hCYP1A2 inhibitor. Naringenin, caffeic acid, and chlorogenic acid did not inhibit hCYP1A2-mediated MROD activity. The 2-h pretreatment of intact yeast cells with psoralen, 5-MOP, and 8-MOP significantly improved cell survival after subsequent 4-h AFB treatment and reduced hCYP1A2-mediated mutagenicity of AFB. Apigenin also significantly decreased mutagenicity. These results suggest that in vivo CYP1A2 inhibition by apiaceous vegetables may be due to the phytochemicals present and imply that apiaceous vegetable intake may be chemopreventive by inhibiting CYP1A2-mediated carcinogen activation.

The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins.

The purpose of our investigation was to identify the transcriptional basis for ultrastructural and functional specialization of human atria and ventricles. Using exploratory microarray analysis (Affymetrix U133A+B), we detected 11,740 transcripts expressed in human heart, representing the most comprehensive report of the human myocardial transcriptome to date. Variation in gene expression between atria and ventricles accounted for the largest differences in this data set, as 3.300 and 2.974 transcripts showed higher expression in atria and ventricles, respectively. Functional classification based on Gene Ontology identified chamber-specific patterns of gene expression and provided molecular insights into the regional specialization of cardiomyocytes, correlating important functional pathways to transcriptional activity: Ventricular myocytes preferentially express genes satisfying contractile and energetic requirements, while atrial myocytes exhibit specific transcriptional activities related to neurohumoral function. In addition, several pro-fibrotic and apoptotic pathways were concentrated in atrial myocardium, substantiating the higher susceptibility of atria to programmed cell death and extracellular matrix remodelling observed in human and experimental animal models of heart failure. Differences in transcriptional profiles of atrial and ventricular myocardium thus provide molecular insights into myocardial cell diversity and distinct region-specific adaptations to physiological and pathophysiological conditions. Moreover, as major functional classes of atrial- and ventricular-specific transcripts were common to human and murine myocardium, an evolutionarily conserved chamber-specific expression pattern in mammalian myocardium is suggested.

Atrial fibrillation is associated with increased expression of ventricular myosin isoforms in atrial myocardium, regarded as part of a dedifferentiation process. Whether reexpression of ventricular isoforms in atrial fibrillation is restricted to transcripts encoding for contractile proteins is unknown. Therefore, this study compares atrial mRNA expression in patients with permanent atrial fibrillation to atrial mRNA expression in patients with sinus rhythm and to ventricular gene expression using Affymetrix U133 arrays. In atrial myocardium, we identified 1434 genes deregulated in atrial fibrillation, the majority of which, including key elements of calcium-dependent signaling pathways, displayed downregulation. Functional classification based on Gene Ontology provided the specific gene sets of the interdependent processes of structural, contractile, and electrophysiological remodeling. In addition, we demonstrate for the first time a prominent upregulation of transcripts involved in metabolic activities, suggesting an adaptive response to increased metabolic demand in fibrillating atrial myocardium. Ventricular-predominant genes were 5 times more likely to be upregulated in atrial fibrillation (174 genes upregulated, 35 genes downregulated), whereas atrial-specific transcripts were predominantly downregulated (56 genes upregulated, 564 genes downregulated). Overall, in fibrillating atrial myocardium, functional classes of genes characteristic of ventricular myocardium were found to be upregulated (eg, metabolic processes), whereas functional classes predominantly expressed in atrial myocardium were downregulated (eg, signal transduction and cell communication). Therefore, dedifferentiation with adoption of a ventricular-like signature is a general feature of the fibrillating atrium.

Kv4.3 channels conduct transient outward K(+) currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K(+) channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K(+) currents.

To obtain region- and disease-specific transcription profiles of human myocardial tissue, we explored mRNA expression from all four chambers of eight explanted failing [idiopathic dilated cardiomyopathy (DCM), n=5; ischemic cardiomyopathy (ICM), n=3], and five non-failing hearts using high-density oligonucleotide arrays (Affymetrix U95Av2). We performed pair-wise comparisons of gene expression in the categories (1) atria versus ventricles, (2) disease-regulated genes in atria and (3) disease-regulated genes in ventricles. In the 51 heart samples examined, 549 genes showed divergent distribution between atria and ventricles (272 genes with higher expression in atria, 277 genes with higher expression in ventricles). Two hundred and eighty-eight genes were differentially expressed in failing myocardium compared to non-failing hearts (19 genes regulated in atria and ventricles, 172 regulated in atria only, 97 genes regulated in ventricles only). For disease-regulated genes, down-regulation was 4.5-times more common than up-regulation. Functional classification according to Gene Ontology identified specific biological patterns for differentially expressed genes. Eleven genes were validated by RT-PCR showing a good correlation with the microarray data. Our goal was to determine a gene expression fingerprint of the heart, accounting for region- and disease-specific aspects. Recognizing common gene expression patterns in heart failure will significantly contribute to the understanding of heart failure and may eventually lead to the development of pathway-specific therapies.

We have investigated the effects of the ethacrynic acid derivative 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), an inhibitor of the volume-sensitive anion channel (VSAC), on electrical activity and insulin secretion in rat pancreatic beta-cells. DCPIB inhibited whole-cell VSAC currents in beta-cells with IC50 values of 2.2 and 1.7 microM for inhibition of outward and inward currents, respectively. DCPIB also inhibited the VSAC at the single channel level in cells activated by glucose. In intact cells, DCPIB caused a net increase in beta-cell input conductance and evoked an outward current that was sensitive to inhibition by tolbutamide, suggesting KATP channel activation. However, no KATP channel activation was evident under conventional whole-cell conditions, suggesting that the drug might activate the channel in intact cells via an indirect mechanism, possibly involving nutrient metabolism. DCPIB suppressed glucose-induced electrical activity in beta-cells, hyperpolarised the cell membrane potential at a substimulatory glucose concentration and prevented depolarisation when the glucose concentration was raised to stimulatory levels. The suppression of electrical activity by DCPIB was associated with a marked inhibition of glucose-stimulated insulin release from intact islets. It is concluded that DCPIB inhibits electrical and secretory activity in the beta-cell as a combined result of a reciprocal inhibition of VSAC and activation of KATP channel activities, thus producing a marked hyperpolarisation of the beta-cell membrane potential.

Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.

The HCN4 gene encodes a hyperpolarization-activated cation current contributing to the slow components of the pacemaking currents I(f) in the sinoatrial node and I(h) or I(q) in the thalamus. Heterologous expression studies of individual HCN channels have, however, failed to reproduce fully the diversity of native I(f/h/q) currents, suggesting the presence of modulating auxiliary subunits. Consistent with this is the recent description of KCNE2, which is highly expressed in the sinoatrial node, as a beta-subunit of rapidly activating HCN1 and HCN2 channels. To determine whether KCNE2 can also modulate the slow component of native I(f/h/q) currents, we co-expressed KCNE2 with HCN4 in Xenopus oocytes and in Chinese hamster ovary (CHO) cells and analysed the resulting currents using two-electrode voltage-clamp and patch-clamp techniques, respectively. In both cell types, co-expressed KCNE2 enhanced HCN4-generated current amplitudes, slowed the activation kinetics and shifted the voltage for half-maximal activation of currents to more negative voltages. In contrast, the related family members KCNE1, KCNE3 and KCNE4 did not change current characteristics of HCN4. Consistent with these electrophysiological results, the carboxy-terminal tail of KCNE2, but not of other KCNE subunits, interacted with the carboxy-terminal tail of HCN4 in yeast two-hybrid assays. KCNE2, by modulating I(f) or I(h) currents, might thus contribute to the electrophysiological diversity of known pacemaking currents in the heart and brain.

Objectives: The purpose of the study was to characterize the ionic and molecular mechanisms in the very early phases of electrical remodeling in a rabbit model of rapid atrial pacing (RAP)., Background: Long-term atrial fibrillation reduces L-type Ca(2+) (I(Ca,L)) and transient outward K(+) (I(to)) currents by transcriptional downregulation of the underlying ionic channels. However, electrical remodeling starts early after the onset of rapid atrial rates. The time course of ion current and channel modulation in these early phases of remodeling is currently unknown., Methods: Rapid (600 beats/min) right atrial pacing was performed in rabbits. Animals were divided into five groups with pacing durations between 0 and 96 h. Ionic currents were measured by patch clamp techniques; messenger ribonucleic acid (mRNA) and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot, respectively., Results: L-type calcium current started to be reduced (by 47%) after 12 h of RAP and continued to decline as pacing continued. Current changes were preceded or paralleled by decreased mRNA expression of the Ca(2+) channel beta subunits CaB2a, CaB2b, and CaB3, whereas significant reductions in the alpha(1) subunit mRNA and protein expression began 24 h after pacing onset. Transient outward potassium current densities were not altered within the first 12 h, but after 24 h, currents were reduced by 48%. Longer pacing periods did not further decrease I(to). Current changes were paralleled by reduced Kv4.3 mRNA expression. Kv4.2, Kv1.4, and the auxiliary subunit KChIP2 were not affected., Conclusions: L-type calcium current and I(to) are reduced in early phases of electrical remodeling. A major mechanism appears to be transcriptional downregulation of underlying ion channels, which partially preceded ion current changes.