Tomohiko Ai

Drug-induced long-QT syndrome associated with a subclinical SCN5A mutation.

Background—Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K+ channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na+ channel mutation in an individual who exhibited drug-induced LQTS. Methods and Results—An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na+ channel (SCN5A). The L1825P channel heterologously expressed in tsA-201 cells showed Na+ current with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A-associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na+ channel features characteristic of Brugada syndrome. Peak Na+ current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. Conclusions—This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.

Small-Conductance Calcium-Activated Potassium Channel and Recurrent Ventricular Fibrillation in Failing Rabbit Ventricles

Rationale: Fibrillation/defibrillation episodes in failing ventricles may be followed by action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (SVF). Objective: We hypothesized that activation of apamin-sensitive small-conductance Ca2+-activated K+ (SK) channels is responsible for the postshock APD shortening in failing ventricles. Methods and Results: A rabbit model of tachycardia-induced heart failure was used. Simultaneous optical mapping of intracellular Ca2+ and membrane potential (Vm) was performed in failing and nonfailing ventricles. Three failing ventricles developed SVF (SVF group); 9 did not (no-SVF group). None of the 10 nonfailing ventricles developed SVF. Increased pacing rate and duration augmented the magnitude of APD shortening. Apamin (1 &mgr;mol/L) eliminated recurrent SVF and increased postshock APD80 in the SVF group from 126±5 to 153±4 ms (P<0.05) and from 147±2 to 162±3 ms (P<0.05) in the no-SVF group but did not change APD80 in nonfailing group. Whole cell patch-clamp studies at 36°C showed that the apamin-sensitive K+ current (IKAS) density was significantly larger in the failing than in the normal ventricular epicardial myocytes, and epicardial IKAS density was significantly higher than midmyocardial and endocardial myocytes. Steady-state Ca2+ response of IKAS was leftward-shifted in the failing cells compared with the normal control cells, indicating increased Ca2+ sensitivity of IKAS in failing ventricles. The Kd was 232±5 nmol/L for failing myocytes and 553±78 nmol/L for normal myocytes (P=0.002). Conclusions: Heart failure heterogeneously increases the sensitivity of IKAS to intracellular Ca2+, leading to upregulation of IKAS, postshock APD shortening, and recurrent SVF.

Alpha-1-Syntrophin Mutation and the Long QT Syndrome: a disease of sodium channel disruption

Background— Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-α1 (SNTA1) is known to interact with the cardiac sodium channel (hNav1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNav1.5 by secondarily disturbing sodium channel function. Methods and Results— Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc≥480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G- SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNav1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation ( V h) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1. Conclusion— SNTA1 is a new susceptibility gene for LQTS. A257G- SNTA1 can cause gain-of-function of Nav1.5 similar to the LQT3. Received January 28, 2008; accepted May 12, 2008.Background— Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-α1 (SNTA1) is known to interact with the cardiac sodium channel (hNav1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNav1.5 by secondarily disturbing sodium channel function. Methods and Results— Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc≥480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G-SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNav1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation (Vh) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1. Conclusion— SNTA1 is a new susceptibility gene for LQTS. A257G-SNTA1 can cause gain-of-function of Nav1.5 similar to the LQT3.

Long QT syndrome with compound mutations is associated with a more severe phenotype: A Japanese multicenter study

BACKGROUND Long QT syndrome (LQTS) can be caused by mutations in the cardiac ion channels. Compound mutations occur at a frequency of 4% to 11% among genotyped LQTS cases. OBJECTIVE The purpose of this study was to determine the clinical characteristics and manner of onset of cardiac events in Japanese patients with LQTS and compound mutations. METHODS Six hundred three genotyped LQTS patients (310 probands and 293 family members) were divided into two groups: those with a single mutation (n = 568) and those with two mutations (n = 35). Clinical phenotypes were compared between the two groups. RESULTS Of 310 genotyped probands, 26 (8.4%) had two mutations in the same or different LQTS-related genes (compound mutations). Among the 603 LQTS patients, compound mutation carriers had significantly longer QTc interval (510 ± 56 ms vs 478± 53 ms, P = .001) and younger age at onset of cardiac events (10 ± 8 years vs 18 ± 16 years, P = .043) than did single mutation carriers. The incidence rate of cardiac events before age 40 years and use of beta-blocker therapy among compound mutation carriers also were different than in single mutation carriers. Subgroup analysis showed more cardiac events in LQTS type 1 (LQT1) and type 2 (LQT2) compound mutations compared to single LQT1 and LQT2 mutations. CONCLUSION Compound mutation carriers are associated with a more severe phenotype than single mutation carriers.

Novel KCNJ2 Mutation in Familial Periodic Paralysis With Ventricular Dysrhythmia

Background—Mutations in the KCNJ2 gene, which codes cardiac and skeletal inward rectifying K+ channels (Kir2.1), produce Andersen’s syndrome, which is characterized by periodic paralysis, cardiac arrhythmia, and dysmorphic features. Methods and Results—In 3 Japanese family members with periodic paralysis, ventricular arrhythmias, and marked QT prolongation, polymerase chain reaction/single-strand conformation polymorphism/DNA sequencing identified a novel, heterozygous, missense mutation in KCNJ2, Thr192Ala (T192A), which was located in the putative cytoplasmic chain after the second transmembrane region M2. Using the Xenopus oocyte expression system, we found that the T192A mutant was nonfunctional in the homomeric condition. Coinjection with the wild-type gene reduced the current amplitude, showing a weak dominant-negative effect. Conclusions—T192, which is located in the phosphatidylinositol-4,5-bisphosphate binding site and also the region necessary for Kir2.1 multimerization, is a highly conserved amino acid residue among inward-rectifier channels. We suggest that the T192A mutation resulted in the observed electrical phenotype.

Heterogeneous upregulation of apamin-sensitive potassium currents in failing human ventricles.

Background We previously reported that IKAS are heterogeneously upregulated in failing rabbit ventricles and play an important role in arrhythmogenesis. This study goal is to test the hypothesis that subtype 2 of the small‐conductance Ca2+ activated K+ (SK2) channel and apamin‐sensitive K+ currents (IKAS) are upregulated in failing human ventricles. Methods and Results We studied 12 native hearts from transplant recipients (heart failure [HF] group) and 11 ventricular core biopsies from patients with aortic stenosis and normal systolic function (non‐HF group). IKAS and action potential were recorded with patch‐clamp techniques, and SK2 protein expression was studied by Western blotting. When measured at 1 μmol/L Ca2+ concentration, IKAS was 4.22 (median) (25th and 75th percentiles, 2.86 and 6.96) pA/pF for the HF group (n=11) and 0.98 (0.54 and 1.72) pA/pF for the non‐HF group (n=8, P=0.008). IKAS was lower in the midmyocardial cells than in the epicardial and the endocardial cells. The Ca2+ dependency of IKAS in HF myocytes was shifted leftward compared to non‐HF myocytes (Kd 314 versus 605 nmol/L). Apamin (100 nmol/L) increased the action potential durations by 1.77% (−0.9% and 7.3%) in non‐HF myocytes and by 11.8% (5.7% and 13.9%) in HF myocytes (P=0.02). SK2 protein expression was 3‐fold higher in HF than in non‐HF. Conclusions There is heterogeneous upregulation of IKAS densities in failing human ventricles. The midmyocardial layer shows lower IKAS densities than epicardial and endocardial layers of cells. Increase in both Ca2+ sensitivity and SK2 protein expression contributes to the IKAS upregulation.

CFTR Gating I: Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel (ΔR-CFTR)

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

Apamin-Sensitive Potassium Current Modulates Action Potential Duration Restitution and Arrhythmogenesis of Failing Rabbit Ventricles

Background—Apamin-sensitive K currents (IKAS) are upregulated in heart failure. We hypothesize that apamin can flatten action potential duration restitution (APDR) curve and can reduce ventricular fibrillation duration in failing ventricles. Methods and Results—We simultaneously mapped membrane potential and intracellular Ca (Cai) in 7 rabbit hearts with pacing-induced heart failure and in 7 normal hearts. A dynamic pacing protocol was used to determine APDR at baseline and after apamin (100 nmol/L) infusion. Apamin did not change APD80 in normal ventricles, but prolonged APD80 in failing ventricles at either long (≥300 ms) or short (⩽170 ms) pacing cycle length, but not at intermediate pacing cycle length. The maximal slope of APDR curve was 2.03 (95% confidence interval, 1.73–2.32) in failing ventricles and 1.26 (95% confidence interval, 1.13–1.40) in normal ventricles at baseline (P=0.002). After apamin administration, the maximal slope of APDR in failing ventricles decreased to 1.43 (95% confidence interval, 1.01–1.84; P=0.018), whereas no significant changes were observed in normal ventricles. During ventricular fibrillation in failing ventricles, the number of phase singularities (baseline versus apamin, 4.0 versus 2.5), dominant frequency (13.0 versus 10.0 Hz), and ventricular fibrillation duration (160 versus 80 s) were all significantly (P<0.05) decreased by apamin. Conclusions—Apamin prolongs APD at long and short, but not at intermediate pacing cycle length in failing ventricles. IKAS upregulation may be antiarrhythmic by preserving the repolarization reserve at slow heart rate, but is proarrhythmic by steepening the slope of APDR curve, which promotes the generation and maintenance of ventricular fibrillation.

Block of pancreatic ATP-sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline

1 We investigated the effect of cibenzoline (a class Ia antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP‐sensitive K+ channels (KATP) in single pancreatic β cells of the same species, using radioimmunoassay and patch clamp techniques. 2 Micromolar cibenzoline had a dose‐dependent insulinotrophic action with an EC50 of 94.2±46.4 μm;. The compound inhibited the activity of the KATP channel recorded from a single β‐cell in a concentration‐dependent manner. The IC50 was 0.4 μm in the inside‐out mode and 5.2 μm in the cell‐ attached mode, at pH 7.4. 3 In the cell‐attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 μm at pH 6.2 to 0.9 μm at pH 8.4. On the other hand, the action of cibenzoline in the excised inside‐out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane. 4 In the inside‐out mode, micromolar ADP reactivated the cibenzoline‐blocked KATP channels in a manner similar to that by which ADP restored ATP‐dependent block of the channel. 5 The binding of [3H]‐glibenclamide to pancreatic islets was inhibited by glibenclamide but not by cibenzoline. In contrast, the [3H]‐cibenzoline binding was displaced by unlabelled cibenzoline but not by glibenclamide. It is concluded that cibenzoline blocks pancreatic KATP channels via a binding site distinct from the sulphonylurea receptor.

A ZASP Missense Mutation, S196L, Leads to Cytoskeletal and Electrical Abnormalities in a Mouse Model of Cardiomyopathy

Background—Dilated cardiomyopathy (DCM) is a primary disease of the heart muscle associated with sudden cardiac death secondary to ventricular tachyarrhythmias and asystole. However, the molecular pathways linking DCM to arrhythmias and sudden cardiac death are unknown. We previously identified a S196L mutation in exon 4 of LBD3-encoded ZASP in a family with DCM and sudden cardiac death. These findings led us to hypothesize that this mutation may precipitate both cytoskeletal and conduction abnormalities in vivo. Therefore, we investigated the role of the ZASP4 mutation S196L in cardiac cytoarchitecture and ion channel biology. Methods and Results—We generated and analyzed transgenic mice with cardiac-restricted expression of the S196L mutation. We also performed cellular electrophysiological analysis on isolated S196L cardiomyocytes and protein-protein interaction studies. Ten month-old S196L mice developed hemodynamic dysfunction consistent with DCM, whereas 3-month-old S196L mice presented with cardiac conduction defects and atrioventricular block. Electrophysiological analysis on isolated S196L cardiomyocytes demonstrated that the L-type Ca2+ currents and Na+ currents were altered. The pull-down assay demonstrated that ZASP4 complexes with both calcium (Cav1.2) and sodium (Nav1.5) channels. Conclusions—Our findings provide new insight into the mechanisms by which mutations of a structural/cytoskeletal protein, such as ZASP, lead to cardiac functional and electric abnormalities. This work represents a novel framework to understand the development of conduction defects and arrhythmias in subjects with cardiomyopathies, including DCM.