Tetsushi Furukawa

Meta-analysis identifies six new susceptibility loci for atrial fibrillation

Atrial fibrillation is a highly prevalent arrhythmia and a major risk factor for stroke, heart failure and death. We conducted a genome-wide association study (GWAS) in individuals of European ancestry, including 6,707 with and 52,426 without atrial fibrillation. Six new atrial fibrillation susceptibility loci were identified and replicated in an additional sample of individuals of European ancestry, including 5,381 subjects with and 10,030 subjects without atrial fibrillation (P < 5 × 10−8). Four of the loci identified in Europeans were further replicated in silico in a GWAS of Japanese individuals, including 843 individuals with and 3,350 individuals without atrial fibrillation. The identified loci implicate candidate genes that encode transcription factors related to cardiopulmonary development, cardiac-expressed ion channels and cell signaling molecules.

Nontranscriptional regulation of cardiac repolarization currents by testosterone.

Background—Women have longer QTc intervals than men and are at greater risk for arrhythmias associated with long QTc intervals, such as drug-induced torsade de pointes. Recent clinical and experimental data suggest an important role of testosterone in sex-related differences in ventricular repolarization. However, studies on effects of testosterone on ionic currents in cardiac myocytes are limited. Methods and Results—We examined effects of testosterone on action potential duration (APD) and membrane currents in isolated guinea pig ventricular myocytes using patch-clamp techniques. Testosterone rapidly shortened APD, with an EC50 of 2.1 to 8.7 nmol/L, which is within the limits of physiological testosterone levels in men. APD shortening by testosterone was mainly due to enhancement of slowly activating delayed rectifier K+ currents (IKs) and suppression of L-type Ca2+ currents (ICa,L), because testosterone failed to shorten APD in the presence of an IKs inhibitor, chromanol 293B, and an ICa,L inhibitor, nisoldipine. A nitric oxide (NO) scavenger and an inhibitor of NO synthase 3 (NOS3) reversed the effects of testosterone on APD, which suggests that NO released from NOS3 is responsible for the electrophysiological effects of testosterone. Electrophysiological effects of testosterone were reversed by a blocker of testosterone receptors, a c-Src inhibitor, a phosphatidylinositol 3-kinase inhibitor, and an Akt inhibitor. Immunoblot analysis revealed that testosterone induced phosphorylation of Akt and NOS3. Conclusions—The nontranscriptional regulation of IKs and ICa,L by testosterone is a novel regulatory mechanism of cardiac repolarization that can potentially contribute to the control of QTc intervals by androgen.

Differences in transient outward currents of feline endocardial and epicardial myocytes.

Whole-cell voltage-clamp experiments were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to test the hypothesis that the differences in the amplitude of transient outward current (Ito) contribute to the difference in action potential configuration between endocardial and epicardial myocytes. In the control state, action potentials recorded from epicardial cells demonstrated a prominent notch between phases 1 and 2, and membrane current recordings displayed a prominent Ito, whereas in endocardial cells the notch in action potentials and Ito were small. External application of 4-aminopyridine (2 mM) reduced the amplitudes of notch and Ito in epicardial cells but not in endocardial cells. After application of 4-aminopyridine (2 mM) and caffeine (5 mM), the notch and Ito were abolished completely in both endocardial and epicardial cells. The first component of Ito (Ito1) was present in all epicardial cells studied (n = 20); it was absent in 12 of the 20 endocardial cells, and a small Ito1 was present in the remaining eight endocardial cells. The mean amplitude of Ito1 was significantly greater in epicardial than in endocardial cells. At a test voltage of +80 mV, the amplitude of Ito1 was 102.0 +/- 47.7 pA/pF in epicardial cells and 3.3 +/- 3.3 pA/pF in endocardial cells (p less than 0.01). The second component of Ito (Ito2) was present in all endocardial (n = 30) and epicardial (n = 30) cells studied. The amplitude of Ito2 was significantly greater in epicardial than in endocardial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cardiac ATP-regulated potassium channels in differential responses of endocardial and epicardial cells to ischemia.

Epicardial cells are more susceptible to the electrophysiological effects of ischemia than are endocardial cells. To explore the ionic basis for the differential electrophysiological responses to ischemia at the two sites, we used patch-clamp techniques to study the effects of ATP depletion on action potential duration and the ability of ATP-regulated K+ channels in single cells isolated from feline left ventricular endocardial and epicardial surfaces. During ATP depletion by treatment with 1 mM cyanide (CN-), shortening of action potential durations was significantly greater in epicardial cells than in endocardial cells. Thirty minutes after initiating exposure to 1 mM CN-, action potential duration at 90% repolarization was reduced to 0.70 +/- 0.12 of the control value for endocardial cells versus 0.39 +/- 0.18 for epicardial cells (p less than 0.01), and action potential duration at 20% repolarization was reduced to 0.72 +/- 0.13 for endocardial cells versus 0.12 +/- 0.09 for epicardial cells (p less than 0.01). In both endocardial and epicardial cells, the shortening of action potential by CN- treatment was partially reversed by 0.3 microM glibenclamide; the magnitude of reversal, however, was much greater in epicardial cells. After exposure to 1 mM CN-, the activity of ATP-regulated K+ channels in cell-attached membrane patches was significantly greater in epicardial cells than in endocardial cells. To study the dose-response relation between ATP concentration and open-state probability of the channels, intracellular surfaces of inside-out membrane patches containing ATP-regulated K+ channels were exposed to various concentrations of ATP (10-1,000 microM). The concentration of ATP that produced half-maximal inhibition of the channel was 23.6 +/- 21.9 microM in endocardial cells and 97.6 +/- 48.1 microM in epicardial cells (p less than 0.01). These data indicate that ATP-regulated K+ channels are activated by a smaller reduction in intracellular ATP in epicardial cells than in endocardial cells. The differential ATP sensitivity of ATP-regulated K+ channels in endocardial and epicardial cells may be responsible for the differential shortening in action potentials during ischemia at the two sites.

Progesterone regulates cardiac repolarization through a nongenomic pathway: an in vitro patch-clamp and computational modeling study.

Background— Female sex is an independent risk factor for torsade de pointes in long-QT syndrome. In women, QT interval and torsade de pointes risk fluctuate dynamically during the menstrual cycle and pregnancy. Accumulating clinical evidence suggests a role for progesterone; however, the effect of progesterone on cardiac repolarization remains undetermined. Methods and Results— We investigated the effects of progesterone on action potential duration and membrane currents in isolated guinea pig ventricular myocytes. Progesterone rapidly shortened action potential duration, which was attributable mainly to enhancement of the slow delayed rectifier K+ current (IKs) under basal conditions and inhibition of L-type Ca2+ currents (ICa,L) under cAMP-stimulated conditions. The effects of progesterone were mediated by nitric oxide released via nongenomic activation of endothelial nitric oxide synthase; this signal transduction likely takes place in the caveolae because sucrose density gradient fractionation experiments showed colocalization of the progesterone receptor c-Src, phosphoinositide 3-kinase, Akt, and endothelial nitric oxide synthase with KCNQ1, KCNE1, and CaV1.2 in the caveolae fraction. We used computational single-cell and coupled-tissue action potential models incorporating the effects of progesterone on IKs and ICa,L; the model reproduces the fluctuations of cardiac repolarization during the menstrual cycle observed in women and predicts the protective effects of progesterone against rhythm disturbances in congenital and drug-induced long-QT syndrome. Conclusions— Our data show that progesterone modulates cardiac repolarization by nitric oxide produced via a nongenomic pathway. A combination of experimental and computational analyses of progesterone effects provides a framework to understand complex fluctuations of QT interval and torsade de pointes risks in various hormonal states in women.

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.

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of ×40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- ≥ Br- ≫ I- ≥ F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2ΔNT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2ΔNT-injected oocytes (-5.9 ± 0.4 μA), followed by ClC-2-injected oocytes (-4.3 ± 0.6 μA), and smallest in water-injected oocytes (-0.2 ± 0.2 μA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2ΔNT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to a hyposmolar environment.In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of x 40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- > or = Br- >> I- > or = F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2 delta NT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2 delta NT-injected oocytes (-5.9 +/- 0.4 microA), followed by ClC-2-injected oocytes (-4.3 +/- 0.6 microA), and smallest in water-injected oocytes (-0.2 +/- 0.2 microA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 +/- 0.2%) > ClC-2-injected oocytes (5.3 +/- 0.5%) > ClC-2 delta NT-injected oocytes (1.1 +/- 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopus oocytes appear to act for volume regulation when exposed to a hyposmolar environment.

Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias

Various types of Cl- currents have been recorded in cardiac myocytes from different regions of the heart and in different species. With few exceptions, most of these currents are not active under basal conditions, but are activated under the influence of various agonists and by physical stress. These channels are distributed nonuniformly, depending on the cell type, tissue and region of the heart. Therefore, Cl- current activation may influence membrane potential and impulse formation differently in different cells, and may play a role in arrhythmogenesis. Among these Cl- currents, the protein kinase A-activated Cl- current (I Cl.PKA), the stretch- or swelling-activated Cl- current (I Cl.SWELL) and the Ca(2+)-activated Cl current (I Cl.Ca) comprise the major anion currents that modify cardiac electrical activity. These currents exhibit outward-going rectification, or are predominantly activated at depolarized voltages and, thus, contribute significantly to shortening of the action potential duration but little to diastolic depolarization. The action potential shortening by Cl- current activation may not only perpetuate reentry by shortening the refractory period in a reentry pathway, but may also prevent the development of early afterdepolarization and triggered activity caused by the prolongation of action potentials. I Cl.Ca contributes to delayed afterdepolarization at diastolic potentials in Ca(2+)-overloaded cells. Another factor limiting the influence of Cl- currents on diastolic potentials is the presence of a predominantly opposing background K+ current, except at the nodal regions that lack these K+ channels, or under conditions of decreased K+ conductance. Therefore, the contribution of Cl- currents to the genesis of arrhythmias may depend on their association with the conductance of other ions, especially that of K+.

Acute effects of oestrogen on the guinea pig and human IKr channels and drug-induced prolongation of cardiac repolarization

Female gender is a risk factor for drug‐induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether‐a‐go‐go‐related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender‐differences in drug‐induced QT prolongation and baseline QTC intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17β‐oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug‐sensitivity of the hERG (IKr) channel in Langendorff‐perfused guinea pig hearts, patch‐clamped guinea pig cardiomyocytes and culture cells over‐expressing hERG. We found that physiological concentrations of E2 partially suppressed IKr in a receptor‐independent manner. E2‐induced modification of voltage‐dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug‐binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QTC prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031‐sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031‐induced QTC prolongation, and may provide a novel mechanism for the higher susceptibility to drug‐induced arrhythmia in women.

Integrating Genetic, Transcriptional, and Functional Analyses to Identify 5 Novel Genes for Atrial Fibrillation

Background— Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. Methods and Results— To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13–1.23; P=6.5×10−16), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06–1.14; P=2.2×10−8), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08–1.16; P=5.7×10−11), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06–1.14; P=9.8×10−9). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26–1.39; P=2.0×10−25) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08–1.16; P=3.9×10−9). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10−19), GJA1 (P=2.66×10−6), and TBX5 (P=1.36×10−5). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). Conclusions— We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.